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• W2060667309 abstract "If, encoded by thehyperpolarization-activated cyclicnucleotide-modulated (HCN) channel family, is a key player in cardiac and neuronal pacing. Although HCN channels structurally resemble voltage-gated K+ (Kv) channels, their structure-function correlation is much less clear. Here we probed the functional importance of the HCN1 S3-S4 linker by multiple substitutions of its residues. Neutralizing Glu235, an acidic S3-S4 linker residue conserved in all hyperpolarization-activated channels, by Ala substitution produced a depolarizing activation shift (V½ = −65.0 ± 0.7 versus −70.6 ± 0.7 mV for wild-type HCN1); the charge-reversed mutation E235R shifted activation even more positively (−56.2 ± 0.5 mV). Increasing external Mg2+ mimicked the progressive rightward shifts of E235A and E235R by gradually shifting activation (V½ = 1 < 3 < 10 < 30 mm); ΔV½ induced by 30 mmMg2+ was significantly attenuated for E235A (+7.9 ± 1.2 versus +11.3 ± 0.9 mV for wild-type HCN1) and E235R (+3.3 ± 1.4 mV) channels, as if surface charges were already shielded. Consistent with an electrostatic role, the energetic changes associated with ΔV½ resulting from various Glu235 substitutions (i.e. Asp, Ala, Pro, His, Lys, and Arg) displayed a strong correlation with their charges (ΔΔG = −2.1 ± 0.3 kcal/mol/charge;r = 0.94). In contrast, D233E, D233A, D233G, and D233R did not alter activation gating. D233C (in C318S background) was also not externally accessible when probed with methanethiosulfonate ethylammonium (MTSEA). We conclude that the S3-S4 linker residue Glu235 influences activation gating, probably by acting as a surface charge. If, encoded by thehyperpolarization-activated cyclicnucleotide-modulated (HCN) channel family, is a key player in cardiac and neuronal pacing. Although HCN channels structurally resemble voltage-gated K+ (Kv) channels, their structure-function correlation is much less clear. Here we probed the functional importance of the HCN1 S3-S4 linker by multiple substitutions of its residues. Neutralizing Glu235, an acidic S3-S4 linker residue conserved in all hyperpolarization-activated channels, by Ala substitution produced a depolarizing activation shift (V½ = −65.0 ± 0.7 versus −70.6 ± 0.7 mV for wild-type HCN1); the charge-reversed mutation E235R shifted activation even more positively (−56.2 ± 0.5 mV). Increasing external Mg2+ mimicked the progressive rightward shifts of E235A and E235R by gradually shifting activation (V½ = 1 < 3 < 10 < 30 mm); ΔV½ induced by 30 mmMg2+ was significantly attenuated for E235A (+7.9 ± 1.2 versus +11.3 ± 0.9 mV for wild-type HCN1) and E235R (+3.3 ± 1.4 mV) channels, as if surface charges were already shielded. Consistent with an electrostatic role, the energetic changes associated with ΔV½ resulting from various Glu235 substitutions (i.e. Asp, Ala, Pro, His, Lys, and Arg) displayed a strong correlation with their charges (ΔΔG = −2.1 ± 0.3 kcal/mol/charge;r = 0.94). In contrast, D233E, D233A, D233G, and D233R did not alter activation gating. D233C (in C318S background) was also not externally accessible when probed with methanethiosulfonate ethylammonium (MTSEA). We conclude that the S3-S4 linker residue Glu235 influences activation gating, probably by acting as a surface charge. If or Ih, encoded by thehyperpolarization-activated cyclicnucleotide-modulated (HCN) 1The abbreviations used are: HCNhyperpolarization-activated cyclicnucleotide-gatedKvvoltage-gated K+WTwild-typeMTSEAmethanethiosulfonate ethylammonium or the so-called pacemaker channel gene family, is a key contributor to spontaneous rhythmic activity in cardiac and neuronal cells (1DiFrancesco D. Annu. Rev. Physiol. 1993; 55: 455-472Crossref PubMed Scopus (673) Google Scholar, 2Pape H.C. Annu. Rev. Physiol. 1996; 58: 299-327Crossref PubMed Scopus (982) Google Scholar, 3DiFrancesco D. J. Physiol. (Lond.). 1981; 314: 377-393Crossref Scopus (238) Google Scholar, 4DiFrancesco D. J. Physiol. (Lond.). 1981; 314: 359-376Crossref Scopus (268) Google Scholar, 5Brown H.F. Giles W. Noble S.J. J. Physiol. (Lond.). 1977; 271: 783-816Crossref Scopus (79) Google Scholar, 6Yanagihara K. Irisawa H. Pfluegers Arch. 1980; 388: 255-260Crossref PubMed Scopus (50) Google Scholar, 7Gauss R. Seifert R. Kaupp U.B. Nature. 1998; 393: 583-587Crossref PubMed Scopus (379) Google Scholar, 8Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (787) Google Scholar, 9Santoro B. Liu D.T. Yao H. Bartsch D. Kandel E.R. Siegelbaum S.A. Tibbs G.R. Cell. 1998; 93: 717-729Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar). Although HCN channels structurally resemble voltage-gated K+ (Kv) channels (for instance, both are tetramers made up of monomeric subunits consisting of six membrane-spanning segments) (7Gauss R. Seifert R. Kaupp U.B. Nature. 1998; 393: 583-587Crossref PubMed Scopus (379) Google Scholar, 8Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (787) Google Scholar, 9Santoro B. Liu D.T. Yao H. Bartsch D. Kandel E.R. Siegelbaum S.A. Tibbs G.R. Cell. 1998; 93: 717-729Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar, 10Xue T. Marban E. Li R.A. Circ. Res. 2002; 90: 1267-1273Crossref PubMed Scopus (66) Google Scholar), a distinguishing functional feature that discriminates pacemaker channels from the Kv counterparts is their signature “backward” gating (i.e. activation upon hyperpolarization rather than depolarization). The molecular basis of this unique gating phenotype is unknown. Recently, it has been suggested that the voltage-sensing mechanism of the sea urchin sperm HCN (i.e. SPIH or spHCN) and Kv channels may be conserved (11Mannikko R. Elinder F. Larsson H.P. Nature. 2002; 419: 837-841Crossref PubMed Scopus (164) Google Scholar). In any case, the structure-function correlation of HCN channels is much less defined compared with the well studied Kv channels. Comparison of these related yet functionally distinct ion channels should provide important insights into the unique behavior of HCN channels. hyperpolarization-activated cyclicnucleotide-gated voltage-gated K+ wild-type methanethiosulfonate ethylammonium Previous studies of Kv channels have demonstrated that the S3-S4 linker influences activation gating (12Gonzalez C. Rosenman E. Bezanilla F. Alvarez O. Latorre R. J. Gen. Physiol. 2000; 115: 193-208Crossref PubMed Scopus (66) Google Scholar, 13Gonzalez C. Rosenman E. Bezanilla F. Alvarez O. Latorre R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9617-9623Crossref PubMed Scopus (58) Google Scholar, 14Sorensen J.B. Cha A. Latorre R. Rosenman E. Bezanilla F. J. Gen. Physiol. 2000; 115: 209-222Crossref PubMed Scopus (34) Google Scholar, 15Mathur R. Zheng J. Yan Y. Sigworth F.J. J. Gen. Physiol. 1997; 109: 191-199Crossref PubMed Scopus (56) Google Scholar). By analogy to Kv channels, it is possible that the S3-S4 linker (defined as residues 229–237 here, HCN1 numbering) of HCN channels also contributes to activation gating. However, this idea has not been tested. In this study, we probed the functional importance of the S3-S4 linker of HCN1 channels by multiple substitutions of its residues. We found that the acidic linker residue Glu235, conserved among all known hyperpolarization-activated channels (Fig.1), prominently influences HCN gating. Glu235 is also largely responsible for the charge-shielding effects of external Mg2+. Novel insights into the structural and functional roles of the S3-S4 linker of HCN channels are discussed. A preliminary report has appeared (16Henrikson C.A. Dong P. Marban E. Li R.A. Circulation. 2002; 106: II-226Google Scholar). Mouse HCN1 (kindly provided by Drs. S. A. Siegelbaum and B. Santoro) was subcloned into the pGHE expression vector (9Santoro B. Liu D.T. Yao H. Bartsch D. Kandel E.R. Siegelbaum S.A. Tibbs G.R. Cell. 1998; 93: 717-729Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar). Mutations were created using PCR with overlapping mutagenic primers. The desired mutations were confirmed by DNA sequencing. cRNA was transcribed fromNheI-linearized DNA using T7 RNA polymerase (Promega, Madison, WI). HCN1 channel constructs were heterologously expressed and studied in Xenopus oocytes. Briefly, stage IV–VI oocytes were surgically removed from female frogs anesthetized by immersion in 0.3% 3-aminobenzoic acid ethyl ester, followed by digestion with 1 mg/ml collagenase (type IA) in OR-2 containing 88 mmNaCl, 2 mm KCl, 1 mm MgCl2, and 5 mm HEPES (pH 7.6) for 30–60 min. Isolated oocytes were injected with cRNA (50 or 100 ng/cell) and stored in 96 mmNaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, and 5 mm HEPES (pH 7.6) supplemented with 50 μg/ml gentamycin, 5 mm pyruvate, and 0.5 mm theophylline. Two-electrode voltage-clamp recordings were performed at room temperature (23–25 °C) using a Warner OC-725B amplifier 1–2 days after cRNA injection as described (10Xue T. Marban E. Li R.A. Circ. Res. 2002; 90: 1267-1273Crossref PubMed Scopus (66) Google Scholar). Because Xenopusoocytes are essentially a mass of yolk surrounded by an outer fenestrated membrane, membrane potential could be accurately measured only to within approximately ±2 mV. Furthermore, it was assumed that ion conductance had reached steady-state under our experimental conditions. The regular recording bath solution contained 97.8 mm KCl, 2 mm NaCl, 10 mm HEPES, and 1 mm MgCl2 (pH 7.5). The MgCl2concentration was increased in certain experiments as indicated. The voltage dependence of HCN channel activation was assessed by plotting tail currents measured immediately after pulsing to −140 mV as a function of the preceding 3-s test pulse voltage normalized to the maximum tail current recorded. Data were fit to the Boltzmann functions using the Marquardt-Levenberg algorithm in a nonlinear least-squares procedure: m∞ = 1/(1 + exp((Vt − V½)/k)), where Vt is the test potential;V½ is the half-point of the relationship; andk = RT/zF is the slope factor, where R, T, z, and F have their usual meanings. Changes in free energy (ΔΔG) associated with steady-state activation shifts caused by amino acid substitution were calculated using the following equation: ΔΔG =RT(V½(mutant)/k(mutant)−V½(WT)/kWT). For simplification of kinetic analysis, the time constants for activation (τact) and deactivation (τdeact) were estimated by fitting macroscopic and tail currents, respectively, with a monoexponential function. However, it should be noted that the onset of HCN1 currents shows sigmoidicity and that tail currents also exhibited an initial delay. The mechanism underlying such complex kinetic behavior of HCN channels is not understood; further analysis using multiple exponential components was beyond the scope of the this study. For estimating open and closed rates of channels, the bell-shaped distribution of τact and τdeactwas fitted to the following relation: τ = 1/(α0e−Vm/Vo+ β0eVm/Vo), where α0 and β0 reflect the open and closed rates at zero voltage, respectively. Data are presented as mean ± S.E. Statistical significance was determined using unpaired Student'st test with p < 0.05 representing significance. We first characterized wild-type (WT) HCN1 channels by examining their activation gating properties. Stepping the transmembrane potential to voltages below −40 mV activated typical time-dependent inward currents whose time constants (τact) became faster with progressive hyperpolarization (Fig. 2, A and D). The midpoint (V½) and slope factor (k) derived from the steady-state activation curve were −70.6 ± 0.7 mV and 9.5 ± 0.5 (n = 6), respectively (Fig.2B). We also studied the deactivation properties of WT channels by examining the voltage dependence of the rate of tail current decay following maximum channel opening by hyperpolarizing to −140 mV (Fig. 2, C and D). Unlike τact, the deactivation time constant (τdeact) became faster with increasing depolarization. Plotting these time constants together against the test potential revealed that the voltage dependence of τact and τdeact had a bell-shaped form (Fig. 2D); α0 and β0 for WT HCN1 channels derived from this curve were (3.6 ± 0.5) × 10−1 and (2.3 ± 0.2) × 10 s−1, respectively (see “Experimental Procedures”). The peak of the τ curve coincided with the steady-state activation midpoint. To investigate the functional roles of the S3-S4 linker in HCN1 activation, we first mutated Glu235, an acidic residue conserved in all hyperpolarization-activated channels (cf.Fig. 1), to Ala and Arg for net charge changes of +1 and +2 at this channel site, respectively. The charge-neutralized substitution E235A produced a significant depolarizing shift in steady-state channel activation without altering the slope factor (Fig.3C; see also Fig. 4). Interestingly, the charge-reversed mutation E235R shifted activation even more positively (Fig. 3D), highly suggestive of an electrostatic role of residue 235. Similar to E235A, the slope factor of E235R channels was also not altered (Figs. 3D and4C).Figure 4Effects of various Glu235mutations on HCN1 activation gating. A, representative currents through E235D, E235P, E235H, and E235K channels. Band C, summary of steady-state activation midpoints and slope factors, respectively, of various Glu235 mutants (i.e. E235D, E235A, E235P, E235H, E235R, and E235K).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because HCN channels are not embedded in the membrane in an orientation opposite to that of depolarization-activated channels (17Xue T. Li R.A. J. Biol. Chem. 2002; 277: 46233-46242Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), the S3-S4 linker should be on the extracellular side. We hypothesized that if Glu235 is externally accessible and its effects on channel activation are electrostatic, screening surface charges should effectively shield its anionic charge and thereby produce activation shifts similar to those observed with the E235A and E235R mutants. Consistent with this notion, increasing external Mg2+ gradually shifted steady-state activation in the depolarizing direction (V½= 70.6 ± 0.7 mV (n = 6), −68.9 ± 1.1 mV (n = 3), −66.0 ± 0.4 mV (n = 3), and −60.1 ± 1.5 mV (n = 8) for 1, 3, 10, and 30 mm Mg2+, respectively) (Fig. 3B), mimicking the progressive rightward shifts caused by mutations E235A and E235R. As extra support for the external electrostatic role of residue 235, Fig. 3 (C and D) shows that the response of E235A and E235R channels to Mg2+ was significantly attenuated. 30 mm Mg2+ induced only a 7.9 ± 1.2 mV (n = 5) depolarizing shift of the steady-state activation midpoint for E235A channels (versus ΔV½ = 11.3 ± 0.9 mV (n = 8) for WT channels; p < 0.05), whereas that for E235R channels was virtually not shifted at all (ΔV½ = 3.3 ± 1.4 mV (n = 3); p > 0.05), as if surface charges were already shielded. Activation shifts of WT, E235A, and E235R HCN1 channels caused by 30 mm Mg2+ are summarized and compared in Fig. 3E. If the S3-S4 linker residue Glu235 influences HCN1 activation by electrostatic means, substitutions of residue 235 by amino acids other than Ala and Arg that also render its charge neutralized or reversed should produce rightward activation shifts similar to those of E235A and E235R channels. In complete accordance with this notion, the activation midpoints of E235K, E235H, and E235P channels were all displaced in the depolarizing direction (Fig. 4B). The charge-reversed mutations (E235R, E235K, and E235H) generally produced more pronounced activation shifts than did the charge-neutralized counterparts (E235P and E235A), although E235P was more positively shifted than E235H. In contrast to these charge-altered substitutions, the charge-conserved substitution E235D displayed gating properties indistinguishable from those of WT channels. None of the Glu235 mutations significantly altered the slope factor (p > 0.05) (Fig.4C). To determine whether a correlation between steady-state activation and the charge of residue 235 (Q235) exists, we plotted the energetic changes (ΔΔG) associated with the activation shifts resulting from the various substitutions studied relative to WT channels (i.e. Glu235) against their own charges. Fig. 5Aindicates that ΔΔG and Q235 were linearly correlated (r = 0.94) with a slope dependence of 2.1 ± 0.3 kcal/mol/charge. Taken collectively, our results were consistent with an external electrostatic role of residue 235. In comparison with WT channels, all Glu235 mutants displayed bell-shaped τ curves, except with peaks shifted in the depolarizing direction that paralleled the corresponding ΔV½(see Fig. 5B (inset) for an example). Neither α0 nor β0 (derived from τ curves) displayed any obvious correlation with the charge of residue 235. Fig.5B shows that, whereas β0 was not affected by the side chain volume at all, there was a trend that α0tended to accelerate with increasing side chain bulk, but the correlation was weak (r = 0.44). We also investigated the effects of substituting another anionic residue, Asp233, within the same linker (cf. Fig. 1). In contrast to Glu235mutations, however, none of the D233A, D233G, D233E, and D233R channels exhibited gating properties different from those of WT HCN1 (Fig.6, A–C) despite the close proximity of residues 233 and 235. These results indicate that the functional changes observed with Glu235 substitutions are site-specific. We also probed the external accessibility of residue 233 by examining the sensitivity of D233C channels (in the background of C318S to eliminate the intrinsic sensitivity of WT channels to MTS compounds) (17Xue T. Li R.A. J. Biol. Chem. 2002; 277: 46233-46242Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) to the hydrophilic sulfhydryl modifier MTSEA. Like C318S channels, D233C/C318S channels were not reactive to external application of 2.5 mm MTSEA (Fig.6D). E235C/C318S did not lead to functional expression of measurable currents, rendering the assessment of its sensitivity to MTS agents not possible. Combining the S3-S4 linker mutations D233A and E235A with the S4-S5 linker substitution W270A, whose equivalent mutation in HCN2 (i.e. W323A) is known to shift activation positively (18Chen J. Mitcheson J.S. Tristani-Firouzi M. Lin M. Sanguinetti M.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11277-11282Crossref PubMed Scopus (133) Google Scholar), produced an activation shift more positive than either of the individual mutations, indicating that the effects from these single substitutions were largely additive (Fig.7). These results suggest that the external S3-S4 linker and the cytoplasmic S4-S5 linker are likely to exert their effects on activation via mechanisms that are independent of each other.Figure 7Effects of W270A and D233A/E235A/W270A on HCN1 activation gating. A, representative currents through W270A and D233A/E235A/W270A channels. B, steady-state activation curves of the same channel constructs inA. W270A alone already produced significant depolarizing activation shifts compared with WT channels. The triple mutation D233A/E235A/W270A shifted activation even more positively, suggesting the S3-S4 and S4-S5 linkers operate independently.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Previous studies have demonstrated that the extracellular S3-S4 linker is a determinant of activation in various K+ and Ca2+ channels (12Gonzalez C. Rosenman E. Bezanilla F. Alvarez O. Latorre R. J. Gen. Physiol. 2000; 115: 193-208Crossref PubMed Scopus (66) Google Scholar, 13Gonzalez C. Rosenman E. Bezanilla F. Alvarez O. Latorre R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9617-9623Crossref PubMed Scopus (58) Google Scholar, 14Sorensen J.B. Cha A. Latorre R. Rosenman E. Bezanilla F. J. Gen. Physiol. 2000; 115: 209-222Crossref PubMed Scopus (34) Google Scholar, 15Mathur R. Zheng J. Yan Y. Sigworth F.J. J. Gen. Physiol. 1997; 109: 191-199Crossref PubMed Scopus (56) Google Scholar, 19Nakai J. Adams B.A. Imoto K. Beam K.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1014-1018Crossref PubMed Scopus (88) Google Scholar). Our results indicate that the S3-S4 linker of HCN channels also influences activation gating. The charge-neutralizing and charge-reversing mutations of linker residue 235 positively shifted steady-state activation in a charge-dependent fashion (as reflected by the linear ΔΔG versus Q235 relationship). Surface charge shielding by external Mg2+ mimics our charge-changing mutations by shifting the WT activation curve in the depolarizing direction; such Mg2+-induced shifts were reduced by mutation E235A, as if the channels were already partially screened by this charge-neutralized substitution. The charge-screening effects of Mg2+ were further attenuated by E235R, although there was a modest residual response (ΔV½ ∼ 3 mV) to the addition of 30 mm Mg2+. Although the activation shift and the decreased Mg2+ effect caused by the neutralization of Glu235 could be most easily explained by residue 235 functioning as a surface charge, the additional decreased Mg2+ effect caused by the charge-reversed E235R mutation could result from the screening of an additional negative surface charge of the channel by the substituted arginine. Indeed, this notion is consistent with our finding that neutralizing Glu235eliminated ∼50% of the Mg2+ effect and that Mg2+ can screen only negative surface charges. Further studies are required to identify this additional endogenous surface charge. Theoretically, change of a surface charge should produce concomitant shifts in the voltage dependence of both steady-state activation and gating kinetics. Indeed, this was observed with our Glu235 mutants (Fig. 5B, inset), consistent with the effect of a surface charge. Taken collectively, our results suggest that Glu235 is largely responsible for the surface charge-shielding effects of Mg2+, probably at an externally accessible position (although the Mg2+ effects could be indirect). Interestingly, the attenuated charge-screening effects of external Mg2+ on Glu235 mutant channels mirror the abolition of surface charge-shielding effects of H+ observed with the Shaker mutant, whose S3-S4 linker (including the acidic residues Glu333, Glu334, Glu335, and Asp336) has been deleted (14Sorensen J.B. Cha A. Latorre R. Rosenman E. Bezanilla F. J. Gen. Physiol. 2000; 115: 209-222Crossref PubMed Scopus (34) Google Scholar). Mechanistically, it is tempting to speculate that the S3-S4 linker of HCN1 channels does not undergo significant conformational changes during activation (e.g. movements in the direction opposite to that of the positively charged S4) (15Mathur R. Zheng J. Yan Y. Sigworth F.J. J. Gen. Physiol. 1997; 109: 191-199Crossref PubMed Scopus (56) Google Scholar) because of its apparent lack of contribution to the effective gating charge (as reflected by the relatively unchanged slope factors of Glu235 mutants). Because Glu235 is separated by only two residues from the first basic S4 residue (i.e.Lys238), this native glutamate may serve as a surface charge that influences HCN activation by shaping the local electric field sensed by the positively charged S4 (thereby shifting the steady-state V½). Theoretically, neutralization of a negative surface charge will always have the same effect, independent of the voltage-sensing mechanism, by altering the transmembrane voltage gradient: the voltage dependence should be shifted to more depolarized potentials such that a stronger depolarization will restore the original voltage profile. In this regard, the S3-S4 linker does not need to directly participate in gating to exert its effects. However, based on the data presented, we cannot exclude the possibility that the S3-S4 linker could undergo major conformational changes (e.g. horizontal movements would not alter the slope factor). Although α0 and β0 of HCN1 were not drastically altered by Glu235 mutations, other S3-S4 linker residues may alter the energy barriers separating the channel transitions required for channel openings (i.e. gating kinetics). Clearly, additional experiments are needed to further explore the functional role of this channel region. It should be noted that the gating parameters studied here can also be modulated by other mechanisms. For instance, it has been demonstrated that cAMP binding to the cyclic nucleotide-binding domain of HCN channels shifts the voltage dependence of steady-state activation to more depolarizing potentials and accelerates activation kinetics by relieving an intrinsic inhibitory influence of the cyclic nucleotide-binding domain on basal gating (20Wainger B.J. DeGennaro M. Santoro B. Siegelbaum S.A. Tibbs G.R. Nature. 2001; 411: 805-810Crossref PubMed Scopus (402) Google Scholar). Although mutating the S3-S4 linker and/or Mg2+ application may have indirect or long-range allosteric effects on this modulatory mechanism, it is reassuring that a highly correlated linear relationship was observed with multiple Glu235 mutations carrying different charges; indeed, the isoform used in our study, HCN1, is less sensitive to allosteric modulation by the cyclic nucleotide-binding domain than HCN2 (8Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (787) Google Scholar, 9Santoro B. Liu D.T. Yao H. Bartsch D. Kandel E.R. Siegelbaum S.A. Tibbs G.R. Cell. 1998; 93: 717-729Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar, 20Wainger B.J. DeGennaro M. Santoro B. Siegelbaum S.A. Tibbs G.R. Nature. 2001; 411: 805-810Crossref PubMed Scopus (402) Google Scholar, 21Ludwig A. Zong X. Stieber J. Hullin R. Hofmann F. Biel M. EMBO J. 1999; 18: 2323-2329Crossref PubMed Scopus (314) Google Scholar, 22Chen S. Wang J. 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Alternatively, Asp233 could be farther away from the S4 segment, thereby minimizing the effects of charge neutralization at this channel site. Clearly, additional experiments are needed to distinguish between these possibilities. Previous studies with depolarization-activated K+ channels have established that channel regions other than the S4 segments are also involved in the process of voltage sensing (25Papazian D.M. Timpe L.C. Jan Y.N. Jan L.Y. Nature. 1991; 349: 305-310Crossref PubMed Scopus (431) Google Scholar, 26Liman E.R. Hess P. Weaver F. Koren G. Nature. 1991; 353: 752-756Crossref PubMed Scopus (234) Google Scholar, 27Logothetis D.E. Kammen B.F. Lindpaintner K. Bisbas D. Nadal-Ginard B. Neuron. 1993; 10: 1121-1129Abstract Full Text PDF PubMed Scopus (46) Google Scholar, 28Logothetis D.E. Movahedi S. Satler C. Lindpaintner K. Nadal-Ginard B. Neuron. 1992; 8: 531-540Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 29Aggarwal S.K. MacKinnon R. 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