Am J Physiol Cell Physiol Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 284: C127-C144, 2003. First published October 9, 2002; doi:10.1152/ajpcell.00250.2002
0363-6143/03 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
284/1/C127    most recent
00250.2002v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (11)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Takahata, T.
Right arrow Articles by Ishikawa, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takahata, T.
Right arrow Articles by Ishikawa, T.
Vol. 284, Issue 1, C127-C144, January 2003

SK4/IK1-like channels mediate TEA-insensitive, Ca2+-activated K+ currents in bovine parotid acinar cells

T. Takahata, M. Hayashi, and T. Ishikawa

Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although Ca2+-activated K+ (KCa) channels distinct from maxi-K+ channels have been suggested to contribute to muscarinically stimulated K+ currents in salivary acinar cells, the molecular nature of the channels is unclear. Using electrophysiological and RT-PCR techniques, we have now investigated the involvement of SK4/IK1-like channels in native KCa currents in bovine parotid acinar (BPA) cells. Ca2+-dependent K+ efflux from perfused bovine parotid tissues was not inhibited by a maxi-K+ channel blocker, tetraethylammonium (TEA). Whole cell recordings from BPA cells showed a TEA-insensitive KCa conductance, which was highly permeable to Rb+. In inside-out macropatches, TEA-insensitive Rb+ currents were activated by Ca2+ with half-maximal values of 0.4 µM. 1-Ethyl-2-benzimidazolinone (1-EBIO) increased the Ca2+ sensitivity of the currents. The calmodulin antagonists trifluoperazine, calmidazolium, and W-7 inhibited the Ca2+-activated Rb+ currents. In outside-out macropatches, Ca2+-activated Rb+ currents were inhibited by Ba2+, quinine, clotrimazole, and charybdotoxin but not by d-tubocrarine or apamin. RT-PCR analysis showed transcripts of SK4/IK1 in BPA cells. These results collectively suggest that SK4/IK1-like channels mediate the native KCa currents in BPA cells.

patch clamp; salivary secretion; HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS WELL ESTABLISHED that Ca2+-activated K+ (KCa) channels play a critical role in muscarinically stimulated fluid and electrolyte secretion in salivary glands (7). As in other secretory epithelia, the K+ channels in the basolateral membrane of acinar cells allow K+ to leave the cell and establish the apical (and basolateral) cell membrane potential more negative than the Nernst potential for anions such as Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, thereby providing a driving force for the sustained electrogenic anion efflux across the apical membrane. The K+ channels responsible for carrying the muscarinically stimulated outward K+ current during secretion have been investigated by using patch-clamp methods in salivary acinar cells in various species. The earliest patch-clamp studies first identified the presence of tetraethylammonium (TEA)-sensitive, large-conductance, voltage- and Ca2+-dependent K+ channels (BKCa or maxi-K+ channels) in the basolateral membrane of acinar cells (36). In cell-attached patch studies, maxi-K+ channels have been shown to be activated by addition of ACh or carbachol to the bathing solution (13, 37, 41, 53).

It has been suggested, however, by a number of other studies using whole cell patch-clamp techniques or K+ (and Rb+) efflux measurements that KCa channels, distinct from maxi-K+ channels, may also contribute significantly to the outward K+ current in the acinar cells of various salivary glands including rat parotid (29, 46), sheep parotid (20, 55), bovine parotid (34), rat mandibular (26, 27), and mouse mandibular glands (22). These studies have shown that a maxi-K+ channel blocker, TEA, is largely ineffective in inhibiting muscarinically or Ca2+-evoked K+ conductance. Available data for the TEA-insensitive, Ca2+-activated whole cell currents suggest that the channels are blocked by quinine and Ba2+ and have a significant conductance for Rb+ (20, 21, 26). A cell-attached patch study on mouse mandibular acinar cells has also shown the presence of a TEA-insensitive, 40-pS K+ channel that is activated by addition of ACh to the bathing solution, conductive for Rb+, and blocked by quinine (22). However, the molecular nature of the TEA-insensitive KCa channels responsible for muscarinically stimulated K+ currents have still remained unknown.

Recent molecular studies have identified four members (SK1-3 and SK4/IK1) of KCa channels that form Ca2+-activated, small- to intermediate-conductance K+ channels in various excitable and nonexcitable tissues. SK1-3 channels are predominantly expressed in excitable tissues (50), whereas SK4/IK1 channels are expressed in peripheral nonexcitable cells including secretory epithelia (24, 30, 31, 52), erythrocytes (49), and lymphocytes (32, 35). Intriguingly, Jensen et al. (30) reported that the highest level of transcripts of hSK4/IK1 was expressed in salivary glands. These finding prompted us to hypothesize that SK4/IK1 channels may mediate the native TEA-insensitive KCa currents in salivary acinar cells. In addition, the heterologously and naturally expressed SK4/IK1 currents have been shown to be relatively insensitive to TEA (Kd = 30-40 mM) (15, 31, 35), conductive for Rb+ (15, 30), and blocked by Ba2+ (15, 31), the properties compatible with described properties of native salivary KCa currents mentioned above. However, there is no direct functional and molecular evidence to suggest that SK4/IK1-like channels contribute to native KCa currents in salivary acinar cells.

The main aim of the present work thus was to characterize the biophysical and pharmacological properties of the native TEA-insensitive KCa currents in bovine parotid acinar (BPA) cells to provide a basis for the comparison to the described properties of expressed SK4/IK1 channels. In a K+ efflux study, we have first confirmed that ACh- and the Ca2+ ionophore A-23187-evoked K+ efflux from perfused bovine parotid tissue is not inhibited by TEA. Using the whole cell patch-clamp technique, we have then shown the presence of a TEA-insensitive KCa conductance with a significant Rb+ permeability in BPA cells. In inside-out and outside-out macropatches excised from basolateral membrane of BPA cells, we have demonstrated that biophysical and pharmacological properties of TEA-insensitive KCa currents strikingly resemble those of expressed SK4/IK1 currents described to date. Finally, RT-PCR analysis has confirmed the presence of the transcripts of SK4/IK1 in bovine parotid cells. To our knowledge, this is the first functional evidence for the involvement of SK4/IK1-like channels in native KCa currents in salivary acinar cells.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

K+ efflux study. Bovine parotid tissue was obtained from a local slaughterhouse (Hokkaido Ebetsu Meat Inspection Center, Ebetsu, Japan). The animals were both males and females. The tissue was put in a standard NaCl-rich solution on ice immediately after removal from the animals and then transported to the laboratory. Parotid tissue was dissected free of fat and connective tissue and sliced with a blade. The perfusion method used in the present study was similar to that described for rat parotid acinar cells previously (57). In brief, the perfusion column consisted of a 1-ml micropipette tip sealed with cotton wool. Approximately 250 mg of sliced bovine parotid tissues were placed between layers of Bio-Gel P-2 resin (Bio-Rad, Hercules, CA) dissolved in a standard perfusion solution on the column. The column, the top of which was covered with silicone cap, was kept in a water bath at 37°C. The sliced tissues were perfused at a flow rate of 1 ml/min with the standard NaCl-rich solution of the following composition (in mM): 110 NaCl, 25 NaHCO3, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, and 10 Na-acetate. The solution was equilibrated with 95% O2-5% CO2, and pH was 7.4. In a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solution, NaHCO3 (25 mM) was replaced with equimolar Na-glutamate, and the solution was equilibrated with 100% O2. The tissues were preperfused for at least 15 min before the start of each experiment.

The effluent of the perfusate was collected every 30 s and subjected to measurement of K+ concentration with a flame photometer (Corning 480) as described previously (27). Net efflux of K+ from the tissues (JK) was calculated by the following equation
J<SUB>K</SUB><IT>=F</IT>(C<SUB>o</SUB> − C<SUB>i</SUB>)
where F is flow rate and C is K+ concentration. Subscripts o and i indicate output and input solution of the column, respectively. Efflux of K+ from the tissues was assigned a positive value.

Patch-clamp experiments. The parotid glands were obtained as mentioned in K+ efflux study. Isolated acini and acinar cells were prepared as described for sheep parotid acinar cells previously (28). Briefly, small pieces of gland, trimmed of fat and connective tissue, were minced finely with scissors and incubated in a Mg2+- and Ca2+-free bathing solution containing collagenase (type II, 500 U/ml; Worthington, Freehold, NJ) for 30 min at 37°C in a shaking water bath. After gentle trituration with a 10-ml pipette, the medium was then replaced with a fresh collagenase-containing solution, and the tissue fragments were incubated once again for 30 min. The fragments were then sieved through a 148-µm nylon mesh. The suspension was then washed twice, resuspended in a standard NaCl-rich bathing solution, and stored at room temperature or at 4°C until used.

The cell preparations were pipetted on to a coverslip and transferred to a chamber mounted on an Olympus inverted microscope. Current recordings were made using the standard whole cell, inside-out, and outside-out configurations of the patch-clamp technique (18). The patch-clamp pipettes, which were pulled from glass capillaries (LG16; Dagan, Minneapolis, MN) using a vertical puller (model PP-830; Narishige, Tokyo, Japan), had resistances of about 2-5 MOmega when filled with a standard K-glutamate-rich solution. An Axopatch-1D patch-clamp amplifier (Axon Instruments, Foster City, CA) was used to measure the membrane currents. The reference electrode was a Ag-AgCl electrode, which was connected to the bath via an agar bridge filled with a standard NaCl-rich bathing solution. The amplifier was driven by pCLAMP6 software to allow the delivery of voltage-step protocols with concomitant digitization of the current. The membrane currents were filtered through an internal four-pole Bessel filter at 1 kHz and sampled at 2 kHz. Current-voltage (I-V) relationships were studied by using 10-mV voltage pulses, each of 400-ms duration, delivered at voltages ranging between -120 and 50 mV, and voltage pulses were separated by 3 s, during which the membrane potential was held at either -60 or 0 mV. As an alternative to voltage steps, voltage ramps were applied. Typically, the command voltage was varied from -120 or -80 mV to +50 mV over a duration of 800 ms every 10 s. The capacitance transient current in experiments where the membrane conductance was not activated was compensated by using the Axopatch-1D amplifier. In these experiments, the whole cell capacitance and the series resistance (Rs) were 22.2 ± 2.3 pF (n = 28) and 26.1 ± 2.5 MOmega (n = 26), respectively. The Rs was not electronically compensated during the experiments, and the potentials reported here have not been corrected for the Rs. The whole cell currents were not corrected for leakage. It was difficult to estimate the Rs properly in the experiments where the membrane conductance was activated by an increase in cytosolic Ca2+. In any case, the conductances of currents in the nanoampere range will be underestimated as a result of the voltage decrease across the Rs. We therefore performed excised inside-out or outside-out macropatch experiments to overcome the errors in the whole cell experiments and to characterize the currents more accurately. The pipette potential was corrected for the liquid junction potentials between pipette solution and the bath solution and between the bath solution and the agar bridge as described by Barry and Lynch (2) and Neher (38).

The experimental solutions are detailed in Table 1. The free Ca2+ concentrations of the pipette and bath solutions were calculated from an equation that takes into account the concentrations of Mg2+, Ca2+, EGTA (96% purity), and pH (40), and the appropriate amount of CaCl2 was added to the solution.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Experimental solutions in patch-clamp experiments

The relationship between membrane current and intracellular Ca2+ concentration ([Ca2+]i) at each membrane voltage was fit to the Hill equation
Y=Y<SUB>max</SUB>/[1 + (<IT>K</IT><SUB>d</SUB>/[Ca<SUP>2+</SUP>]<SUB>i</SUB>)<SUP><IT>n</IT><SUB>H</SUB></SUP>] (1)
where Y is K+ channel current, Ymax is the maximum current, Kd is the apparent dissociation constant, and nH is the Hill coefficient. In the context of this equation, the Hill coefficient controls the steepness of the relationship between K+ channel activation (current) and [Ca2+]i.

To analyze titration curves for Ba2+ inhibition of the current, we used the ratio I/I0 of current measured in the presence of Ba2+ (I) to that in its absence (I0), described by the following equation:
I/I<SUB>0</SUB>=K<SUB>i</SUB>(<IT>V</IT>)<IT>/</IT>(<IT>K</IT><SUB>i</SUB>(<IT>V</IT>) + A) (2)
where Ki(V) and A are the voltage-dependent inhibitory constant and the concentration of Ba2+, respectively.

In the case of a voltage-dependent block, Ki(V) has also been expressed by Woodhull (54) as a Boltzmann relationship with respect to the voltage as,
K<SUB>i</SUB>(<IT>V</IT>)<IT>=K</IT><SUB>i</SUB>(0) exp(<IT>z′FV/RT</IT>) (3)
where Ki(0) is the inhibitory constant at 0 mV, z' is a slope parameter, V is voltage, and F, R, and T have their conventional meanings. z' is equal to the product of the actual valence of the blocking ion z and the fraction of the membrane potential (or electrical distance) delta  acting on the ion.

Patch-clamp experiments were performed at room temperature (about 20°C). Bath solution changes were accomplished by gravity feed from reservoirs. The results were reported as means ± SE of several independent experiments (n), where n refers to the number of cells patched.

Statistical significance was evaluated by using the two-tailed paired and unpaired Student's t-test as appropriate. A value of P < 0.05 was considered significant.

Chemicals employed were of reagent grade. A-23187, TEA-Cl, clotrimazole, HEPES, EGTA, W-7-HCl [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, HCl], calmidazolium chloride (compound R24571), and trifluoperazine dihydrochloride were obtained from Sigma (Tokyo, Japan). ACh-Cl, atropine sulfate, quinine hydrochloride, and d-tubocrarine chloride were from Wako Chemicals (Osaka, Japan). 1-Ethyl-2-benzimidazolinone (1-EBIO) was from Tocris (Avonmouth, UK), and charybdotoxin and apamin were from Peptide Institute (Osaka, Japan).

RT-PCR. BPA cells were prepared as described in Patch-clamp experiments. Male Sprague-Dawley rats (200-380 g) were killed immediately by cervical dislocation, the submandibular glands were removed rapidly, and acinar cells were isolated as described for BPA cells. RT-PCR experiments were performed with mRNA extracted from bovine parotid and rat submandibular acinar cells prepared by using TRIzol reagent (GIBCO BRL, Tokyo, Japan) and a BioMag mRNA purification kit (Polysciences, Warrington, PA) following the manufacturer's instructions. First-strand cDNA was generated from mRNA using Superscript II RT (GIBCO BRL). The specific oligonucleotide primers for the PCR were 5'-CCTCCTACCGCAGCATCG-3' (sense) and 5'-TCCATCATGAAGTTGTGCAC-3' (antisense). The size of the expected fragments was 382 bp. The PCR reactions were performed with Taq polymerase (GIBCO BRL). The PCR conditioning was as follows: 35 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 1 min. As a control, beta -actin cDNA was amplified using the primers 5'-GACTACCTCATGAAGATCCT-3' (sense) and 5'-CCACATCTGCTGGAAGGTGG-3' (antisense), and a 510-bp product was obtained. Each PCR reaction was performed at least three times, and products were visualized by loading a 2-µl sample on a 1% agarose gel using a 100-bp DNA ladder marker (TOYOBO, Tokyo, Japan) as a standard. The fragments were first subcloned into pGEM-T Easy vector (Promega, Tokyo, Japan) and sequenced.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Characteristics of ACh- or A-23187-evoked K+ efflux from bovine parotid tissue. Stimulation of salivary glands with muscarinic agonists is well known to cause a large net K+ efflux due to activation of KCa channels on the basolateral membrane of the acinar cells (7). To examine whether TEA-insensitive KCa channels distinct from the maxi-K+ channels may play a crucial role in muscarinically stimulated K+ efflux in BPA cells, we measured net K+ flux to and from the perfused segments of the bovine parotid gland tissue using flame photometry. Figure 1A shows a typical example of the experiments indicating the dynamic changes of net K+ movement induced by ACh (10 µM). Stimulation with ACh for 3 min induced a transient net K+ efflux followed by a sustained net influx of K+ after the cessation of stimulation. When ACh was administered during two consecutive stimulations, with a 15-min period of recovery, the total amounts of net K+ efflux during the two stimulations were not different. These ACh-induced responses were completely inhibited by the muscarinic receptor antagonist atropine (1 µM) (data not shown). The dose-response relationship of the effect of ACh on net K+ efflux is also shown in Fig. 1A. Because stable and reproducible responses were obtained at a concentration of 10 µM ACh, we used this concentration in the remaining experiments.


View larger version (31K):
[in this window]
[in a new window]
  		Fig. 1.   A: time course of ACh (10 µM)-induced net K+ efflux from perfused bovine parotid fragments. Fragments were stimulated twice with ACh for 3 min. Inset: dose-response relation for the effect of ACh on net K+ efflux. Total net K+ efflux during the 2nd stimulation with various concentrations of ACh normalized to that during the 1st stimulation with ACh (10 µM) is plotted as a function of ACh concentration. Values are means ± SE of 4 experiments. B: effect of removal of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (25 mM)/CO2 (5%) from perfusing solution on ACh-induced net K+ efflux. Fragments were stimulated 3 times with ACh for 3 min in the presence and absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 in the perfusing solution. C: effect of removal of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 from perfusing solution on A-23187 (3 µM)-induced net K+ efflux. Perfusion of Ca2+-free solution containing 0.5 mM EGTA was begun before addition of A-23187. Net K+ efflux was evoked by addition of CaCl2 (3 mM) to standard perfusing solution in the presence and absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 in perfusing solution. D: summary of effect of removal of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 from perfusing solution on ACh (open bars)- or A-23187 (filled bars)-induced response. Total net K+ efflux during the 2nd or 3rd stimulation (in the absence or presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2, respectively) with ACh or A-23187 was expressed as a percentage of that of observed during the 1st stimulation with ACh or A-23187 (control). Values are means ± SE of 5 experiments. E and F: effect of tetraethylammonium (TEA; 10 mM) on the ACh (E)- or A-23187 (F)-induced response. Insets: total net K+ efflux during the 2nd (in presence of TEA) or 3rd stimulation with ACh was expressed as a percentage of that of observed during the 1st stimulation with ACh (control). Values are means ± SE of 7 experiments. F, inset: total net K+ efflux during the 2nd (in presence of TEA) or 3rd stimulation with A-23187 was expressed as a percentage of that of observed during the 1st stimulation with A-23187 (control). Values are means ± SE of 5 experiments.

The ACh-induced K+ efflux appeared to be dependent on cytosolic Ca2+ concentration for the following reasons. First, in experiments where tissues were stimulated twice with ACh under conditions in which CaCl2 had been removed from the perfusing solution (with addition of 0.5 mM EGTA), the first stimulation with ACh (10 µM) evoked a small and transient net K+ efflux, whereas the second stimulation with ACh induced a much smaller net K+ efflux (17.1 ± 2.1%, n = 5, of the control response). When CaCl2 (1 mM) was reintroduced into the perfusing solution, ACh was again able to evoke net K+ efflux. Second, the Ca2+ ionophore A-23187 (3 µM) with added Ca2+ (3 mM) was able to mimic the ACh-induced response (see also Fig. 1F).

We next examined the effect of removal of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 from the perfusing solution on the ACh-induced net K+ efflux. Figure 1B shows an example of the experiments demonstrating the absolute HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 requirement for ACh-induced net K+ efflux in bovine parotid fragments. The HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 dependency of the net K+ efflux in bovine parotid fragments was confirmed in experiments where the effect of removal of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 from the perfusing solution was examined on A-23187-induced net K+ efflux (Fig. 1C). The HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 requirement for ACh- and A-23187-induced net K+ efflux in bovine parotid fragments is summarized in Fig. 1D. In the control experiments, replacement of 25 mM Cl- with equimolar glutamate in the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 in the perfusing solution had no effect on the ACh-induced response (data not shown).

To assess the involvement of maxi-K+ channels in a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2-dependent, Ca2+-activated K+ efflux pathway, we next examined the effect of TEA (10 mM) on the ACh- and A-23187-induced net K+ efflux. As shown in Fig. 1, E and F, TEA (10 mM) did not inhibit the ACh- or A-23187-induced response. Quinine (1 mM) reduced the ACh-induced K+ efflux to 40.2 ± 4.5% (n = 4; P < 0.002) of the control response. Ba2+ (1 mM) had a tendency to inhibit the ACh-induced K+ efflux so that it reduced the efflux to 63.5 ± 11.2% (n = 5; P = 0.06) of the control response.

Characteristics of TEA-insensitive, Ca2+-activated whole cell K+ currents in BPA cells. Using the conventional whole cell patch-clamp technique, we next examined whether BPA cells would contain a TEA-insensitive KCa conductance. Figure 2A shows representative recordings of whole cell currents from single BPA cells dialyzed with the standard K-glutamate-rich pipette solution having 10-7 M free Ca2+. The bath solution was a standard Na-glutamate-rich solution. Under these experimental conditions, the steady-state whole cell I-V relationship of BPA cells had both outwardly and inwardly rectifying components (see also Fig. 2C). The outwardly rectifying current became evident at potentials more positive than -22 mV, had a characteristically noisy time course, and activated over 20-30 ms following depolarization. The outwardly rectifying conductance was strongly inhibited by TEA (10 mM), a maxi-K+ channel blocker. The current was carried by K+, because replacement of the K+ in the pipette solution by Cs+ or Na+ abolished the outward current (data not shown). The inwardly rectifying component was evident at potentials more negative than -82 mV, was characteristically noise free, and activated rapidly. The inwardly rectifying component was completely blocked by a low concentration of Ba2+ (0.1 mM) (data not shown). Our unpublished results suggest that the conductance is mediated by an inwardly rectifying K+ channel, Kir2.1 (M. Hayashi, S. Komazaki, and T. Ishikawa, unpublished observations).


View larger version (25K):
[in this window]
[in a new window]
  		Fig. 2.   A and B: representative whole cell recordings from a single bovine parotid acinar (BPA) cell with standard K-glutamate [pCa = 7 (A) or pCa = 6 (B)] pipette solution and standard Na-glutamate bath solution before and after addition of TEA (10 mM) to the bath. The cells were held at -62 mV and stepped for intervals of 400 ms to potentials ranging between -122 and +48 mV in 10-mV steps. C: steady-state current-voltage (I-V) relationships for TEA-insensitive whole cell currents from cells dialyzed with pipette solutions having pCa = 7 (open circle ; n = 4) and pCa = 6 (; n = 20), respectively. Vm, membrane potential.

Figure 2B shows representative recordings of the whole cell currents before and after addition of TEA (10 mM) to the bath solution for a single BPA cell dialyzed with the standard pipette solution with 10-6 M free Ca2+. In the presence of TEA (10 mM), depolarizing and hyperpolarizing voltage steps from -62 mV rapidly elicited whole cell currents with a reversal potential (-67.2 ± 1.4 mV, n = 14) close to the Nernst potential for K+ (-87.3 mV). Under these experimental conditions, the average current amplitude at -2 mV was 1,452.2 ± 288.2 pA (n = 13). The I-V relationships corresponding to the two different free Ca2+ concentrations (i.e., 10-7 and 10-6 M) are shown in Fig. 2C. In contrast to the cells dialyzed with pipette solutions with 10-7 free Ca2+, cells dialyzed with pipette solutions containing 10-6 M free Ca2+ exhibited a large whole cell outward current whose I-V relationship was nearly linear over a wide voltage range under a physiological Na+/K+ gradient. The reversal potential of the TEA-insensitive current was shifted from -80.1 ± 5.8 mV (n = 4) to +1.4 ± 0.7 mV (n = 12) with a slope of 44.2 mV per e-fold change when external K+ concentration was varied between 2 and 150 mM (Fig. 3A). We then performed experiments where K+ (150 mM) in the bath solution was replaced with equimolar Rb+ and found little, if any, change in reversal potential (0.0 ± 0.9 mV, n = 4) (Fig. 3B), suggesting that Rb+ had an equal permeability relative to K+. We also performed additional experiments in which cells were dialyzed in the standard Na-glutamate-rich bathing solution containing TEA (10 mM) with a pipette solution in which K+ was totally replaced with Rb+, Cs+, or Na+. In independent experiments for Cs+ or Na+, the average current amplitude at a holding potential of -3 or 0 mV was 16.6 ± 11.2 pA (n = 7) or -20.0 ± 70.0 pA (n = 7), respectively. In contrast with Cs+ or Na+ substitution experiments, when the cells were dialyzed with a Rb-glutamate-rich pipette solution, a large outward conductance was observed (Fig. 3C). In these experiments, the current amplitude at -2 mV and the reversal potential were 3,442.9 ± 917.7 pA (n = 7) and -66.6 ± 2.8 mV (n = 7), respectively. Taken together, these results suggest that the sequence of the relative monovalent cation permeabilities for the TEA-insensitive KCa conductance is K+ = Rb+ Na+ = Cs+.


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 3.   A: semilogarithmic plot of the reversal potential of TEA (10 mM)-insensitive whole cell currents as a function of extracellular K+ concentration ([K+]o). The cells were dialyzed with pipette solutions having pCa = 6. The continuous line shows the linear regression fit to the data. Values are means ± SE of 4-14 experiments. B: steady-state I-V relationships for TEA-insensitive whole cell currents from cells bathed in 150 mM K-glutamate-rich (open circle ) or 150 mM Rb-glutamate-rich () solution. The cells were dialyzed with pipette solutions having pCa = 6. Values are means ± SE of 4 experiments. C: steady-state I-V relationships for whole cell currents from cells dialyzed with Rb-glutamate-rich (; mean of 7 experiments) or Cs-glutamate-rich (open circle ; mean of 7 experiments) pipette solution having pCa = 6. The bath solution was the standard Na-glutamate solution. Error bars representing SE were omitted when so small as to lie within symbols. D: dependency of TEA-insensitive whole cell current activation on free Ca2+ concentrations ([Ca2+]i) in the pipette solution. Whole cell current amplitudes at -2 mV are plotted against [Ca2+]i in the pipette solution. Values are means ± SE of 4-20 experiments.

We next examined the Ca2+ dependency of the TEA-insensitive K+ currents in experiments in which cells were dialyzed with pipette solutions having different, buffered free Ca2+ concentrations (Fig. 3D). In contrast to the cells dialyzed with pipette solutions having 10-6 or 3 × 10-7 M free Ca2+ concentrations, cells dialyzed with pipette solutions containing either 10-7 or 10-8 M free Ca2+ concentrations exhibited only small whole cell outward currents. The average current amplitudes at -2 mV obtained from independent experiments with 10-7 and 10-8 M free Ca2+ were 184.5 ± 52.9 pA (n = 4) and 96.1 ± 26.6 pA (n = 6), respectively.

Ca2+-dependent activation of Rb+ currents in excised inside-out macropatches obtained from basolateral membrane of BPA cells. We further characterized the TEA-insensitive KCa conductance in inside-out or outside-out macropatches excised from basolateral membrane of acinar cells. In preparing dispersed cells from the bovine parotid gland, we could easily collect intact acini in which the cell orientation was undisturbed and routinely obtain excised patches. In these experiments, we used Rb+ as a charge carrier because the contribution of the conductance through inwardly rectifying K+ channels can be minimized (M. Hayashi, S. Komazaki, and T. Ishikawa, unpublished observations). We also included TEA (10 mM) in the pipette or the bath solution to block the maxi-K+ channels in inside-out or outside-out patch experiments, respectively. Figure 4A shows the representative tracings of currents elicited by 400-ms voltage steps from inside-out macropatches excised from basolateral membrane of acinar cells. Voltage-step commands evoked large, time-independent currents only when Ca2+ was included in the (bath) internal solution. Under these conditions, average current amplitudes of Ca2+ (1 µM)-activated Rb+ currents at -75 and -55 mV were -481.5 ± 48.4 pA (n = 41) and -371.2 ± 40.2 pA (n = 41), respectively. In separate experiments, as shown in Fig. 4B, we confirmed the monovalent cation selectivity of the Ca2+ (1 µM)-activated currents under these conditions. When the bathing solution was replaced from an N-methyl-D-glucamine (NMDG)-glutamate-rich to a Na-glutamate-rich solution, the reversal potential of the currents was not changed. However, K+ substitution for Na+ caused a shift of the reversal potential to close to zero. Figure 4C shows an example of inside-out macropatch experiments where free Ca2+ concentration in the fluid bathing the cytosolic surface of the patch was varied between <1 nM and 10 µM. Figure 4D plots Rb+ inward current amplitude at -55 mV against free Ca2+ concentration in the fluid bathing the cytosolic surface of the patch, demonstrating a steep Ca2+-dependent activation of the current. To provide a quantitative description of the Ca2+ dependence, we fit the Hill equation (see MATERIALS AND METHODS) to these data and obtained an apparent Kd of 0.43 ± 0.05 µM and a Hill coefficient of 2.54 ± 0.29 (n = 5) at -55 mV. Figure 4E also shows the plots of Kd and Hill coefficient against membrane potential and demonstrates that Ca2+ activation of the currents is independent of membrane potential. The mean Kd values and Hill coefficients were 0.46 ± 0.06 µM and 3.43 ± 0.51 at -95 mV, 0.47 ± 0.05 µM and 2.96 ± 0.41 at -75 mV, 0.47 ± 0.06 µM and 2.71 ± 0.34 at -35 mV, and 0.45 ± 0.06 µM and 2.52 ± 0.21 at -15 mV, respectively (n = 5).


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 4.   A: representative tracings of currents elicited by voltage steps from an inside-out macropatch excised from basolateral membrane of a BPA cell in the presence (left) or absence (right) of internal Ca2+ (1 µM). The membrane patch was held at +5 mV and stepped for intervals of 400 ms to potentials ranging between -95 and +55 mV in 10-mV steps. The pipette contained a Rb-glutamate-rich solution having 10 mM TEA, and the bath contained the standard NMDG-glutamate solution. B: current traces elicited by 800-ms voltage ramps from -75 to +55 mV from an inside-out macropatch excised from basolateral membrane of a BPA cell. The traces were obtained in the bathing solutions containing the indicated monovalent cations (K+, Na+, and NMDG+) in the presence of 1 µM free Ca2+. The pipette contained a Rb-glutamate-rich solution having 10 mM TEA. C: representative I-V relationships for currents in the presence of different [Ca2+]i (black-triangle, <1 nM; , 0.1 µM; , 0.3 µM; open circle , 1 µM; , 10 µM). The pipette contained a Rb-glutamate-rich solution having 10 mM TEA, and the bath solution was NMDG-glutamate rich. D: Ca2+ concentration response for TEA-insensitive Rb+ currents measured at -55 mV normalized by the response to Ca2+ (1 µM) [I/ICa2+ (1 µM)] plotted as a function of [Ca2+]i. Data were fit with the Hill equation (Eq. 1), yielding a Kd = 0.43 µM and a Hill coefficient = 2.54. Values are means ± SE of 5 experiments. E: voltage independence of Kd. Kd values (top) and Hill coefficient (bottom) plotted as functions of the membrane potential. Values are means ± SE of 5 experiments.

Pharmacological characteristics of TEA-insensitive, Ca2+-activated Rb+ currents in excised basolateral membrane macropatches. We next investigated the effect of K+ channel blockers on the TEA-insensitive, Ca2+-activated Rb+ currents in outside-out macropatches. The pipette contained a Rb-glutamate-rich solution with a 1 µM free Ca2+ concentration, and the bath contained a Rb-Cl-rich solution with TEA (10 mM). The addition of quinine (1 mM) to the bath inhibited Ca2+-activated Rb+ currents (Fig. 5A), and the inhibition was reversible and voltage independent (data not shown). Extracellular Ba2+ (1 and 10 mM) also reduced the currents in a concentration-dependent manner. In contrast to quinine, Ba2+ inhibited the currents in a voltage-dependent manner with the block increasing with hyperpolarization (Fig. 5B). The addition of Ba2+ (1 and 10 mM) to the bathing solution reduced the current amplitude at -65 mV to 79.2 ± 7.6% (P = 0.07) and 50.3 ± 7.2% (P < 0.01) of the control level (n = 5), respectively (Fig. 5B). We next analyzed the voltage dependence of the Ba2+ block (Fig. 5C). The data for 10 mM Ba2+ were fitted by using Eq. 2 (see MATERIALS AND METHODS) to determine the Kd at each potential. The following Kd values were estimated: 1.06 ± 0.12 mM at -125 mV, 2.05 ± 0.43 mM at -115 mV, 3.13 ± 0.62 mM at -105 mV, 4.75 ± 0.98 mM at -95 mV, 6.30 ± 1.18 mM at -85 mV, and 9.67 ± 1.86 mM at -75 mV (n = 3). These data were fitted by using Eq. 3 (see MATERIALS AND METHODS) with a Kd(0) of 266.71 ± 95.79 mM and a slope (delta ) of 0.53 ± 0.03 (n = 3).


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 5.   A: effect of quinine on Ca2+ (1 µM)-activated Rb+ currents in outside-out macropatches. Representative I-V relationships in the absence (black-triangle, control) and presence of quinine (, 0.1 mM; , 1 mM) obtained from an outside-out macropatch are shown (left). The pipette contained a Rb-glutamate-rich solution having 1 µM free Ca2+, and the bath contained a RbCl-rich solution containing 10 mM TEA. Quinine was applied to the bathing solution. open circle , Data obtained in a Rb+-free, NMDG-Cl-rich bath solution containing 10 mM TEA. Bar graph shows normalized current in the presence of quinine (0.1 and 1 mM) (right). Normalized current was determined by comparing the current amplitude at -65 mV. In each experiment, current was recorded in a Rb+-free, NMDG-Cl-rich solution and subtracted from each total current to calculate the Ca2+-activated Rb+ currents. *P < 0.05, **P < 0.01. B: effect of Ba2+ (, 0.1 mM; , 1 mM; and , 10 mM) on Ca2+ (1 µM)-activated Rb+ currents in outside-out macropatches. Representative I-V relationships in the absence (black-triangle, control) and presence of blocker obtained from an outside-out macropatch are shown (left). The pipette and the bath contained a Rb-glutamate-rich solution having 1 µM free Ca2+ and a RbCl-rich solution containing 10 mM TEA, respectively. open circle , Data obtained in a Rb+-free, NMDG-Cl-rich bath solution containing 10 mM TEA. Bar graph shows normalized current in the presence of Ba2+ (1 and 10 mM) (right). Normalized current was determined by comparing the current amplitude at -65 mV. In each experiment, current was recorded in a Rb+-free, NMDG-Cl-rich solution and subtracted from each total current to calculate the Ca2+-activated Rb+ currents. **P < 0.01. C: voltage dependence of Kd values for Ba2+ (10 mM) block of Ca2+ (1 µM)-activated Rb+ currents. Data were fitted with an equation derived from Eqs. 2 and 3 (see MATERIALS AND METHODS). Kd values were plotted on a semilogarithmic scale. In each experiment, current was recorded in a Rb+-free, NaCl-rich solution containing 10 mM TEA and subtracted from each total current to calculate the Ca2+-activated Rb+ currents. The continuous line shows the linear regression fit to the data. Values are means ± SE of 3 experiments.

We also examined several other compounds for their ability to block TEA-insensitive, Ca2+-activated Rb+ currents in outside-out macropatches. Figure 6, A and B, shows an example of experiments using clotrimazole or charybdotoxin, a known blocker of SK4/IK1 channel, respectively. Extracellular application of clotrimazole (0.1 and 1 µM) reduced the Rb+ currents in a dose-dependent manner (Fig. 6A). We also found that charybdotoxin (100 nM) strongly blocked the Rb+ currents (Fig. 6B). Clotrimazole- or charybdotoxin-induced inhibition of the currents was not voltage dependent (data not shown). In contrast to these inhibitors, neither apamin (100 nM) nor d-tubocrarine (100 µM), a known SK(1-3) channel blocker did reduce the currents (Fig. 6, C and D).


View larger version (38K):
[in this window]
[in a new window]
  		Fig. 6.   A: effect of external clotrimazole (0.1 and 1 µM) (A), charybdotoxin (100 nM) (B), apamin (100 nM) (C), or d-tubocrarine (100 µM) (D) on Ca2+ (1 µM)-activated Rb+ currents in outside-out macropatches. Representative instantaneous I-V relationships in the absence and presence of the blockers are shown. Insets: bar graphs showing normalized current in the presence of each blocker. Normalized current was determined by comparing the current amplitude at -65 mV in the absence (C) or presence of each blocker. In each experiment, current was recorded in a Rb+-free, NMDG-Cl-rich solution and subtracted from each total current to calculate the Ca2+-activated Rb+ currents. Values are means ± SE of 5 (A, D) or 6 (B, C) experiments. *P < 0.05, **P < 0.01.

We next examined the effect of 1-EBIO on TEA-insensitive, Ca2+-activated Rb+ currents in excised inside-out patches. 1-EBIO has been shown to activate heterologously expressed SK4/IK1 channels (30, 43, 51). Figure 7A illustrates the response of the Rb+ currents to cytoplasmic application of 1-EBIO (100 µM) at 0.3 µM free Ca2+. 1-EBIO induced robust activation of Rb+ currents at 0.3 µM free Ca2+. However, 1-EBIO did not induce currents in a Ca2+-free solution (<1 nM) and was not able to further increase the amplitude of the Rb+ currents when applied at a saturating free Ca2+ concentration of 1 µM (Fig. 7, B and C). These results suggest that 1-EBIO shifted the Ca2+ dependency of channel activation without changing the maximum level.


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 7.   A and B: effect of 1-ethyl-2-benzimidazolinone (1-EBIO; 100 µM) on TEA (10 mM)-insensitive Rb+ currents activated by 0.3 (A) or 1 µM Ca2+ (B). Representative traces of the currents elicited by 800-ms voltage ramps from -75 to +55 mV obtained from an inside-out macropatch are shown. The pipette contained a Rb-glutamate-rich solution having 10 mM TEA, and the bath contained a NMDG-glutamate-rich solution. C: Ca2+ concentration response for the native TEA-insensitive Rb+ currents in the absence (open circle ) and presence of 1-EBIO (). The current measured at -55 mV normalized by the response to Ca2+ (1 µM) in the absence of 1-EBIO is plotted as a function of [Ca2+]i. Values are means ± SE of 3-6 experiments, except for data point at 0.1 µM free Ca2+ obtained from a single experiment.

Calmodulin antagonists inhibit TEA-insensitive, Ca2+-activated Rb+ currents in BPA cells. To examine the role of calmodulin (CaM) in the Ca2+ activation of the currents, we next investigated the effects of structurally unrelated Ca2+/CaM antagonists, trifluoperazine (TFP), calmidazolium, and W-7, on the TEA-insensitive, Ca2+-activated Rb+ currents in inside-out macropatches. Representative current traces are shown in Fig. 8, A-C. In each experiment, Ca2+-independent current was recorded and subtracted from each total current to calculate the Ca2+-activated Rb+ currents. As shown, these compounds inhibited TEA-insensitive, Ca2+(1 µM)-activated Rb+ currents. When bath-applied during a recording, TFP (100 µM), calmidazolium (10 µM), and W-7 (50 µM) reduced the current to 43.0 ± 20.9% (n = 9; P < 0.0001), 4.7 ± 5.5% (n = 4; P < 0.0001), and 64.8 ± 25.3% (n = 6; P < 0.02) of the control currents at -55 mV, respectively. In contrast to the effects of calmidazolium, TFP and W-7 partially blocked the currents, although one experiment (of 9 experiments for TFP and of 6 experiments for W-7) showed a complete inhibition by these blockers. Inhibitory effects of TFP and W-7 were reversible, but the calmidazolium-induced inhibition was not. We also analyzed the voltage dependence of the inhibition, which is summarized in Fig. 8, D-F, by plotting the current amplitude as a function of membrane potential. None of these CaM antagonists showed a voltage-dependent block.


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 8.   Effects of the calmodulin antagonists trifluoperazine (TFP; 100 µM), calmidazolium (10 µM), and W-7 (50 µM) on TEA (10 mM)-insensitive Rb+ currents activated by 1 µM Ca2+. A-C: representative instantaneous I-V relationships of currents in inside-out macropatches in the absence and presence of TFP (A), calmidazolium (B), or W-7 (C). Ramp command voltages were applied from -75 to +55 mV. D-F: average current in the presence of TFP (D), calmidazolium (E), or W-7 (F) expressed as a percentage of control current at each membrane potential between -75 and +25 mV. Values are means ± SE of 4-9 experiments.

RT-PCR study. Because the above-described electrophysiological properties of native TEA-insensitive KCa currents identified in BPA cells are strikingly similar to those of expressed the SK4/IK1 channels, we next performed RT-PCR with mRNA of freshly isolated BPA cells to examine whether the cells contain the molecular candidate of the K+ channels. Because complete sequences for SK4/IK1 for Rattus norvegicus and Homo sapiens (but not for Bos taurus) have been published, we performed RT-PCR with specific primers for hSK4/IK1 and rSK4/IK1 (described in detail in MATERIALS AND METHODS). Under the experimental conditions chosen, we could detect the expected 382-bp amplicon. The results are summarized in Fig. 9A. The sequence analysis of the PCR fragment showed that the sequence was almost identical to those reported in human (GenBank accession nos. NM_002250 and AF000972), rat (NM_023021), and mouse SK4/IK1 (NM_008433) so that it shared 94, 87, and 90% nucleotide identity with them and 99, 96, and 98% similarity at the amino acid level, respectively. Because it has been shown that rat submandibular acinar cells exhibit the TEA-insensitive KCa currents, we used these same primers and performed RT-PCR with mRNA of freshly isolated rat submandibular acinar cells, also detecting the expected 382-bp amplicon (Fig. 9B), which was confirmed by sequence analysis to be the described rSK4/IK1 (NM_023021).


View larger version (73K):
[in this window]
[in a new window]
  		Fig. 9.   RT-PCR studies of BPA cells (A) and rat submandibular acinar cells (B). Amplified PCR products generated using gene-specific primers (described in MATERIALS AND METHODS) for SK4/IK1 and beta -actin were fractionated on 1% agarose gels, and size markers were used to indicate the size of the experimental fragments. Lane M, 100-bp DNA ladder size standard. RT, reverse transcriptase.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Muscarinically and Ca2+-evoked K+ efflux in BPA cells are mediated by TEA-insensitive, Ca2+-activated K+ channels distinct from maxi-K+ channels. To our knowledge, there is only one study of muscarinic agonist, carbachol-activated Ca2+-dependent K+ efflux from bovine parotid tissue using 86Rb+ as a K+ marker (34). The 86Rb+ efflux study showed that carbachol-induced 86Rb+ loss from bovine parotid was not blocked by 5 mM TEA. However, because described properties of maxi-K+ channels in salivary acinar cells do have a very low conductance for Rb+ (12), the involvement of maxi-K+ channels in mediating Ca2+-dependent K+ efflux might not have been assessed correctly. Therefore, we performed the present K+ flux measurements by using tissue fragments to ascertain whether maxi-K+ channels play an important role in Ca2+-dependent K+ efflux during muscarinically stimulated fluid secretion in bovine parotid as shown in other salivary glands (27). Two observations provided consistently negative answers. Neither ACh (10 µM)- nor Ca2+ ionophore A-23187 (3 µM)-induced net K+ efflux from perfused bovine parotid tissues was inhibited by TEA (10 mM) (Fig. 1, E and F). Because the concentration of TEA used in the present study is shown to completely block the maxi-K+ channels in BPA cells (data not shown), as also shown in various tissues, these results should reveal that a major part of the Ca2+-dependent K+ efflux, most probably across the basolateral membrane, is largely independent of maxi-K+ channels. This conclusion was further supported by the present whole cell patch-clamp studies demonstrating a TEA-insensitive KCa conductance in BPA cells (Fig. 2B). The view that the TEA (10 mM)-insensitive whole cell currents described in this study are mediated by KCa channels is strongly supported by the following observations: 1) the current was K+ selective over Na+ and Cs+ (Figs. 2C and 3, A and C), and 2) the current was activated by cytosolic Ca2+ (Fig. 3D). Although the localization of the TEA-insensitive KCa channels to the basolateral membrane or the apical membrane of the acinar cells cannot be decided from the whole cell patch-clamp experiments, the present study in macropatches excised from the basolateral membrane provides direct evidence for the basolateral localization of the channels. Our results do not exclude the possibility that the channels are also functionally expressed in apical membrane as well, however.

ACh- or A-23187-evoked net K+ efflux from the bovine parotid tissue was totally dependent on the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 in the perfusate (Fig. 1, B and C). These results are in good agreement with the previous 86Rb+ flux study with bovine parotid (34), which showed that carbachol-induced 86Rb+ loss in a nominally HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2-free solution was significantly less than in the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2. Interestingly, that study also showed that the 86Rb+ loss was inhibited by the anion channel blocker diphenylamino-2-carboxylate and unaffected by the complete replacement of extracellular Cl- with gluconate (34). Furthermore, a previous study with sheep parotid acinar cells also demonstrated that ACh caused a large, transient decrease in cytosolic pH, which was largely reduced in the absence of extracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2, blocked by the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate, and unaffected when extracellular Cl- was replaced by gluconate (47). Taken together with these previous data, assuming that the currently accepted model of salivary secretion would be applicable to bovine parotid secretion, the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2-dependent K+ efflux could be interpreted as a model showing that the secretion is driven by electrogenic apical HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux via anion channels that is electrically balanced by a simultaneous basolateral K+ efflux via Ca2+-activated K+ channels distinct from maxi-K+ channels. Although there is no direct electrophysiological evidence for the presence of a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-permeable anion channel in BPA cells at this time, it is interesting to note that a Ca2+-activated anion conductance in rat submandibular acinar cells has been shown to be permeable to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (25). Further patch-clamp studies are indeed required to answer the question of whether such a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-permeable anion conductance is also present in BPA cells.

The inhibition of ACh- or A23187-evoked net K+ efflux upon removal of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 could also be, at least in part, due to an inhibition of the TEA-insensitive KCa channels by changes in intracellular pH, because a transient intracellular acidification induced by ACh is known to be inhibited in a nominally HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2-free buffer in various salivary acinar cells including ruminant parotid acinar cells (7, 47). In fact, endogenously expressed SK4/IK1-like channels in T84 cells, human colonic adenocarcinoma cells, have been reported to be highly sensitive to cytosolic pH (9).

Comparison of native bovine parotid KCa currents with heterologously expressed SK4/IK1 currents and other native SK4/IK1-like currents. Biophysical properties of the native TEA-insensitive, Ca2+-activated K+ (Rb+) currents in BPA cells had the following features: 1) weakly inward rectifying I-V relationships in symmetrical Rb+ conditions and nearly linear I-V relationships in physiological Na+/K+ (Rb+) gradient (Figs. 2C, 3C, and 5A), 2) time- and voltage-independent activation (Fig. 4A), 3) a permeability (PX/PK) sequence estimated from reversal potentials indicating K+ = Rb+ Na+, NMDG+ (Figs. 3, B and C, and 4B) activation by submicromolar free Ca2+ (Figs. 3D and 4D). These characteristics of the native currents are in common with those of the naturally and heterologously expressed SK4/IK1 currents characterized at the single-channel and whole cell level (15, 24, 30, 31, 32, 35, 51) as follows: 1) inwardly rectified single channel (10-54 pS) and whole cell I-V relationships in symmetrical K+ conditions and nearly linear I-V relationships in physiological Na+/K+ gradient, 2) time- and voltage-independent activation of the whole cell currents and single-channel gating, 3) a permeability (PX/PK) sequence for whole cell current indicating K+ = Rb+ > Cs+ Na+, Li+, NMDG+, and 4) activation by submicromolar free Ca2+ concentration with an EC50 of 0.1-0.5 µM. Although we have not provided evidence for a similarity of the native channels to the expressed SK4/IK1 at the single-channel level in the present study, our preliminary inside-out patch experiments have shown an inwardly rectifying, Rb+-permeable, Ca2+-activated, intermediate-conductance (40-50 pS) K+ channel. Further studies are indeed required to identify a single-channel conductance that mediates the currents identified in the present study.

The pharmacological properties of the native TEA-insensitive, Ca2+-activated Rb+ currents in BPA cells had the following features: the native currents were blocked by clotrimazole (100 nM) (Fig. 6A) and charybdotoxin (100 nM) (Fig. 6B), but not by apamin (100 nM) and d-tubocrarine (0.1 mM) (Fig. 6, C and D). Clotrimazole has been reported to block expressed SK4/IK1 and native SK4/IK1-like currents (1, 4, 24, 30, 32, 35, 44) but not SK3-like currents (5). Charybdotoxin is shown to block with high affinity expressed SK4/IK1 currents (24, 30, 31, 32, 35) and SK4/IK1-like currents in the native human T lymphocyte and in red blood cells (Gardos channel) (4, 15, 32, 39, 44), whereas other SK channels are not affected (14, 19, 48). Conversely, apamin and d-tubocrarine have been shown to block the cloned SK1-3 channels with different affinities (16, 17, 24, 33, 45, 48), whereas there is no effect on heterologously expressed SK4/IK1 (31, 32, 35) and native SK4/IK1-like currents (3, 15, 32). Thus the pharmacological profiles of the native KCa currents in BPA cells are also similar to those of expressed SK4/IK1 channels but not to those of SK1-3 channels.

Among the blockers tested in the present study, only extracellular Ba2+ induced a voltage-dependent block of the TEA-insensitive, Ca2+-activated Rb+ currents in excised outside-out macropatches (Fig. 5, B and C). As expected for an ion binding within the membrane electrical field (54), the Kd (0 mV) for Ba2+ and the slope (delta ) of Ba2+ block were estimated to be 267 mM and delta  = 0.53, respectively. These results suggest that the Ba2+ binding site is located at an electrical distance half the way across the ion conductive pathway from the outside. A similar voltage-dependent block of Ba2+ has been reported for naturally expressed SK4/IK1-like whole cell currents in human T lymphocytes. The block of the currents by extracellular Ba2+ was also as steep as expected from the movement of a single divalent cation about 75% into the membrane field (delta  = 0.74) (15). To our knowledge, voltage-dependent block of Ba2+ has not been reported for the heterologously expressed SK4/IK1 channels, although there is a report showing that extracellular application of 1 mM Ba2+ reduced whole cell outward K+ currents by 88% elicited by membrane depolarization to +80 mV from the holding potential of -80 mV in Chinese hamster ovary (CHO) cells stably expressing hSK4/IK1 (31). Ca2+ (1 µM)-activated hSK4/IK1-like K+ currents in T84 cells, in which transcripts of hSK4/IK1 are expressed, have been shown to be blocked by intracellular Ba2+ but not by extracellular Ba2+ (9, 23).

We also extended the pharmacological study to include 1-EBIO, which has been shown to increase both the heterologously expressed SK4/IK1 (30, 32, 35, 43, 51, 52) and other native SK4/IK1-like currents (10, 32, 52). As expected from the striking resemblance between native bovine KCa and expressed SK4/IK1 currents, the native Ca2+-activated Rb+ currents were modulated by 1-EBIO (Fig. 7). We also showed in excised inside-out patches that 1-EBIO (100 µM) enhanced the currents induced by a low Ca2+ activity (0.3 µM), but it had no effect at a lower Ca2+ activity (<0.1 µM), and it did not further increase the currents at a higher Ca2+ activity (1 µM) (Fig. 7, B and C), suggesting that the activation of the native TEA-insensitive, Ca2+-activated Rb+ currents by 1-EBIO is Ca2+ dependent and likely due to a shift in Ca2+ sensitivity. Similar 1-EBIO modulations have been reported for the cloned SK4/IK1 channels (43, 51).

There is strong evidence that Ca2+ sensitivity of SK4/IK1 channels is mediated by CaM (11, 32, 56). It has also been proposed that 1-EBIO interacts with the intracellular COOH terminus of SK4/IK1 channels, most likely the CaM binding domain, and stabilizes the Ca2+-CaM channel interaction that drives channel opening (42). Therefore, 1-EBIO modulation and the steepness of intracellular Ca2+ dependence of native KCa currents in BPA cells may together suggest a cooperative mechanism of channel activation via a Ca2+-CaM channel interaction. In support of this view, all three CaM antagonists consistently induced a reduction of the native KCa currents in a voltage-independent manner (Fig. 8). We cannot exclude the possibility, however, that these drugs inhibited the currents via a Ca2+/CaM-independent mechanism. In fact, the published information on the effect of CaM inhibitors on SK4/IK1 channels is contradictory. Khanna et al. (32) found in conventional whole cell configuration that both expressed hSK4/IK1 currents in CHO cells and native human T-lymphocyte SK4/IK1-like currents were sensitive to the CaM antagonists W7, calmidazolium, and TFP. Conversely, Fanger et al. (11) showed that these antagonists were ineffective in blocking whole cell currents at physiological membrane potentials recorded from native T lymphocytes and hIK1-transfected COS-7 cells. Von Hahn et al. (51) found in inside-out patches that TFP and W-7 reversibly inhibited expressed rSK4 currents in Xenopus oocytes but that calmidazolium had no effect even at 10 µM. However, native IKCa channels (Gardos channels) of human erythrocytes have been shown not to be inhibited by these CaM antagonists in cell-attached and in excised inside-out patches (8). Further studies are thus needed to elucidate the role of CaM in Ca2+-dependent activation of the native SK4/IK1-like currents.

Physiological role of SK4/IK1-like channels in BPA and other salivary acinar cells. Unlike other well-studied salivary glands, little is known about the secretory mechanism in bovine parotid by which large volumes of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-rich saliva are produced not only in response to a secretomotor stimulus but also at rest (6). Because it is well established that muscarinic stimulation of salivary gland to drive fluid and electrolyte secretion is tightly coupled to [Ca2+]i (7), it is reasonable to conclude that the SK4/IK1-like channels identified in the present work may be responsible for muscarinically stimulated secretion but not for resting secretion by bovine parotid gland. This view is supported by several lines of evidence. First, the SK4/IK1-like channels described here exhibited the sensitivity needed to become active during a rise in cytosolic free Ca2+ concentration above 10-7 M (Figs. 3D and 4D). On the other hand, when BPA cells were dialyzed with the standard K-glutamate-rich pipette solution containing 10-7 M free Ca2+, or when the cytosolic surfaces of excised inside-out patches were exposed to the bathing solution containing 10-7 M free Ca2+, the SK4/IK1-like channels were almost inactive (Figs. 3D and 4D). Under these conditions, the whole cell K+ conductance in these cells was dominated by outwardly and inwardly rectifying components, attributable to maxi-K+ channels and inwardly rectifying K+ channels, respectively (Fig. 2A) (M. Hayashi, S. Komazaki, and T. Ishikawa, unpublished observations). Second, the high Rb+ conductivity (Fig. 3, B and C) and charybdotoxin sensitivity (Fig. 6B) of the channels are also in keeping with previous 86Rb+ efflux data in a bovine parotid mince preparation (34), where muscarinic agonist (carbachol)-induced 86Rb+ loss was inhibited by Leiurus quinquestriatus venom, which contains charybdotoxin (34). Finally, quinine and Ba2+ sensitivities of the channels are in line with the present K+ efflux data indicating that quinine (1 mM) and Ba2+ (1 mM) reduced the ACh-induced K+ efflux. In the present study, we found that Ba2+ (1 mM) had a tendency to inhibit the ACh-induced K+ efflux, but the effect was not statistically significant (P = 0.06). Given that extracellular Ba2+ blocks the SK4/IK1-like currents in a voltage-dependent manner (Fig. 5, B and C), Ba2+ may not necessarily block the K+ efflux significantly at physiological membrane potentials in intact tissues. In fact, extracellular Ba2+ (1 mM) only reduced the current amplitude at -65 mV to 79.2 ± 7.6% of the control level (P = 0.07) (Fig. 5B).

An important question arising from the present study concerns the role of SK4/IK1-like channels in mediating Ca2+-activated K+ currents in other salivary acinar cells. As mentioned previously, electrophysiological profiles of the SK4/IK1-like currents in BPA cells have properties similar to those of Ca2+- or ACh-activated whole cell K+ conductance reported in other native salivary cells such as rat and mouse submandibular acinar cells (21, 26) and in sheep parotid acinar cells (20). Furthermore, our RT-PCR analysis has also shown transcripts of rSK4/IK1 in rat submandibular acinar cells (Fig. 9B). The recent finding that human parotid acinar cells exhibit charybdotoxin-sensitive, carbachol-induced whole cell K+ currents (41) is also tempting to postulate for a possible role of SK4/IK1 channels in mediating muscarinically stimulated K+ currents in human as well. Therefore, it is highly possible that SK4/IK1-like channels are commonly involved in mediating K+ currents during muscarinically evoked secretion by salivary glands in various species.


    ACKNOWLEDGEMENTS

We thank Dr. K. Yoshimura for valuable advice on the perfusion method and A. Inagaki for help in analyzing data and for comments on the manuscript.


    FOOTNOTES

This study was supported by grants from the Ito Foundation, Northern Advancement Center for Science & Technology Foundation, and in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sport and Culture of Japan. M. Hayashi was supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists.

Address for reprint requests and other correspondence: T. Ishikawa, Laboratory of Physiology, Dept. of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido Univ., Sapporo 060-0818, Japan (E-mail: torui{at}vetmed.hokudai.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published October 9, 2002;10.1152/ajpcell.00250.2002

Received 30 May 2002; accepted in final form 11 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alvarez, J, Montero M, and Garcia-Sancho J. High affinity inhibition of Ca2+-dependent K+ channels by cytochrome P-450 inhibitors. J Biol Chem 267: 11789-11793, 1992[Abstract/Free Full Text].

2.   Barry, PH, and Lynch JW. Liquid junction potentials and small cell effects in patch-clamp analysis. J Membr Biol 121: 101-117, 1991[Web of Science][Medline].

3.   Brugnara, C, Armsby CC, De Franceschi L, Crest M, Euclaire MF, and Alper SL. Ca2+-activated K+ channels of human and rabbit erythrocytes display distinctive patterns of inhibition by venom peptide toxins. J Membr Biol 147: 71-82, 1995[Web of Science][Medline].

4.   Brugnara, C, De Franceschi L, and Alper SL. Ca2+-activated K+ transport in erythrocytes. Comparison of binding and transport inhibition by scorpion toxins. J Biol Chem 268: 8760-8768, 1993[Abstract/Free Full Text].

5.   Carignani, C, Roncarati R, Rimini R, and Terstappen GC. Pharmacological and molecular characterisation of SK3 channels in the TE671 human medulloblastoma cell line. Brain Res 939: 11-18, 2002[Web of Science][Medline].

6.   Church, DC. Salivary function and production. In: The Ruminant Animal. Digestive Physiology and Nutrition, edited by Church DC.. Englewood Cliffs, NJ: Prentice Hall, 1988, p. 117-124.

7.   Cook, DI, Van Lennep EW, Roberts ML, and Young JA. Secretion by the Major Salivary Glands. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by Johnson LR, Alpers DH, Christensen J, Jacobson ED, and Walsh JH.. New York: Raven, 1994, p. 1061-1117.

8.   Del Carlo, B, Pellegrini M, and Pellegrino M. Calmodulin antagonists do not inhibit IKCa channels of human erythrocytes. Biochim Biophys Acta 1558: 133-141, 2002[Medline].

9.   Devor, DC, and Frizzell RA. Calcium-mediated agonists activate an inwardly rectified K+ channel in colonic secretory cells. Am J Physiol Cell Physiol 265: C1271-C1280, 1993[Abstract/Free Full Text].

10.   Devor, DC, Singh AK, Frizzell RA, and Bridges RJ. Modulation of Cl- secretion by benzimidazolones. I. Direct activation of a Ca2+-dependent K+ channel. Am J Physiol Lung Cell Mol Physiol 271: L775-L784, 1996[Abstract/Free Full Text].

11.   Fanger, CM, Ghanshani S, Logsdon NJ, Rauer H, Kalman K, Zhou J, Beckingham K, Chandy KG, Cahalan MD, and Aiyar J. Calmodulin mediates calcium-dependent activation of the intermediate conductance KCa channel, IKCa1. J Biol Chem 274: 5746-5754, 1999[Abstract/Free Full Text].

12.   Gallacher, DV, Maruyama Y, and Petersen OH. Patch-clamp study of rubidium and potassium conductances in single cation channels from mammalian exocrine acini. Pflügers Arch 401: 361-367, 1984[Web of Science][Medline].

13.   Gallacher, DV, and Morris AP. A patch-clamp study of potassium currents in resting and acetylcholine-stimulated mouse submandibular acinar cells. J Physiol 373: 379-395, 1986[Abstract/Free Full Text].

14.   Grissmer, S, Lewis RS, and Cahalan MD. Ca2+-activated K+ channels in human leukemic T cells. J Gen Physiol 99: 63-84, 1992[Abstract/Free Full Text].

15.   Grissmer, S, Nguyen AN, and Cahalan MD. Calcium-activated potassium channels in resting and activated human T lymphocytes. Expression levels, calcium dependence, ion selectivity, and pharmacology. J Gen Physiol 102: 601-630, 1993[Abstract/Free Full Text].

16.   Grunnet, M, Jensen BS, Olesen SP, and Klaerke DA. Apamin interacts with all subtypes of cloned small-conductance Ca2+-activated K+ channels. Pflügers Arch 441: 544-550, 2001[Web of Science][Medline].

17.   Grunnet, M, Jespersen T, Angelo K, Frøkjaer-Jensen C, Klaerke DA, Olesen SP, and Jensen BS. Pharmacological modulation of SK3 channels. Neuropharmacology 40: 879-887, 2001[Web of Science][Medline].

18.   Hamill, OP, Marty A, Neher E, Sakmann B, and Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391: 85-100, 1981[Web of Science][Medline].

19.   Hanselmann, C, and Grissmer S. Characterization of apamin-sensitive Ca2+-activated potassium channels in human leukaemic T lymphocytes. J Physiol 496: 627-637, 1996[Abstract/Free Full Text].

20.   Hayashi, T, Hirono C, Young JA, and Cook DI. The ACh-induced whole-cell currents in sheep parotid secretory cells. Do BK channels really carry the ACh-evoked whole-cell K+ current? J Membr Biol 144: 157-166, 1995[Web of Science][Medline].

21.   Hayashi, T, Poronnik P, Young JA, and Cook DI. The ACh-evoked, Ca2+-activated whole-cell K+ current in mouse mandibular secretory cells. Whole-cell and fluorescence studies. J Membr Biol 152: 253-259, 1996[Web of Science][Medline].

22.   Hayashi, T, Young JA, and Cook DI. The Ach-evoked Ca2+-activated K+ current in mouse mandibular secretory cells. Single channel studies. J Membr Biol 151: 19-27, 1996[Web of Science][Medline].

23.   Huber, SM, Tschöp J, Braun GS, Nagel W, and Horster MF. Bradykinin-stimulated Cl- secretion in T84 cells. Role of Ca2+-activated hSK4-like K+ channels. Pflügers Arch 438: 53-60, 1999[Web of Science][Medline].

24.   Ishii, TM, Silvia C, Hirschberg B, Bond CT, Adelman JP, and Maylie J. A human intermediate conductance calcium-activated potassium channel. Proc Natl Acad Sci USA 94: 11651-11656, 1997[Abstract/Free Full Text].

25.   Ishikawa, T. A bicarbonate- and weak acid-permeable chloride conductance controlled by cytosolic Ca2+ and ATP in rat submandibular acinar cells. J Membr Biol 153: 147-159, 1996[Web of Science][Medline].

26.   Ishikawa, T, and Murakami M. Tetraethylammonium-insensitive, Ca2+-activated whole-cell K+ currents in rat submandibular acinar cells. Pflügers Arch 429: 748-750, 1995[Web of Science][Medline].

27.   Ishikawa, T, Murakami M, and Seo Y. Basolateral K+ efflux is largely independent of maxi-K+ channels in rat submandibular glands during secretion. Pflügers Arch 428: 516-525, 1994[Web of Science][Medline].

28.   Ishikawa, T, Wegman EA, and Cook DI. An inwardly rectifying potassium channel in the basolateral membrane of sheep parotid secretory cells. J Membr Biol 131: 193-202, 1993[Web of Science][Medline].

29.   Iwatsuki, N, Maruyama Y, Matsumoto O, and Nishiyama A. Activation of Ca2+-dependent Cl- and K+ conductances in rat and mouse parotid acinar cells. Jpn J Physiol 35: 933-944, 1985[Web of Science][Medline].

30.   Jensen, BS, Strøbaek D, Christophersen P, Jørgensen TD, Hansen C, Silahtaroglu A, Olesen SP, and Ahring PK. Characterization of the cloned human intermediate-conductance Ca2+-activated K+ channel. Am J Physiol Cell Physiol 275: C848-C856, 1998[Abstract/Free Full Text].

31.   Joiner, WJ, Wang LY, Tang MD, and Kaczmarek LK. hSK4, a member of a novel subfamily of calcium-activated potassium channels. Proc Natl Acad Sci USA 94: 11013-11018, 1997[Abstract/Free Full Text].

32.   Khanna, R, Chang MC, Joiner WJ, Kaczmarek LK, and Schlichter LC. hSK4/hIK1, a calmodulin-binding KCa channel in human T lymphocytes. Roles in proliferation and volume regulation. J Biol Chem 274: 14838-14849, 1999[Abstract/Free Full Text].

33.   Köhler, M, Hirschberg B, Bond CT, Kinzie JM, Marrion NV, Maylie J, and Adelman JP. Small-conductance, calcium-activated potassium channels from mammalian brain. Science 273: 1709-1714, 1996[Abstract/Free Full Text].

34.   Lee, SI, and Turner RJ. Secretagogue-induced 86Rb+ efflux from bovine parotid is HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> dependent. Am J Physiol Regul Integr Comp Physiol 264: R162-R168, 1993[Abstract/Free Full Text].

35.   Logsdon, NJ, Kang J, Togo JA, Christian EP, and Aiyar J. A novel gene, hKCa4, encodes the calcium-activated potassium channel in human T lymphocytes. J Biol Chem 272: 32723-32726, 1997[Abstract/Free Full Text].

36.   Maruyama, Y, Gallacher DV, and Petersen OH. Voltage and Ca2+-activated K+ channel in baso-lateral acinar cell membranes of mammalian salivary glands. Nature 302: 827-829, 1983[Medline].

37.   Morris, AP, Gallacher DV, Fuller CM, and Scott J. Cholinergic receptor-regulation of potassium channels and potassium transport in human submandibular acinar cells. J Dent Res 66: 541-546, 1987[Abstract/Free Full Text].

38.   Neher, E. Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol 207: 123-131, 1992[Web of Science][Medline].

39.   Ohnishi, ST, Katagi H, and Katagi C. Inhibition of the in vitro formation of dense cells and of irreversibly sickled cells by charybdotoxin, a specific inhibitor of calcium-activated potassium efflux. Biochim Biophys Acta 1010: 199-203, 1989[Medline].

40.   Oiki, S, and Okada Y. Ca-EGTA buffer in physiological solutions. Seitai-no-kagaku 38: 79-83, 1987. (In Japanese.)

41.   Park, K, Case RM, and Brown PD. Identification and regulation of K+ and Cl- channels in human parotid acinar cells. Arch Oral Biol 46: 801-810, 2001[Web of Science][Medline].

42.   Pedarzani, P, Mosbacher J, Rivard A, Cingolani LA, Oliver D, Stocker M, Adelman JP, and Fakler B. Control of electrical activity in central neurons by modulating the gating of small conductance Ca2+-activated K+ channels. J Biol Chem 276: 9762-9769, 2001[Abstract/Free Full Text].

43.   Pedersen, KA, Schrøder RL, Skaaning-Jensen B, Strøbaek D, Olesen SP, and Christophersen P. Activation of the human intermediate-conductance Ca2+-activated K+ channel by 1-ethyl-2-benzimidazolinone is strongly Ca2+-dependent. Biochim Biophys Acta 1420: 231-240, 1999[Medline].

44.   Rittenhouse, AR, Vandorpe DH, Brugnara C, and Alper SL. The antifungal imidazole clotrimazole and its major in vivo metabolite are potent blockers of the calcium-activated potassium channel in murine erythroleukemia cells. J Membr Biol 157: 177-191, 1997[Web of Science][Medline].

45.   Shah, M, and Haylett DG. The pharmacology of hSK1 Ca2+-activated K+ channels expressed in mammalian cell lines. Br J Pharmacol 129: 627-630, 2000[Web of Science][Medline].

46.   Shigetomi, T, Hayashi T, Ueda M, Kaneda T, Tokuno H, Takai A, and Tomita T. Effects of Ca2+ removal and of tetraethylammonium on membrane currents induced by carbachol in isolated cells from the rat parotid gland. Pflügers Arch 419: 332-337, 1991[Web of Science][Medline].

47.   Steward, MC, Poronnik P, and Cook DI. Bicarbonate transport in sheep parotid secretory cells. J Physiol 494: 819-830, 1996[Abstract/Free Full Text].

48.   Strøbaek, D, Jorgensen TD, Christophersen P, Ahring PK, and Olesen SP. Pharmacological characterization of small-conductance Ca2+-activated K+ channels stably expressed in HEK 293 cells. Br J Pharmacol 129: 991-999, 2000[Web of Science][Medline].

49.   Vandorpe, DH, Shmukler BE, Jiang L, Lim B, Maylie J, Adelman JP, de Franceschi L, Cappellini MD, Brugnara C, and Alper SL. cDNA cloning and functional characterization of the mouse Ca2+-gated K+ channel, mIK1. Roles in regulatory volume decrease and erythroid differentiation. J Biol Chem 273: 21542-21553, 1998[Abstract/Free Full Text].

50.   Vergara, C, Latorre R, Marrion NV, and Adelman JP. Calcium-activated potassium channels. Curr Opin Neurobiol 8: 321-329, 1998[Web of Science][Medline].

51.   Von Hahn, T, Thiele I, Zingaro L, Hamm K, Garcia-Alzamora M, Köttgen M, Bleich M, and Warth R. Characterisation of the rat SK4/IK1 K+ channel. Cell Physiol Biochem 11: 219-230, 2001[Web of Science][Medline].

52.   Warth, R, Hamm K, Bleich M, Kunzelmann K, von Hahn T, Schreiber R, Ullrich E, Mengel M, Trautmann N, Kindle P, Schwab A, and Greger R. Molecular and functional characterization of the small Ca2+-regulated K+ channel (rSK4) of colonic crypts. Pflügers Arch 438: 437-444, 1999[Web of Science][Medline].

53.   Wegman, EA, Ishikawa T, Young JA, and Cook DI. Cation channels in basolateral membranes of sheep parotid secretory cells. Am J Physiol Gastrointest Liver Physiol 263: G786-G794, 1992[Abstract/Free Full Text].

54.   Woodhull, AM. Ionic blockage of sodium channels in nerve. J Gen Physiol 61: 687-708, 1973[Abstract/Free Full Text].

55.   Wright, RD, and Blair-West JR. The effects of K+ channel blockers on ovine parotid secretion depend on the mode of stimulation. Exp Physiol 75: 339-348, 1990[Abstract].

56.   Xia, XM, Fakler B, Rivard A, Wayman G, Johnson-Pais T, Keen JE, Ishii T, Hirschberg B, Bond CT, Lutsenko S, Maylie J, and Adelman JP. Mechanism of calcium gating in small-conductance calcium-activated potassium channels. Nature 395: 503-507, 1998[Medline].

57.   Yoshimura, K, and Nezu E. Dynamic changes in the rate of amylase release induced by various secretagogues examined in isolated rat parotid cells by using column perifusion. Jpn J Physiol 41: 443-459, 1991[Web of Science][Medline].

Am J Physiol Cell Physiol 284(1):C127-C144
0363-6143/03 $5.00 Copyright © 2003 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
S. Y. Lee, M. L. Palmer, P. J. Maniak, S. H. Jang, P. D. Ryu, and S. M. O'Grady
P2Y receptor regulation of sodium transport in human mammary epithelial cells
Am J Physiol Cell Physiol, November 1, 2007; 293(5): C1472 - C1480.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
V. G. Romanenko, T. Nakamoto, A. Srivastava, T. Begenisich, and J. E. Melvin
Regulation of membrane potential and fluid secretion by Ca2+-activated K+ channels in mouse submandibular glands
J. Physiol., June 1, 2007; 581(2): 801 - 817.
[Abstract] [Full Text] [PDF]

Home page
 JGPHome page
J. Thompson and T. Begenisich
Membrane-delimited Inhibition of Maxi-K Channel Activity by the Intermediate Conductance Ca2+-activated K Channel
J. Gen. Physiol., January 30, 2006; 127(2): 159 - 169.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
S. Yamaguchi and T. Ishikawa
Electrophysiological characterization of native Na+-HCO3- cotransporter current in bovine parotid acinar cells
J. Physiol., October 1, 2005; 568(1): 181 - 197.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
M. Hayashi, C. Kunii, T. Takahata, and T. Ishikawa
ATP-dependent regulation of SK4/IK1-like currents in rat submandibular acinar cells: possible role of cAMP-dependent protein kinase
Am J Physiol Cell Physiol, March 1, 2004; 286(3): C635 - C646.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
284/1/C127    most recent
00250.2002v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (11)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Takahata, T.
Right arrow Articles by Ishikawa, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takahata, T.
Right arrow Articles by Ishikawa, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online