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To correspondence should be addressed: Inst. of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, 720 Rutland Ave., Ross 844, Baltimore, MD 21205. Tel.: 410-614-0035; Fax: 410-955-7953
* This work was supported in part by National Institutes of Health Grant R01 HL52768 and a research career development award from the Cardiac Arrhythmias Research & Education Foundation, Inc. (to R. A. L.).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. ‡ Supported by NHLBI Training Grant T32-HL07227-26 from the National Institutes of Health. § Holds the Michel Mirowski, M.D. Professorship of Cardiology of The Johns Hopkins University.
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 ( = −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 ( = 1 < 3 < 10 < 30 mm); Δ 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 Δ 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)
). 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) (
), 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 (
). 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.
Previous studies of Kv channels have demonstrated that the S3-S4 linker influences activation gating (
). 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 (
Figure 1Putative transmembrane topology of HCN1.A, the six putative transmembrane segments (S1–S6) of a monomeric HCN subunit. The approximate locations of the S3-S4 residues investigated (i.e. Asp233 and Glu235, HCN1 numbering) are highlighted. The GYG selectivity motif and the cyclic nucleotide-binding domain (CNBD) are also shown. B, sequence comparison of the S3-S4 linkers of HCN1–4 with those of hyperpolarization-activated sea urchin sperm (SPIH) and plant Arabidopsis thaliana (KAT1) channels and depolarization-activatedShaker and HERG K+ channels. Although the S3-S4 linker is generally variable even within the hyperpolarization-activated channel family, Glu235 is absolutely conserved among HCN1–4, SPIH, and KAT1 channels.
). 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.
Electrophysiology, Experimental Protocols, and Data Analysis
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 (
). 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 − )/k)), where Vt is the test potential; 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((mutant)/k(mutant)−(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.
Statistics
Data are presented as mean ± S.E. Statistical significance was determined using unpaired Student'st test with p < 0.05 representing significance.
RESULTS
The S3-S4 Linker Residue Glu235 Influences HCN1 Activation Gating
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 () 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.
Figure 2WT HCN1 activation gating.A, representative traces of hyperpolarization-activated currents through WT HCN1 channels. The electrophysiological protocol used to elicit currents is displayed above. The tail currents recorded at −140 mV (boxed) are magnified to the right. Activation kinetics (τact) were obtained by fitting the initial macroscopic current with a monoexponential function. B, steady-state activation relationship. C, typical tail currents (magnified to the right) obtained by pulsing the oocyte to a series of potentials from −100 to +40 mV after a 3-s prepulse to −140 mV. Fitting tail currents with a monoexponential function allows estimation of the deactivation kinetics (τdeact). D, summary of τact(▪) and τdeact (■). The distribution of τ was bell-shaped. The rate constants α0 and β0, which reflect the open and closed rates, respectively at zero voltage, were derived from the τ curve.
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 3E235A and E235R shift activation and attenuate the charge-shielding effects of Mg2+.A, representative traces of hyperpolarization-activated currents through E235A and E235R HCN1 channels recorded under standard conditions. The same protocol described in the legend to Fig. 1 was used to elicit currents. B, steady-state activation curves of WT HCN1 recorded with 1, 3, 10, and 30 mm external Mg2+. The activation curves displayed a progressive positive shift with increasing Mg2+ concentration.C and D, steady-state activation curves of E235A and E235R, respectively, recorded with 1 (▪) and 30 (▿) mm external Mg2+. Both E235A and E235R mutations produced significant depolarizing activation shifts without altering the slope factor compared with WT channels (– – –; 1 mm Mg2+). E, bar graphsummarizing steady-state shifts in response to 30 mm Mg2+ (versus 1 mm) for WT, E235A, and E235R channels. Notably, activation shifts caused by 30 mm Mg2+ were significantly attenuated for E235A channels and almost abolished for E235R channels. *,p < 0.05.
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).
), 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 (= 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 Δ = 11.3 ± 0.9 mV (n = 8) for WT channels; p < 0.05), whereas that for E235R channels was virtually not shifted at all (Δ = 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.
Glu235 Electrostatically Influences Steady-state Activation from an External Location
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 Δ(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).
Figure 5Energetic and kinetic analyses of various Glu235 mutations.A, energetic changes (ΔΔG) in steady-state activation shifts associated with various Glu235 substitutions relative to WT channels were plotted against their own charges (i.e. Q235). E235H was assigned a charge of 0.8 (estimated from the Henderson-Hasselbalch equation assuming that the pKa of His235 is the same as that of free histidine in solution under our recording conditions). A strong linear correlation (r = 0.94) with a slope dependence of 2.1 ± 0.3 kcal/mol/charge was observed between ΔΔG associated with Glu235 mutations andQ235, consistent with an electrostatic role of residue 235. B, α0 (▪) and β0 (○) of various Glu235 mutants were plotted against the corresponding side chain volume. Theinset shows that the peak of gating kinetics of E235R channels (solid arrow) was shifted in a depolarizing direction relative to that of the WT τ curve (dashed arrow). Other constructs were shifted similarly.
Asp233 Is Not Externally Accessible, and Its Mutations Do Not Alter Gating
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) (
) 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 (
), 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 6Effects of Asp233 mutations on HCN1 activation gating.A, representative records of currents through D233E, D233A, D233G, and D233R channels. Band C, steady-state activation curves and gating kinetics, respectively, of the same channels displayed in A. All of the Asp233 mutants studied displayed gating properties that were not different from those of WT HCN1 channels.D, effects of external application of 2.5 mmMTSEA on D233C/C318S channels. D233C/C318S channels were not reactive to MTSEA indicating that residue 233 is not functionally modified by this sulfhydryl modifier from the extracellular side.
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.
). 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 (Δ ∼ 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 (
Structural and Functional Roles of the HCN1 S3-S4 Linker
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) (
) 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 ). 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 (
). 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 (
Although Glu235 is likely to influence activation gating by acting as an external surface charge of the channel, none of the Asp233 mutations studied altered HCN gating properties. Furthermore, Asp233 was not externally accessible, although it is possible that Cys233 was successfully modified, but without functional changes. These observations could be readily explained by assuming that the HCN S3-S4 linker also forms a helical structure. Such an arrangement of S3-S4 linker residues would place Asp233 on a side of the helix opposite to that of Glu235, shielding the aspartate side chain from the S4 voltage sensor. 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.
Insights into Hyperpolarization-activated Gating
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 (
). This finding led to the conclusion that S4 moves only a short distance during activation, although many other experiments suggest that S4 translocates a large distance during the process of activation gating (
). Despite these discrepancies, the consensus is that S4 moves outward upon depolarization to induce channel opening of Kv channels (and also other depolarization-activated channels).
Using sulfhydryl modification of cysteine-substituted sea urchin sperm SPIH mutant channels, Larsson and co-workers (
) recently provided evidence that the voltage-sensing mechanism of Kv channels is also conserved in this hyperpolarization-activated channel (i.e. outward movements of S4 upon depolarization). Because the voltage-sensing mechanisms are conserved between depolarization- and hyperpolarization-activated channels, the mechanisms that couple the voltage sensor and the activation gate must be different. Although the present study did not address the molecular mechanism underlying the difference in coupling that contributes to the backward gating phenotype of HCN channels, emerging evidence appears to support the notion that Kv and HCN channels share significant structural and functional similarities, e.g. both are tetramers whose fundamental building blocks are six-transmembrane subunits (
); and the S3-S4 linker influences gating, etc. Therefore, it is becoming increasingly apparent that subtle differences in structural design or coupling mechanism (albeit to be identified) may explain their distinctive activation phenotypes (
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