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Am J Physiol Cell Physiol 294: C136-C144, 2008. First published November 7, 2007; doi:10.1152/ajpcell.00340.2007
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Structural and functional determinants in the S5-P region of HCN-encoded pacemaker channels revealed by cysteine-scanning substitutions

Ka-Wing Au,1,2 Chung-Wah Siu,1,3,4 Chu-Pak Lau,1 Hung-Fat Tse,1 and Ronald A. Li1,3,4,5

1Department of Medicine, Queen Mary Hospital, and 2Institute of Cardiovascular Science and Medicine, University of Hong Kong, Hong Kong; 3Stem Cell Program and 4Department of Cell Biology and Human Anatomy, University of California, Davis; and 5Institute of Pediatric Regenerative Medicine, Shriners Hospital for Children of North America, Sacramento, California

Submitted 1 August 2007 ; accepted in final form 23 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are responsible for the membrane pacemaker current that underlies the spontaneous generation of bioelectrical rhythms. However, their structure-function relationship is poorly understood. Previously, we identified several pore residues that influence HCN gating properties and proposed a pore-to-gate mechanism. Here, we systematically introduced cysteine-scanning substitutions into the descending portion of the P loop (residues 339–345) of HCN1-R (where R is resistance to sulfhydryl-reactive agents) channels, in which all endogenous cysteines except C303 have been removed or replaced. F339C, K340C, A341C, M342C, S343C, and M345C did not produce functional currents. Interestingly, the loss of function phenotype of F339C could be rescued by the reducing agent dithiothreitol (DTT). H344C but not HCN1-R and DTT-treated F339C channels were sensitive to blockade by divalent Cd2+ (current with 100 µM Cd2+/control current at –140 mV = 67.6 ± 2.9%, 109.3 ± 3.1%, and 103.8 ± 1.7%, respectively). Externally applied methanethiosulfate ethylammonium, a covalent sulfhydryl-reactive compound, irreversibly modified H344C by reducing the current at –140 mV (to 43.7 ± 6.5%), causing a hyperpolarizing steady-state activation shift (change in half-activation voltage: ~6 mV) and decelerated gating kinetics (by up to 3-fold). Based on these results, we conclude that pore residues 339–345 are important determinants of the structure-function properties of HCN channels and that the side chain of H344 is externally accessible.

hyperpolarization-activated cyclic nucleotide; cysteine mutagenesis; sulfhydryl modification

PACEMAKER CURRENT (If or Ih), encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel gene family (11), is a key contributor to the spontaneous generation of electrical rhythms in certain cardiac and neuronal cells by modulation of the rate of cellular depolarization (6, 10–12, 16, 20, 21, 25, 27, 32). To date, four mammalian HCN channel isoforms (HCN1–4) have been identified, each with a distinct pattern of gene expression and tissue distribution (20, 24–26). The four isoforms have different gating properties and can coassemble with each other to form heteromultimers (8, 28, 31). Functionally, HCN channels nonselectively permeate Na+ and K+ (with a ratio of 1:4 vs. ≤1:100 of K+ channels) and are activated upon hyperpolarization rather than depolarization of classical voltage-gated K+ (Kv) channels. Despite these differences in permeation and gating, however, HCN and Kv channels have highly homologous primary amino acid sequences. Structurally, both are tetramers of monomeric subunits consisting of six membrane-spanning segments (S1–S6), each including a positively charged voltage-sensing S4 segment and a P loop between S5 and S6 that forms part of the ion-conducting pore (Fig. 1). The pore region can be further subdivided into three regions: the S5-P and P-S6 linkers form the extrapore and the descending and ascending limbs of the P loop, where the permeation determinant GYG motif is located, constitute the deeper region of the extracellular pore (Fig. 1). The cytoplasmic side of the pore is believed to consist of S6 segments (22).


Figure 1
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  		Fig. 1. Schematic of hyperpolarization-activated cyclic nucleotide-modulated (HCN1-R, where R indicated resistance to sulfhydryl-reactive agents) channel structure. The 6 putative transmembrane segments (S1–S6) of a monomeric HCN subunit are shown at the bottom. Sequence comparisons of the ascending limb of the S5-S6 P loops of various HCN channels are shown at the top. Endogenous cysteines that were substituted are circled with the replacement amino acid given. The site of truncation is also indicated. The seven residues NH2-terminal to the GYG motif were individually substituted by cysteine in this study. The S5–S6 linker consists of the S5-P, P loop, and P-S6 linker.

 
In our previous studies, we identified several external pore residues that influence the gating properties of HCN channels (i.e., P loop residues A352 and A354 and S5-P residue C318, HCN1 numbering) and their modulation by external K+ (1, 2, 30). Based on these results, a pore-to-gate mechanism has been proposed. Additionally, residue 352 has also been demonstrated to determine the Cl– dependence of HCN channels by allosterically coupling Cl– binding to activation (29). Despite these findings, the structure-function properties of the HCN outer pore remain poorly defined. To obtain a better understanding, in the present study, we systematically introduced single cysteine substitutions into the descending portion of the P loop of HCN1 channels (residues 339–345) followed by an examination of the functional consequences. The susceptibility of substituted channels to such sulfhydryl-reactive agents as Cd2+ and methanethiosulfonate (MTS) compounds was investigated for assessing the accessibility pattern of engineered cysteines.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Molecular biology. Murine HCN1 (kindly provided by Dr. Steven Siegelbaum) (25) was subcloned into the pGHE expression vector. HCN1-R (where R is resistance to sulfhydryl-reactive agents) was also a gift from Dr. Siegelbaum and has been described in detail previously (5). In brief, 11 of 12 endogenous cysteine residues of wild-type (WT) HCN1 were removed or substituted: 6 cysteines were removed by truncating the COOH terminus and 4 of the 6 remaining cysteines were conservatively replaced by serine or threonine (i.e., C55S, C318S, C347S, and C374T) with C298 substituted by isoleucine according to the homologous residue in cyclic nucleotide-gated (CNG) channels. As we and others have previously demonstrated, substitution of C303 with any of the other 19 amino acids did not produce functional channels (5, 30). Therefore, C303 was not mutated in HCN1-R. All cysteine substitutions investigated in this study were generated in the HCN1-R background using the Stratagene QuickChange site-directed mutagenesis kit. The desired mutations were confirmed by DNA sequencing. cRNA was transcribed from NheI-linearized DNA using the Ambion MEGAscript transcription kit.

Oocyte preparation and heterologous expression. Stage IV–VI oocytes were surgically removed from female Xenopus laevis anesthetized by immersion in 0.3% 3-aminobenzoic acid ethyl ester, followed by digestion with 1 mg/ml collagenase (type IA) in OR2 solution containing 88 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES (titrated to pH 7.6 with NaOH) for 30–60 min. Isolated oocytes were stored in ND96 solution containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES (titrated to pH 7.6 with NaOH) supplemented with 50 µg/ml gentamycin, 5 mM pyruvate, and 0.5 mM theophylline. Within 5 h after isolation, each oocyte was injected with 50 ng cRNA (1 ng/1 nl). Oocytes were studied 24–48 h after cRNA injection. The protocol was approved by the University ethical committee with protocol number 05-12026.

Preparation of oocyte membrane fractions and Western blot analysis. Injected and control (uninjected) oocytes were homogenized in ice-cold PBS containing an EDTA-free protease inhibitor cocktail (1 tablet/10 ml, Roche Applied Science) and centrifuged for 10 min at 1,000 g to remove pigment and nuclear and mitochondrial materials. The supernatant was fractioned into cytosolic and membrane fractions by ultracentrifugation at 120,000 g for 1 h at 4°C. The membrane fraction was resuspended in 1x LDS sample buffer and electrophoresed on a 10% polyacrylamide-SDS gel. Membrane proteins were then transferred onto a nitrocellulose membrane, followed by blockade with 5% nonfat dried milk for 1 h. Blocked membranes were washed in Tris-buffered saline-Tween (TBST) and incubated with a purified rabbit polycolonal anti-HCN1 antibody (Alomone Labs, Jerusalem, Israel) at a 1:200 dilution for 4°C overnight, followed by a wash in TBST and an incubation with horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin (Chemicon, Temecula, CA) diluted to 1:10,000 in 5% nonfat dried milk for 1 h at room temperature. Membranes were washed again three times in TBST, and bound secondary antibodies were detected with an ECL detection kit (Chemicon) by autoradiography.

Electrophysiology. Two-electrode voltage-clamp recordings were performed at room temperature using a Warner C-725B amplifier. The measuring electrodes (TW120-6, World Precision Instruments, Sarasota, FL), whose tips were plugged with 1% agarose dissolved in 3 M KCl solution, were pulled using a Narishige PP-83 vertical puller. When backfilled with 3 M KCl solution, the typical electrode resistances were ~2–5 M{Omega}. The recording bath solution contained 96 mM KCl, 2 mM NaCl, 2 mM MgCl2, and 10 mM HEPES (titrated to pH 7.5 with NaOH).

For the steady-state current-voltage (I-V) protocol, whole cell currents were measured at the end of a 3-s pulse from –140 to 0 mV from a holding potential of –30 mV against the test potentials. The voltage dependence of HCN channel activation was assessed by plotting tail currents measured immediately after a pulse to –140 mV as a function of the preceding 3-s test pulse normalized to the tail current at –140 mV. Data were fitted to the Boltzman function using the Marquardt-Levenberg algorithm in a nonlinear least-squares procedure:

Formula
where Vt is the test voltage, V1/2 is the voltage at which 50% activation was achieved or the so-called activation midpoint, and k = RT/zF and is the slope factor of steady-state activation (m{infty}) (where R is the gas constant, T is temperature, z is valence, and F is Faraday's constant) (5, 30). For current kinetics, activation ({tau}act) and deactivation ({tau}deact) time constants were estimated by fitting macroscopic currents with a monoexponential function.

To test for Cd2+ sensitivity, each oocyte expressing a HCN1-R pore cysteine construct was first stimulated by a 2-s voltage pulse from a holding voltage of 0 mV to a test voltage of –140 mV. The steady-state current at –140 mV was used as the initial current magnitude against which currents after CdCl2 exposure were compared. Immediately after the initial test pulse, cells were exposed to varying concentrations of CdCl2 (3 µM–1 mM). Voltage pulses (from 0 to –140 mV, 2-s duration) were then applied every minute until steady-state current blockade was reached. The fractional block by CdCl2 was plotted against [CdCl2] to generate a binding isotherm. Data were fitted to the Hill equation using the Marquardt-Levenberg algorithm in a nonlinear least-squares procedure:

Formula
where Fblocked is the fractional current block, Imin is the minimum fractional current block, Imax is the maximum fractional current block, IC50 is the [CdCl2] that resulted in 50% fractional block, and nH is the Hill coefficient (assumed to be 1).

MTS ethylammonium (MTSEA; Toronto Research Chemicals, Toronto, ON, Canada) powder was dissolved in deionized water at 0.2 M shortly before experiments were performed. The stock solution was stored on ice and used with 1 h. The MTS solution was applied extracellularly after all control data were obtained. The MTS stock solution was diluted with the bath solution to 2 mM and applied to the oocyte immediately. The time course of MTS modification was fit with the following single-exponential equation:

Formula
where F is the fraction of remaining current measured at –140 mV in the presence of MTS, t is the cumulative exposure time, {tau}MTS is the time constant for MTS modification, and S is the steady-state plateau.

All data reported are means ± SE. Statistical significance was determined using an unpaired Student's t-test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
We first examined the effect of our cysteine substitutions on the function of HCN channels by individually expressing WT and various constructs in oocytes. Under control conditions, only WT, HCN1-R, and H344C channels expressed measurable hyperpolarization-activated currents (Fig. 2); F339C, K340C, A341C, M342C, S343C, and M345C channels did not produce functional currents. If the introduced sulfhydryls in the pore are in close proximity, it is possible that they cross-link and thereby restrict the pore for permeation. To test this possibility, we incubated injected oocytes with the reducing agent dithiothreitol (DTT; 2.5 mM) overnight. Interestingly, the loss of function phenotype of F339C channels could be rescued after DTT incubation, as evident by the presence of hyperpolarization-activated currents (Fig. 2). In contrast, K340C, A341C, M342C, S343C, and M345C channels continued to fail to produce measurable currents after DTT treatment. DTT had no detectable effect on WT, HCN1-R, and H344C channels. The corresponding steady-state I-V relationships before and after DTT incubation are shown in Fig. 2B. There were no significant changes in gating properties in the presence and absence of DTT. For instance, V1/2 of H344C channels was not different with and without DTT incubation (–109.1 ± 6.7 and –110.7 ± 5.8 mV, respectively, P > 0.05).


Figure 2
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  		Fig. 2. Raw current tracings of S5-P cysteine-substituted HCN1 constructs. Wild-type (WT), HCN1-R, and uninjected cells are also shown. A: of the seven S5-P mutants, only H344C produced measurable currents; F339C produced measurable current after overnight incubation in DTT. K340C, A341C, M342C, S343C, and M345C did not express before and after DTT incubation. B: steady-state current-voltage (I-V) relationships of WT, HCN1-R and H344C before and after DTT incubation.

 
H344C but not HCN1-R and F339C channels were sensitive to Cd2+ blockade. The divalent cation Cd2+ binds to free reduced sulfhydryls with high affinity and has been extensively used in combination with cysteine-scanning mutagenesis to probe the structure-function properties of ion channels (18). Figure 3 shows the effects of Cd2+ on HCN1-R, F339C (after DTT reduction), and H344C channels. Representative current tracings recorded in the absence and presence of Cd2+ are also shown (Fig. 3A). Consistent with previous reports, HCN1-R channels were insensitive to Cd2+ block (Figs. 3 and 4) (5). The addition of 100 µM Cd2+ to HCN1-R and F339C channels did not lead to any current blockade when the maximum steady-stated currents were measured at –140 mV [current with Cd2+/control current at –140 mV (Icontrol, –140 mV) = 109.3 ± 3.1% (n = 3) and 103.8 ± 1.7% (n = 3), respectively, P > 0.05]. In contrast, 100 µM Cd2+ significantly reduced the current at –140 mV of H344C channels by 32.4 ± 2.9% (n = 5, P < 0.05). Steady-state I-V relationships of HCN1-R, F339C, and H334C recorded in the absence and presence of 100 µM Cd2+ are shown in Fig. 3, B and C. Cd2+ did not shift the V1/2 of HCN1-R, F339C, and H344C channels (Table 1; P > 0.05), indicating that the current reduction observed with H344C channels was not secondary to changes in gating properties. Figure 4 shows the dose-response relationship of current inhibition by Cd2+ block for HCN1-R, F339C, and H334C channels. From these binding curves, the IC50 for H344C was estimated to be 10.8 ± 15 µM. There was no significant concentration dependence for HCN1-R and F339C mutants.


Figure 3
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  		Fig. 3. Effect of Cd2+ on HCN1-R, DTT-treated F339C, and H344C channels. A: representative tracings of whole cell currents through HCN1-R, DTT-treated F339C, and H344C constructs before and after the addition of 100 µM CdCl2. B: steady-state I-V relationships of HCN1-R, DTT-treated F339C, and H344C in the absence ({square}) and presence (bullet) of 100 µM Cd2+. Vm, membrane potential.

 

Figure 4
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  		Fig. 4. A: representative current tracings recorded in the absence and presences of Cd2+ at the concentrations indicated. B: dose-response relationships of HCN1-R, F339C, and H344C channels for Cd2+ block. ICd2+, current with Cd2+ block; I0 @ –140 mV, control current at –140 mV.

 

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  		Table 1. Summary of steady-state changes in pore cysteine channel properties by 100µM CdCl2

 
Effect of MTSEA on permeation and gating in pore cysteine mutants. A potential explanation for the enhanced sensitivity of H344C channels for Cd2+ blockade was that residue 344 is exposed to the aqueous phase of the pore and that Cd2+ binding inhibits permeation. As for F339C, it is either buried in the channel protein or its binding to Cd2+ (with an ionic radius of 1.1 Å) did not lead to blockade due to its remoteness from the permeation pathway. To complement our Cd2+ experiments, we examined the effect of the hydrophilic covalent sulfhydryl modifier MTSEA. Since its size is known (5.8 x 5.8 x 10 Å), successful modification would enable us to estimate the lower limit of the size of the accessible pore region. Figure 5A shows representative records of a family of currents through HCN1-R, F339C, and H344C channels before and after modification by externally applied MTSEA (2 mM). As anticipated, HCN1-R channels were resistant to MTSEA modification, indicating that the endogenous cysteine C303 is either not accessible to MTSEA or that its modification did not result in any functional changes [steady-state current with MTSEA (IMTSEA)/Icontrol,–140 mV = 102.3 ± 4.3%, n = 3] (5, 30). Similar to HCN1-R channels, MTSEA also did not affect F339C channels (IMTSEA/Icontrol,–140 mV = 105.1 ± 2.2%, n = 3). With a stimulation frequency of 0.1 Hz, however, IMTSEA/Icontrol,–140 mV of H344C channels progressively reduced in amplitude in the presence of 2 mM MTSEA. The steady-state block of 43.7 ± 6.5% (n = 3, P < 0.05) was achieved ~5 min with a time constant of 300 ± 30 s. When the stimulation frequency was slowed to 0.03 Hz, it took ~10 min to achieve the steady-state block with a time constant of 600 ± 38 s, although the reduction of IMTSEA/Icontrol,–140 mV (43.7 ± 6.5%, n = 3, P > 0.05) was identical (Fig. 5B).


Figure 5
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  		Fig. 5. H344C channels were sensitive to methanethiosulfate ethylammonium (MTSEA) modification. A: representative tracings of whole cell currents through F339C and H344C constructs before and after modification by MTSEA. External application of 2 mM MTSEA to H344C channels led to current reduction and slowed gating kinetics. In contrast, both HCN1-R and F339C channels were completely insensitive. B: time course of MTSEA modification upon the addition of 2 mM MTSEA (arrow) to H344C channels with a stimulation frequency of 0.1 Hz ({circ}) and 0.03 Hz ({square}) and F339C ({triangleup}) and HCN1-R ({triangledown}) channels both at 0.03 Hz.

 
Reduction of whole cell HCN1 currents by MTSEA could result from changes in the permeation pathway, gating properties, or a combination of both. To distinguish among these possibilities, we studied the steady-state activation properties before and after MTSEA modification. Figure 6 shows that MTSEA resulted in a modest yet significant hyperpolarizing shift of the steady-state activation curve (V1/2 with MTSEA = –122.9 ± 4.8 mV vs. control V1/2 = –116.5 ± 3.3 mV, n = 3, P < 0.05). Of note, the control V1/2 of H344C channels was significantly shifted in the negative direction compared with HCN1-R channels (P < 0.05). MTSEA also slowed both activation and deactivation gating kinetics (Fig. 6, B and C). MTSEA had no effect on the gating properties of HCN1-R and F339 channels.


Figure 6
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  		Fig. 6. Functional effects of MTSEA on H344C channels. A: electrophysiological protocol used for obtaining tail I-V relationships by stepping Vm from –100 to +40 mV with 10-mV increments after a 3-s prepulse to –140 mV. A representative family of tail currents recorded from an oocyte injected with 50 nl of H334C mutant cRNA is shown. V1/2, half-activation voltage. B: normalized activation and deactivation current tracings at –140 and –30 mV, respectively, before and after MTSEA modification as indicated. C: time constants for activation and deactivation in the absence and presence of MTSEA.

 
K340C, A341C, M342C, S343C, and M345C channel proteins were synthesized but not functional. To further explore the basis of the loss of function phenotype of K340C, A341C, M342C, S343C, and M345C channels, we performed Western blot analysis (Fig. 7). For WT and HCN1-R channels, only single bands were detected at ~76 kDa, as expected for the size for the HCN1 monomer both under control (oxidizing) conditions and after DTT incubation. Without DTT treatment, additional bands at ~150 kDa that appeared to be cross-linked dimers were seen for the cysteine-substituted pore constructs F339C, K340C, A341C, M342C, H344C, and M345C. However, these dimers were not seen in S343C channels. Of note, K340C channels expressed only dimers, whereas F339C, A341C, M342C, H344C, and M345C channels displayed both monomers and dimers under control conditions. Interestingly, all dimers disappeared after DTT treatment and only monomers similar to those observed with WT and HCN1-R channels were seen. All constructs displayed bands with intensity comparable with those of WT and HCN1-R channels (with the same cRNA amounts injected), suggesting that protein synthesis and translational efficiency were similar for all of the channels studied. The same pattern was observed in three other experiments independently performed with different batches of Xenopus oocytes.


Figure 7
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  		Fig. 7. Representative Western blot analysis of oocytes injected with WT, HCN1-R, and various cysteine constructs as indicated.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we employed cysteine-scanning substitutions to shed insights into the structure-function properties of the largely unexplored S5-P linker and the P segment intermediately NH2-terminal to the GYG motif that is crucial to ionic permeation (14, 31). Our major findings and their implications are summarized and discussed below.

Residue 344 is exposed to the aqueous phase and externally accessible by Cd2+ and MTSEA. Previously, we reported that the endogenous cysteine C318 in the S5-P linker, located in the outermost rim of the external pore mouth, can be covalently modified by MTS reagents; MTS modification negatively shifted steady-state activation and decelerates gating kinetics (30). Based on these results, we propose that the pore and gate of mammalian HCN channels are allosterically coupled in a manner analogous to the C type and slow inactivation of depolarization-activated K+ and Na+ channels (2, 30). A similar mechanism of pore motion has also been proposed for CNG channels (3, 4, 7, 15, 17, 19, 23). Like C318, C344 can be modified by external MTSEA. MTSEA modification reduced the macroscopic current of both C318 (i.e., WT) and H344C channels. Despite these similarities, WT and H344C channels differ in several aspects from which new insights into the pore can be obtained. For instance, MTSEA reduced the current through WT channels largely by substantially shifting their steady-state activation by ~50 mV (30). Steric hindrance of the pore by the MTS moiety accounted for only ~35% of the inhibition. Furthermore, WT and C318S channels displayed the same sensitivity to Cd2+ blockade. In contrast, MTSEA modification only modestly shifted activation gating of H344C channels (by ~5 mV), indicating that current inhibition is primarily due to pore obstruction by the MTS moiety covalently attached to residue 344. Also, H344C channels were sensitive to Cd2+. Taken together, these observations are consistent with the notion that residue 344 is located in the relatively narrow portion of the external permeation pathway. Interestingly, our MTSEA experiment further suggests that the accessibility of residue 344 is state dependent, also unlike C318 (30). The effects observed with H344C channels are site specific because the same was not seen with F339C channels. Mapping the entire HCN P segment, including the ascending portion of the P loop and the P-S6 linker, will provide a more comprehensive footprint of the pore.

Our results also underscore the importance of residues K340, A341, M342, S343, and M345 structural and functional determinants. When substituted, channel functions are abolished, although the proteins are expressed not differently from WT and HCN1-R channels. Interestingly, F339C, K340C, A341C, M342C, and M345C channels are able to cross-link among themselves to form dimers, which can be reduced to monomers. Of note, the HCN1-R channel contains one remaining endogenous cysteine (i.e., C303) that could also cross-link to disrupt channel functions. However, such an internal disulfide bridge would not lead to the formation of dimers; the possibility of cross-linking individual cysteines from distinct monomers is less likely assuming that the pore structure is analogous to those of K channels that have been characterized (e.g., KcsA) (13). These results shed structural insights into the rather poorly defined HCN pore. For instance, free sulhydryls can cross-link only when they come close into the proximity of each other (with the {alpha}-carbons at particular angles). Furthermore, the formation of disulfide bridge is a dynamic process; completion occurs when both the intersulfhydryl distance and orientation are optimal (9). Since a mixture of dimers and monomers was observed only with F339C, A341C, M342C, and M345C channels but only dimers for K340C channels under control conditions, our results raise the possibility that the intercysteine distance of K340C channels is probably the closest. The lack of measurable functional currents from K340C, A341C, M342C, S343C, and M345C channels, however, did not enable us to assess the accessibility of the corresponding substituted cysteines.

F339C channels are unique in that their loss of function can be rescued by DTT treatment, but the restored currents are sensitive to neither Cd2+ nor MTSEA. While we cannot exclude the possibility that Cd2+ binding or MTSEA modification of C339 does not lead to functional changes due to its distance from the permeation pathway, residue C339 is most likely not exposed given its location between the highly modifiable outermost C318 and C344 that is deeper into the pore. Nevertheless, C339 residues from adjacent monomers may cross-link to form dimers during protein synthesis or trafficking. Further experiments will be needed to dissect the underlying mechanisms.

Based on our results, we conclude that residue 344 is exposed to the aqueous phase. Residues 339–345 from the S5-P linker are important determinants of the structure-function properties of HCN channels.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant R01-HL-72857 (to R. A. Li), the Stem Cell Program of the University of California-Davis School of Medicine (to R. A. Li), and Hong Kong Research Grant Council Grant 7459/04M (to C.-P. Lau, H.-F. Tse, and R. A. Li). C.-W. Siu was supported by a postdoctoral fellowship award from the Croucher Foundation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Li, Univ. of California, Rm. 650, Shriners Hospital, 2425 Stockton Blvd., Sacramento, CA 95817 (e-mail: ronaldli{at}ucdavis.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Azene EM, Sang D, Tsang SY, Li RA. Pore-to-gate coupling of HCN channels revealed by a pore variant that contributes to gating but not permeation. Biochem Biophys Res Commun 327: 1131–1142, 2005.[CrossRef][Web of Science][Medline]

2. Azene EM, Xue T, Li RA. Molecular basis of the effect of potassium on heterologously expressed pacemaker (HCN) channels. J Physiol 547: 349–356, 2003.[Abstract/Free Full Text]

3. Becchetti A, Gamel K. The properties of cysteine mutants in the pore region of cyclic-nucleotide-gated channels. Pflügers Arch 438: 587–596, 1999.[CrossRef][Web of Science][Medline]

4. Becchetti A, Gamel K, Torre V. Cyclic nucleotide-gated channels. Pore topology studied through the accessibility of reporter cysteines. J Gen Physiol 114: 377–392, 1999.[Abstract/Free Full Text]

5. Bell DC, Yao H, Saenger RC, Riley JH, Siegelbaum SA. Changes in local S4 environment provide a voltage-sensing mechanism for mammalian hyperpolarization-activated HCN channels. J Gen Physiol 123: 5–19, 2004.[CrossRef][Web of Science][Medline]

6. Brown HF, Giles W, Noble SJ. Membrane currents underlying activity in frog sinus venosus. J Physiol 271: 783–816, 1977.[Abstract/Free Full Text]

7. Bucossi G, Nizzari M, Torre V. Single-channel properties of ionic channels gated by cyclic nucleotides. Biophys J 72: 1165–1181, 1997.[Web of Science][Medline]

8. Chen S, Wang J, Siegelbaum SA. Properties of hyperpolarization-activated pacemaker current defined by coassembly of HCN1 and HCN2 subunits and basal modulation by cyclic nucleotide. J Gen Physiol 117: 491–504, 2001.[Abstract/Free Full Text]

9. Creighton TE. Protein Folding. New York: Freeman, 1992.

10. DiFrancesco D. A new interpretation of the pace-maker current in calf Purkinje fibres. J Physiol 314: 359–376, 1981.[Abstract/Free Full Text]

11. DiFrancesco D. Pacemaker mechanisms in cardiac tissue. Annu Rev Physiol 55: 455–472, 1993.[CrossRef][Web of Science][Medline]

12. DiFrancesco D. A study of the ionic nature of the pace-maker current in calf Purkinje fibres. J Physiol 314: 377–393, 1981.[Abstract/Free Full Text]

13. Doyle DA, Morais Cabral J, Pfuetzner RA, Kuo A, Gulbis JM, Cohen SL, Chait BT, MacKinnon R. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280: 69–77, 1998.[Abstract/Free Full Text]

14. Er F, Larbig R, Ludwig A, Biel M, Hofmann F, Beuckelmann DJ, Hoppe UC. Dominant-negative suppression of HCN channels markedly reduces the native pacemaker current If and undermines spontaneous beating of neonatal cardiomyocytes. Circulation 107: 485–489, 2003.[Abstract/Free Full Text]

15. Flynn GE, Zagotta WN. Conformational changes in S6 coupled to the opening of cyclic nucleotide-gated channels. Neuron 30: 689–698, 2001.[CrossRef][Web of Science][Medline]

16. Gauss R, Seifert R, Kaupp UB. Molecular identification of a hyperpolarization-activated channel in sea urchin sperm. Nature 393: 583–587, 1998.[CrossRef][Medline]

17. Gavazzo P, Picco C, Eismann E, Kaupp UB, Menini A. A point mutation in the pore region alters gating, Ca2+ blockage, and permeation of olfactory cyclic nucleotide-gated channels. J Gen Physiol 116: 311–326, 2000.[Abstract/Free Full Text]

18. Hille B. Ionic Channels of Excitable Membranes. Sunderland, MA: Sinauer, 2002.

19. Kaupp UB, Seifert R. Cyclic nucleotide-gated ion channels. Physiol Rev 82: 769–824, 2002.[Abstract/Free Full Text]

20. Ludwig A, Zong X, Jeglitsch M, Hofmann F, Biel M. A family of hyperpolarization-activated mammalian cation channels. Nature 393: 587–591, 1998.[CrossRef][Medline]

21. Pape HC. Queer current and pacemaker: the hyperpolarization-activated cation current in neurons. Annu Rev Physiol 58: 299–327, 1996.[CrossRef][Web of Science][Medline]

22. Prole DL, Yellen G. Reversal of HCN channel voltage dependence via bridging of the S4–S5 linker and post-S6. J Gen Physiol 128: 273–282, 2006.[Abstract/Free Full Text]

23. Root MJ, MacKinnon R. Identification of an external divalent cation-binding site in the pore of a cGMP-activated channel. Neuron 11: 459–466, 1993.[CrossRef][Web of Science][Medline]

24. Santoro B, Chen S, Luthi A, Pavlidis P, Shumyatsky GP, Tibbs GR, Siegelbaum SA. Molecular and functional heterogeneity of hyperpolarization-activated pacemaker channels in the mouse CNS. J Neurosci 20: 5264–5275, 2000.[Abstract/Free Full Text]

25. Santoro B, Liu DT, Yao H, Bartsch D, Kandel ER, Siegelbaum SA, Tibbs GR. Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell 93: 717–729, 1998.[CrossRef][Web of Science][Medline]

26. Santoro B, Tibbs GR. The HCN gene family: molecular basis of the hyperpolarization-activated pacemaker channels. Ann NY Acad Sci 868: 741–764, 1999.[CrossRef][Web of Science][Medline]

27. Siu CW, Lieu DK, Li RA. HCN-encoded pacemaker channels: from physiology and biophysics to bioengineering. J Membr Biol 214: 115–122, 2006.[CrossRef][Web of Science][Medline]

28. Ulens C, Tytgat J. Functional heteromerization of HCN1 and HCN2 pacemaker channels. J Biol Chem 276: 6069–6072, 2001.[Abstract/Free Full Text]

29. Wahl-Schott C, Baumann L, Zong X, Biel M. An arginine residue in the pore region is a key determinant of chloride dependence in cardiac pacemaker channels. J Biol Chem 280: 13694–13700, 2005.[Abstract/Free Full Text]

30. Xue T, Li RA. An external determinant in the S5-P linker of the pacemaker (HCN) channel identified by sulfhydryl modification. J Biol Chem 277: 46233–46242, 2002.[Abstract/Free Full Text]

31. Xue T, Marban E, Li RA. Dominant-negative suppression of HCN1- and HCN2-encoded pacemaker currents by an engineered HCN1 construct: insights into structure-function relationships and multimerization. Circ Res 90: 1267–1273, 2002.[Abstract/Free Full Text]

32. Yanagihara K, Irisawa H. Potassium current during the pacemaker depolarization in rabbit sinoatrial node cell. Pflügers Arch 388: 255–260, 1980.[CrossRef][Web of Science][Medline]





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