INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

MANY TYPES OF CELLS UNDERGO spontaneous, localized Ca²⁺ release (Ca²⁺ "sparks" or "puffs"), and local Ca²⁺ concentration can reach levels sufficient to regulate Ca²⁺-dependent conductances in the plasma membrane (13, 20). In the original communication describing Ca²⁺ sparks in smooth muscles, these events were reported to be coupled to spontaneous transient outward currents (STOCs; see Ref. 20), which are due to activation of large-conductance Ca²⁺-activated K⁺ (BK) channels (4). Recent experiments have shown that localized Ca²⁺ transients regulate the open probabilities of other Ca²⁺-dependent conductances, such as Ca²⁺-activated Cl channels (31). In most studies of Ca²⁺ sparks in smooth muscles, localized Ca²⁺ release has been attributed to ryanodine receptors because treatment of cells with ryanodine blocked spontaneous and agonist-enhanced sparks (16, 20, 24, 31). Recently, however, release of Ca²⁺ from inositol 1,4,5-trisphosphate (IP₃) receptor-operated stores has also been shown to be a source of Ca²⁺ spark activity in smooth muscles, and there may be a regenerative relationship between Ca²⁺ release from IP₃ receptors and ryanodine receptors (3, 6).

Normally, enhanced production of IP₃ and subsequent Ca²⁺ release is characteristic of responses to excitatory agonists in smooth muscles. However, localized Ca²⁺ transients mediated by IP₃ receptors may provide a novel means by which Ca²⁺-dependent ionic conductances are activated by inhibitory agonists. In gastrointestinal (GI) smooth muscles, this mechanism might explain the inhibitory actions of ATP, which is thought to be an inhibitory neurotransmitter released from enteric motoneurons (11, 17). Stimulation of GI muscle cells with ATP or the P_2Y receptor agonist 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) leads to activation of small-conductance Ca²⁺-activated K⁺ (SK) channels and hyperpolarization (18, 28). The inhibitory response to ATP in GI muscles appears to involve occupation of P_2Y receptors (9, 29), activation of phospholipase C (PLC), and enhanced production of IP₃ (2, 8, 21). It is possible that localized Ca²⁺ release could provide the link between P_2Y receptors and activation of SK channels.

We previously showed that Ca²⁺ sparks in murine colonic muscles are elicited by IP₃-dependent mechanisms (3), but STOCs, the standard electrophysiological assay of Ca²⁺ sparks, are due to activation of BK channels (see Ref. 20 and review in Ref. 7). BK channels do not mediate the inhibitory responses attributable to ATP in GI muscles, because inhibitors of BK do not block postjunctional inhibitory junction potentials (29). Purinergic inhibitory responses appear to be mainly due to activation of SK channels (18, 28). In the present study, we attempt to relate the P_2Y receptor-IP₃ pathway to activation of SK channels by investigating whether apamin-sensitive STOCs [not blocked by charybdotoxin (ChTX) or iberiotoxin] are present in GI muscles and regulated by purinergic agonists. We use myocytes isolated from the guinea pig taenia coli for these studies, because this is the classic preparation in which apamin-sensitive, enteric inhibitory responses attributed to ATP were first observed (1, 10, 27).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Preparation of smooth muscle cells. Guinea pigs of either sex were killed by CO₂ asphyxiation. Isolated strips (1-2 cm long) of taenia coli were incubated in Ca²⁺-free Hanks' solution (see Solutions), containing 0.12% (wt/vol) collagenase (Worthington Biochemical, Freehold, NJ), 0.2% soybean trypsin inhibitor (type II-S, Sigma, St. Louis, MO), and 0.2% BSA (Sigma). After incubation at 37°C for 15 min with gentle stirring, tissues were washed four times with enzyme-free Hanks' solution. The tissue pieces were then triturated with a wide-bore fire-polished Pasteur pipette to create a cell suspension. The cells were stored at 4°C and used within 6 h.

Current and voltage measurements. Drops of the cell suspensions were placed on a glass coverslip forming the bottom of a 300-µl chamber mounted on an inverted microscope. Cells were allowed to adhere to the coverslip, and then the chamber was perfused at a rate of 3 ml/min. "Giga-seals" were made with fire-polished glass pipettes having tip resistances of 3-4 M. The whole cell, perforated patch (amphotericin B) configuration of the patch-clamp technique was used to record ionic currents under voltage clamp. An Axopatch 200B amplifier with a CV-4 headstage (Axon Instruments, Foster City, CA) was used to measure ionic currents and membrane potentials. Current-clamp experiments (0 current) also were performed on cells with amphotericin-perforated patches. The changes of membrane potential were recorded on a chart recorder (Gould 2200S). A personal computer running pCLAMP software (version 6.0.4, Axon Instruments) was used to collect data.

Solutions and reagents. CaCl₂, KCl, KH₂PO₄, NaCl, NaHCO₃, Na₂HPO₄, sucrose, and glucose were from Fisher Scientific (Fair Lawn, NJ). (±)Bay K 8644, xestospongin C, U-73122, and U-73343 were purchased from Calbiochem and dissolved in DMSO. After dilutions, the final concentration of DMSO was <0.01%. ATP and 2-MeS-ATP were purchased from Sigma. After the nucleotides were dissolved into HEPES-based buffer, the pH was corrected to 7.4. All other chemicals were also purchased from Sigma. Krebs solution contained (in mM) 125 NaCl, 5.9 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 15.5 NaHCO₃, 1.2 Na₂HPO₄, and 11.5 glucose, with pH adjusted to 7.4 by bubbling with 95% O₂ and 5% CO₂. Ca²⁺-free Hanks' solution contained (in mM) 125 NaCl, 5.36 KCl, 15.5 NaHCO₃, 0.336 Na₂HPO₄, 0.44 KH₂PO₄, 10 glucose, 2.9 sucrose, and 11 HEPES, with pH adjusted to 7.4 with NaOH. The bath solution (CaPSS) contained (in mM) 135 NaCl, 5.0 KCl, 2.0 CaCl₂, 1.2 MgCl₂, 10.0 glucose, and 10 HEPES, with pH adjusted to 7.4 with Tris. The pipette solutions contained (in mM) 110 potassium gluconate, 30 KCl, 5 MgCl₂, and 5 HEPES, with pH adjusted to 7.2 with Tris.

Statistical analyses. Data are expressed as means ± SE of n cells. All statistical analyses were performed using SigmaStat 2.0 software (Jandel, San Rafael, CA). We used paired t-tests and one-way repeated measures ANOVA to compare groups of data. In all statistical analyses, P < 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

STOCs in isolated myocytes. Single myocytes from the guinea pig taenia coli were studied with the amphotericin-perforated patch technique. In preliminary studies, we noted that STOCs could be evoked when cells were held at depolarized potentials. STOCs were not resolved at potentials negative to 60 mV. Near 50 mV, small-amplitude STOCs (<10 pA) were recorded. When cells were held at more positive potentials, STOCs of highly variable amplitude and frequency were recorded (Fig. 1A). Amplitude histograms were constructed, and the peak currents of each distribution (1-pA bin size) were plotted as a function of membrane potential (Fig. 1, B and C). The amplitude distributions were bimodal: the mean amplitude of the large-amplitude STOCs was 19 ± 3 pA, and the mean amplitude of small-amplitude STOCs was 5 ± 1 pA, at a holding potential of 30 mV (n = 8). We refer to STOCs of <10 pA as "mini-STOCs" and STOCs of >10 pA as "large STOCs" at holding potentials of 40 mV or 30 mV.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Effects of charybdotoxin (ChTX) on spontaneous transient outward currents (STOCs) in cells studied with amphotericin-permeabilized patch technique. A: control. Representative STOCs recorded at test potentials ranging from 50 to 20 mV. At holding potentials of 30 mV or 40 mV, mini-STOCs were defined as events <10 pA and large STOCs (denoted by *) as events >10 pA. Inset: expanded scale showing STOCs (arrows). At potentials more negative than 40 mV, it was not possible to distinguish between mini-STOCs and large STOCs, so all events are grouped as mini-STOCs. B: current-voltage (I-V) relationship summarizing STOCs from 8 cells. C: representative amplitude histogram of mini- and large STOCs recorded at a holding potential of 30 mV. Amplitude distribution shows a bimodal distribution. D: ChTX (200 nM; traces were recorded after 10-min incubation) abolished activity of large STOCs. Mini-STOCs were still apparent after ChTX (denoted by +). Inset: remaining mini-STOC in presence of ChTX. E: I-V relationship after treatment with ChTX (n = 8). F: representative amplitude histogram (holding potential 30 mV) shows inhibition of large STOCs and reduction in mini-STOCs after ChTX treatment.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Effects of extracellular Ca2+ on STOCs. Same cell was held at 30 mV. A: STOCs recorded under control conditions (i.e., 2 mM external Ca2+ concentration). B: mini-STOCs remaining in presence of 200 nM ChTX for 10 min. C: inhibition of all STOCs after replacement of external Ca2+ in Ca2+-containing physiological saline solution (CaPSS) with Mn2+ (MnPSS). D: summary of results from 5 cells expressing number of events during 100-s recordings. Data are grouped in 10-pA bins. * P < 0.05.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Effects of Bay K 8644 and caffeine on STOCs. Cells were held at holding potential of 40 mV. A: control. STOCs recorded in normal CaPSS. B: after application of Bay K 8644 (2 µM). C: control. STOCs recorded from a different cell in normal CaPSS. D: after application of caffeine (1 mM). Parallel lines denote 2 min or recording omitted between 5th and 6th traces.

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Effects of ryanodine and xestospongin C on STOCs. Cells were held at 30 mV. A: control. STOCs recorded in normal CaPSS. B: after addition of ryanodine (10 µM) for 20 min. C: summary of results showing number of events during 100-s recordings from 4 cells. D: control. STOCs in a different cell in CaPSS. E: after addition of xestospongin C (1 µM) for 20 min. F: summary of results showing number of events during 100-s recordings from 4 cells. * P < 0.05 vs. control.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Effects of apamin on STOCs. Cell was held at 30 mV. A: control. STOCs in CaPSS. B: after addition of apamin (500 nM) for 10 min. C: summary of results showing number of events during 100-s recordings from 5 cells. * P < 0.05 vs. control.

Activation of STOCs by ATP and 2-MeS-ATP. Purinergic stimulation by ATP and 2-MeS-ATP activates SK channels in visceral smooth muscle (18, 28). We tested the effects of these agonists at a holding potential of 40 mV. ATP (1 mM; n = 4) and 2-MeS-ATP (100 µM; n = 5) significantly increased the activity of mini-STOCs (control vs. ATP, from 204 ± 25 to 273 ± 25 events/100 s, a 35% increase; control vs. 2-MeS-ATP, from 174 ± 27 to 257 ± 39 events/100 s, a 50% increase; Fig. 6). Large STOCs also tended to be increased by ATP (from 10 ± 6 to 23 ± 12 events/100 s) and 2-MeS-ATP (from 14 ± 8 to 28 ± 14 events/100 s); however, these responses did not reach levels of statistical significance. We also characterized purinergic activation of STOCs in the presence of ChTX to block BK channels. As above, ChTX (200 nM) significantly attenuated large STOCs and mini-STOCs in these cells (Fig. 7, A and B). Addition of 2-MeS-ATP in the continued presence of ChTX increased the frequency (ChTX vs. ChTX + 2-MeS-ATP, 46 ± 12 vs. 122 ± 15, 222% increase, n = 4) of mini-STOCs (Fig. 7, C and D). Both components of STOCs were greatly attenuated by joint application of apamin and ChTX (i.e., mini-STOCs were reduced from 313 ± 30 to 16 ± 4 events/100 s by apamin and ChTX, a 95% inhibition; Fig. 8, A and B). Large STOCs were completely abolished by apamin and ChTX. In the presence of these drugs, 2-MeS-ATP did not increase the activity of mini-STOCs or large STOCs (Fig. 8, C and D). These data suggest that at least a portion of the response to purinergic stimulation is due to STOCs mediated through activation of apamin-sensitive channels.

View larger version (42K): [in this window] [in a new window]   Fig. 6.   Effects of ATP and 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) on STOCs. Cells were held at 40 mV. A: control STOCs. B: after addition of ATP (1 mM). C: summary of results showing number of events during 100-s recordings from 4 cells. D: control. STOCs from a different cell. E: after addition of 2-MeS-ATP (100 µM). F: summary of results showing number of events during 100-s recordings from 5 cells. * P < 0.05 vs. control.

View larger version (29K): [in this window] [in a new window]   Fig. 7.   Effects of 2-MeS-ATP on STOCs in presence of ChTX. Cell was held at 30 mV. A: control. STOCs in normal CaPSS. B: after addition of ChTX (200 nM) for 10 min. C: 2-MeS-ATP (100 µM) increased number of mini-STOCs in presence of ChTX. D: summary of results showing number of events during 100-s recordings from 4 cells. * P < 0.05.

View larger version (29K): [in this window] [in a new window]   Fig. 8.   Effects of 2-MeS-ATP on STOCs in presence of ChTX and apamin. Cell was held at 30 mV. A: control. STOCs in normal CaPSS. B: after addition of ChTX (200 nM) and apamin (500 nM) for 10 min. C: in presence of ChTX and apamin, 2-MeS-ATP (100 µM) failed to increase STOCs. D: summary of results showing number of events during 100-s recordings from 4 cells. * P < 0.05.

Receptors and second messengers mediating the effect of 2-MeS-ATP on STOCs. We tested whether the enhancements in STOCs by ATP and 2-MeS-ATP were due to activation of purinergic receptors. Pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS; 5 µM), an antagonist of P₂ receptors (19), decreased large STOCs and mini-STOCs at a holding potential of 30 mV (Fig. 9, A and B). 2-MeS-ATP failed to increase the activity of STOCs in the presence of PPADS (Fig. 9, C and D; n = 4).

View larger version (30K): [in this window] [in a new window]   Fig. 9.   Effects of 2-MeS-ATP on STOCs in presence of pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS). Cell was held at 30 mV. A: control. STOCs in normal CaPSS. B: after addition of PPADS (5 µM, 10-min incubation). C: in presence of PPADS, 2-MeS-ATP (100 µM) failed to increase STOCs. D: summary of results showing number of events during 100-s recordings from 4 cells. * P < 0.05.

View larger version (29K): [in this window] [in a new window]   Fig. 10.   Effects of 2-MeS-ATP on STOCs in presence of U-73122. Cell was held at 30 mV. A: control. STOCs in normal CaPSS. B: after addition of U-73122 (1 µM, 10-min incubation). C: 2-MeS-ATP (100 µM) failed to increase STOCs in presence of U-73122. D: summary of results showing number of events during 100-s recordings from 4 cells. * P < 0.05.

View larger version (29K): [in this window] [in a new window]   Fig. 11.   Effects of 2-MeS-ATP on STOCs in presence of U-73343. Cell was held at 30 mV. A: control. STOCs in normal CaPSS. B: after addition of U-73343 (1 µM, 10-min incubation). C: 2-MeS-ATP (100 µM) increased mini-STOCs in presence of U-73343. D: summary of results showing number of events during 100-s recordings from 4 cells. * P < 0.05.

Hyperpolarization induced by purinergic activation. In the current-clamp mode (0 current), we investigated the effects of 2-MeS-ATP on membrane potential. At rest, membrane potential averaged 44.2 ± 3.9 mV (n = 7). In three of seven cells, small fluctuations in resting membrane potential were observed during resting conditions. Application of 2-MeS-ATP (100 µM) to these cells induced transient hyperpolarizations (10 ± 3 mV in amplitude, n = 3; Fig. 12A). There was variability in the durations of the transient hyperpolarizations, which may have reflected differences in the degree of summation of STOCs. In four cells, spontaneous spikelike hyperpolarizations were observed during resting conditions (e.g., frequency averaged 353 ± 29 per 100 s). Addition of ChTX (200 nM) under current-clamp conditions induced a 3 ± 1 mV depolarization (P < 0.05) and decreased the amplitude (to 5 ± 2 mV, P < 0.05) and frequency (to 220 ± 23 per 100 s, P < 0.05; see Fig. 12B) of the transient hyperpolarizations. In the presence of ChTX, 2-MeS-ATP restored the amplitude of the spontaneous transient hyperpolarizations (to 9 ± 2 mV, P < 0.05 compared with amplitude in the presence of ChTX) and increased the frequency (to 580 ± 46, P < 0.05; see Fig. 12B). These events were consistent with the changes in STOC frequency induced by ChTX and 2-MeS-ATP under voltage-clamp conditions. The data suggest that the hyperpolarizations caused by 2-MeS-ATP are mediated, in part, by ChTX-insensitive STOCs.

View larger version (37K): [in this window] [in a new window]   Fig. 12.   Effects of 2-MeS-ATP on resting membrane potentials of current-clamped cells (0 current). A: continuous trace from a representative cell showing transient hyperpolarization after application of 2-MeS-ATP (100 µM; exposure denoted by bar). B: discontinuous recording (10 min were removed between 1st and 2nd traces, as denoted by parallel lines at end of 1st trace) demonstrating effects of 2-MeS-ATP after prior addition of ChTX (200 nM). ChTX (black bar over 1st and 2nd traces) reduced amplitude and frequency of transient hyperpolarizations that occurred spontaneously in this cell and produced a small depolarization in membrane potential. In presence of ChTX, 2-MeS-ATP (shaded bar in 2nd trace) increased frequency of transient hyperpolarizations and restored membrane potential. Dotted line denotes average resting potential during control phase of recording.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

STOCs have been used as an assay of localized Ca²⁺ release (4, 20), and quantitative analysis has shown a high degree of correlation between Ca²⁺ spark amplitude and STOC amplitude (22). In some smooth muscle cells, however, Ca²⁺ sparks are coupled to more than a single Ca²⁺-dependent conductance (31), and the present study indicates that this is also the case in guinea pig taenia coli smooth muscle cells. Records of STOCs from taenia coli myocytes showed typical large-amplitude STOCs and frequent low-amplitude (<10 pA) STOC-like events. All STOCs were Ca²⁺ dependent; they were enhanced by Bay K 8644 and disappeared when cells were incubated for extended periods in low external Ca²⁺ or after intracellular stores were depleted by addition of caffeine. The data indicated that Ca²⁺ release from IP₃-sensitive stores may be the trigger for STOCs in taenia coli myocytes. When BK channels were blocked by ChTX or iberiotoxin (by addition of >15 times the dissociation constant for smooth muscle BK channels; see Refs. 15, 23), STOC-like activity persisted, but the remaining events were of much smaller amplitude than the events attributable to BK. Mini-STOCs were partially blocked by apamin, suggesting that at least a portion of the current was due to SK channels. These data suggest that at least two Ca²⁺-dependent conductances are activated by localized Ca²⁺ release in myocytes from the taenia coli.

The mini-STOCs were relatively difficult to resolve in relation to STOCs caused by BK channels. This is likely due to the fact that the conductance of SK channels is comparatively low (i.e., 5.3 pS, see Ref. 18). Thus >40 SK channels would need to open at the same time to produce the conductance equivalent to 1 BK channel. Because STOCs are typically attributed to the nearly simultaneous increase in open probability of many BK channels, a very large number of SK channels would need to be clustered near sites of Ca²⁺ sparks to produce STOCs of an amplitude equivalent to BK STOCs. Thus it is logical that STOCs due to SK channels are of much smaller amplitude and typically obscured by the much larger amplitude BK STOCs. Typically, openings of SK channels might enhance the amplitude of BK STOCs or produce what would appear to be increased baseline noise in current records. The need to use depolarized potentials to resolve the small currents associated with mini-STOCs increases contamination from BK channels. Therefore, resolution of STOCs due to SK channels required blocking of BK channels.

It was not possible to separate STOCs due to SK and BK channels purely on the basis of amplitude. A significant portion (~80%) of the mini-STOCs were inhibited by ChTX, suggesting that smaller clusters of BK channels or clusters at some distance from the primary spark sites may contribute to the low-amplitude population of STOCs. Apamin inhibited the small-amplitude STOCs by ~20%. Therefore, the mini-STOCs appear to represent outward currents due to openings of both BK and apamin-sensitive SK channels.

The mechanisms by which ATP increases cell membrane conductance and causes hyperpolarization of GI smooth muscles have been unclear. Previous studies showed that ATP, UTP, and the P_2Y agonist 2-MeS-ATP activated an outward current under whole cell recording conditions, and this was attributed to an increase in the open probability of SK channels (18, 28). In the present study, we show that 2-MeS-ATP increases the frequency of STOCs, and the data suggest that this occurs via occupation of P_2Y receptors (PPADS), activation of PLC (U-73122), and increased IP₃ production (xestospongin C). The increase in STOCs in response to 2-MeS-ATP suggests that IP₃-dependent amplification of Ca²⁺ release (either an increase in the number of sparking sites or in the frequency of sparking from established sites) is the main mechanism responsible for P_2Y receptor-mediated stimulation of outward current and hyperpolarization. Experiments performed in current clamp clearly demonstrate the link between purinergic stimulation and hyperpolarization. In isolated cells, hyperpolarization transients were quantal in nature and most likely the result of the STOCs recorded under voltage clamp. In intact tissues, the quantal hyperpolarization transients occurring in many coupled cells would tend to summate temporally and spatially, yielding continuous, analog hyperpolarization responses.

We found that PPADS (P₂ receptor blocker) and U-73122 (PLC blocker) also reduce the frequency of spontaneous STOCs. These data could be interpreted as nonspecific effects of these compounds on SK channels. Previous studies argue against the idea that PPADS blocks SK channels. We found that PPADS decreased the open probability of SK channels in murine colon, but these channels could be activated by releasing internal stores of Ca²⁺ with caffeine (18). PPADS has been shown to be a partial antagonist of IP₃ receptors (26), and this may explain why this compound reduced spontaneous STOC activity. The nonspecific effects of U-73122 were controlled for by also testing U-73343, a nonactive, structurally similar analog of U-73122. The inactive analog had no effect on STOC occurrence. It is likely that U-73122 reduced basal production of IP₃ in the cells, and this might explain the reduction in STOC frequency in response to this compound.

In studies of mouse colonic myocytes, we showed that spontaneous Ca²⁺ sparks are caused by release of Ca²⁺ from IP₃ receptor-operated stores (3). Purinergic stimulation increased Ca²⁺ sparks and tended to organize local release events into Ca²⁺ waves. Ca²⁺ sparks and Ca²⁺ waves never elevated global Ca²⁺ to the threshold for contraction. A similar enhancement in Ca²⁺ spark frequency in response to 2-MeS-ATP is also likely in taenia coli, because we noted an increase in STOCs when cells were exposed to this compound.

Ca²⁺ release is associated with activation of SK and BK channels in myocytes from the taenia coli. This raises the question of why tissue responses to purinergic stimuli or enteric inhibitory neural inputs are reduced by apamin but unaffected by inhibition of BK channels (30). As was recently pointed out by ZhuGe and co-workers (31), the ionic conductances activated by Ca²⁺ sparks are strongly affected by the voltage range in which cells are held or are operating during physiological conditions. These authors found that BK STOCs were favored at more depolarized potentials, but Ca²⁺-activated Cl currents (spontaneous transient inward currents) were more frequently observed at more negative potentials. These results are partly explained by the driving forces responsible for outward K⁺ currents and inward Cl currents. However, BK channels are both voltage and Ca²⁺ dependent, and the open probability of these channels is very low at the relatively negative resting membrane potentials of GI muscles (i.e., negative to 50 mV). SK channels are not voltage dependent, and open probability is increased by low concentrations of Ca²⁺ (18). Therefore, the weighting of purinergic enteric inhibitory responses toward SK channels may be partially due to the voltage range of resting membrane potentials of GI muscles. Stimulating cells with 2-MeS-ATP yielded increases mainly in mini-STOCs, suggesting that Ca²⁺ release via IP₃ receptor-operated stores occurs in close proximity to clusters of SK channels. Therefore, it is possible that part of the discrimination between activation of SK and BK channels is due to tight coupling between receptors, second messenger pathways, IP₃ receptor-operated stores, and SK channels. It would be extremely interesting to know whether the clustering of cellular effector proteins are postjunctional specializations associated with sites of neural innervation in intact muscles.

In summary, the present study suggests a mechanism by which purinergic neurotransmission causes hyperpolarization and reduces the excitability of GI smooth muscles. The data are consistent with the following. Occupation of P_2Y receptors activates PLC and increases production of IP₃. Rather than generally increasing global cytoplasmic Ca²⁺, IP₃ generated via this mechanism enhances localized Ca²⁺ transients. This leads to enhanced production of STOCs, which are mainly caused by activation of SK channels. STOCs cause hyperpolarization transients that summate and cause the hyperpolarization responses in intact muscles. Hyperpolarization of GI muscles lowers excitability and decreases the net open probability of L-type Ca²⁺ channels, leading to a net inhibitory effect.