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J. Biol. Chem., Vol. 279, Issue 16, 16832-16846, April 16, 2004
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From the Ion Channel Laboratory, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada
Received for publication, January 16, 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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70% of total If over voltages ranging from +20 to -150 mV in high K+ solutions. If activated at more depolarized potentials and the activation curve was less steep, whereas deactivation was significantly slowed, consistent with the idea that the mutation inhibited channel closing. The data suggest that the mutation produced allosteric effects on the activation gate (S6 segment) and/or on voltage-sensing elements. We also found that decreases in the ratio of external K+/Na+ further disrupted channel closing in the mutant channel. Finally, our data suggest that the structures involved in producing Iinst are similar between the HCN1 and HCN2 isoforms and that excess HCN protein on the plasma membrane of CHO cells relative to native cells is not responsible for Iinst. The data are consistent with Iinst flowing through a "leaky" closed state but do not rule out flow through a second configuration of recombinant HCN channels or up-regulated endogenous channels/subunits. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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We subsequently examined the role of the selectivity filter and the positively charged S4 region in producing Iinst. This current was not affected by a mutation in the selectivity filter (HCN2 G404S), extracellular perfusion of Cs+, or a mutation in the S4 segment (S306Q) unlike If, which was reduced or eliminated by these mutations as well as by Cs+ (14). Finally, the amplitude of Iinst, but not If, could be predicted on the basis of independent flow of Na+ and K+. Thus, If and Iinst were both mixed cation conductances, but these could be structurally and functionally separated. These results leave open the question of whether the Cs+-insensitive Iinst flows through the same pore as If (for example through a "leaky" closed state) or whether it flows through a second pore that is found within the same channel, formed by a second configuration of HCN channel subunits or associated with HCN channels in the form of up-regulated endogenous channels or subunits.
In the present study, we continued to examine regions of HCN channels potentially involved in regulating Iinst in order to understand how this current is generated. A recent study has shown that mutations of residue Tyr331 of the S4-S5 linker in HCN2 produced large instantaneous currents and reduced If, suggesting a disruption of channel closure (10). However, the nature of the instantaneous currents was not determined in those studies, and thus it was not clear whether the up-regulated current had properties which were similar to Iinst or If. Here, we found that Y331D up-regulated an instantaneous current when expressed in CHO cells, but its properties were more like If than like Iinst. We also found that If amplitude was reduced, the activation curve of the remaining If was shifted to more positive voltages and was less steep, and If deactivation was slowed in the mutant channel. Together, the data support the idea that the Y331D mutation disrupted channel closing through local allosteric effects on the activation gate (S6 segment) and/or on the voltage-sensing elements. Interestingly, our data also suggest that decreasing the ratio of external K+ versus Na+ further disrupted the closing of the mutant channel possibly through a separate "foot in the door" mechanism. Finally, we found that the HCN1 isoform, when expressed on the plasma membrane of CHO cells in amounts similar to native HCN channels, produced Iinst, which was proportional in size to If. These data suggest that the structural elements involved in producing Iinst are similar between HCN1 and HCN2 and that excess expression of HCN subunits on the plasma membrane of CHO cells, relative to native cells, is not responsible for producing Iinst. The data are consistent with Iinst flowing through a leaky closed state but do not rule out flow through a second configuration of recombinant HCN channels or up-regulated endogenous channels/subunits.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Tissue Culture—CHO-K1 cells were obtained from ATCC (Manassas, VA), maintained in Ham's F-12 medium supplemented with antibiotics and 10% fetal bovine serum (Invitrogen) and incubated at 37 °C with 5% CO2. Cells were plated onto glass coverslips and 1 day after splitting were transiently co-transfected with mammalian expression vectors encoding wild-type and/or mutant mHCN2 channels (2 µg/35-mm dish) along with a green fluorescent protein (GFP) reporter plasmid (0.3 µg/dish) using the FuGENE 6 transfection reagent (Roche Applied Science).
Electrophysiology and Analysis—Cells expressing GFP were chosen for whole-cell recordings 24-48 h after transfection or co-transfection with mHCN2 and mHCN1 constructs. The pipette solution contained 130 mM potassium aspartate, 10 mM NaCl, 0.5 mM MgCl2, 1 mM EGTA, 5 mM HEPES, pH adjusted to 7.4 with KOH. The extracellular solution contained varying concentrations of NaCl, KCl, and NMG (see figure legends for each experimental condition), 1.8 mM CaCl2, 0.5 mM MgCl2, 5 mM HEPES, pH adjusted to 7.4 with NaOH. For solution changes, a 200-µl bath was completely exchanged and perfused (0.5-1 ml/min) for at least 1 min prior to collecting data. Whole-cell patch clamp currents were recorded using an Axopatch 200B amplifier and Clampex software (Axon Instruments, Union City, CA) at room temperature (20-22 °C). Currents were not leak-subtracted, and capacitance compensation was not used. Patch clamp pipettes were pulled from borosilicate glass and were fire-polished before use (pipette resistance is 2.5-4.5 megaohms). Data were filtered at 2 kHz and were analyzed using Clampfit (Axon Instruments), Origin (Microcal, Northampton, MA) and Excel (Microsoft, Seattle, WA) software. Current densities were determined by dividing measured currents by the capacitance, which was estimated by the ClampEx software from the time constant of the current elicited by a 2-mV test pulse at the beginning of whole-cell recording.
In order to determine the voltage dependence of block by Cs+ of instantaneous current in cells expressing HCN2 Y331D (15), the following equation was used,
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where I and ICs represent the HCN2 Y331D instantaneous current in control conditions and after Cs+, [Cs] is Cs+ concentration (2 mM), ko is the dissociation constant of Cs+ binding to the block site at zero voltage, and 
is the "electrical" distance of the block site from the external membrane surface. Equation 1 was used to the fit the data in Fig. 6, A and C, and values for ko and were obtained (see legend to Fig. 6). 
2 values were also obtained, indicating how well the points were fit. This analysis was also used to examine Cs+ block of If in CHO cells expressing wild-type HCN2.
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 | (Eq. 2) |
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To assess rates of If activation and deactivation, time constants were generated using a single exponential fitting procedure. An initial delay occurred prior to both activation and deactivation, which was not well described by a single exponential function and therefore was not used in our fits. Student's paired or unpaired t test was used to determine significance when comparisons were made between groups of cells (p < 0.05).
Immunocytochemistry and Confocal Microscopy—At 24-48 h post-transfection, cells on coverslips were washed with PBS and fixed in 2% paraformaldehyde in PBS for 5 min. The cells were then washed with PBS, permeabilized using 0.2% Triton X-100, and blocked with 10% normal goat serum (NGS). After one wash with PBS containing 1% NGS, the cells were incubated with a rabbit polyclonal antibody to HCN2 (Alomone Labs, Jerusalem, Israel) at a dilution of 1:400 in PBS with 1% NGS for 48 h at 4 °C. The antibody was removed, and cells were again washed with PBS. Cells were then incubated with a donkey anti-rabbit antibody tagged with cyanine 3 (Jackson Laboratories, West Grove, PA) at a dilution of 1:200 in PBS with 1% NGS for 1 h at room temperature in the dark. The antibody was removed, cells were washed in PBS, and the coverslips with cells were mounted on slides using Permount (Fisher). Using an inverted Zeiss TurboPascal confocal microscope, serial sections were taken in 0.8-1.0 µM steps using a x 63 oil immersion objective lens and an excitation wavelength of 543 nm.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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We anticipated three possible scenarios upon transfection of HCN2 Y331D in CHO cells. 1) If the instantaneous current were significantly larger than Iinst measured in cells expressing the wild-type channel and sensitive to Cs+, then this would suggest that the mutation favored an "If-like" open state. 2) If the instantaneous current were significantly larger than Iinst measured in cells expressing the wild-type channel but insensitive to Cs+, then this would suggest that the mutation favored an "Iinst-like" state. 3) If the instantaneous current were not significantly larger than Iinst measured in cells expressing the wild-type channel and were insensitive to Cs+, then this would suggest that the mutation did not affect Iinst. We expected that the instantaneous currents would be carried by Na+ and K+ in any of the above scenarios. Based on the possibility that Iinst results from a leaky closed state, we hypothesized that the Y331D mutation would increase Iinst and thus that this current would be significantly up-regulated by the mutation, insensitive to Cs+, approximately linear with respect to changes in voltage and carried by Na+ and K+ (11, 14).
A comparison of the S3 segment, the S4 segment, and the S4-S5 linker among mouse HCN1 and HCN2, sea urchin (Strongylocentrotus purpuratus) HCN, and the recently crystallized KvAP (16) shows the position of the HCN2 Y331D mutation (Fig. 1). Using the whole-cell patch clamp approach, we determined the densities of If and instantaneous currents recorded from cells expressing wild-type HCN2 and HCN2 Y331D in order to control for variability in cell surface area. Cells expressing HCN2 Y331D produced significantly less If and significantly more instantaneous current than did cells expressing wild-type HCN2, as shown in representative current traces (Figs. 2A and 3A). The increase in instantaneous current in cells expressing HCN2 Y331D is apparent in I-V curves generated at each of three varying concentrations of extracellular K+ or Na+ examined (Figs. 2, B-D, and 3, B-D). The increase in instantaneous current amplitude due only to the Y331D mutation is shown in I-V curves that represent the difference in instantaneous currents between cells expressing HCN2 and HCN2 Y331D (Figs. 2E and 3E).
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Interestingly, the I-V curves for Iinst in cells expressing wild-type HCN2 were linear, whereas the I-V curves for the instantaneous current in cells expressing HCN2 Y331D rectified in the inward direction especially at the elevated extracellular K+ and Na+ concentrations. Inward rectification of instantaneous currents was also observed in experiments in Xenopus oocytes expressing HCN2 Y331D (10). The rectification suggested that the mixed cation instantaneous currents in cells expressing HCN2 Y331D and HCN2 were different. Previous studies using HEK cells expressing mouse HCN2 also showed that the fully activated If had greater inward rectification at 30 mM K+ as compared with 5.4 mM K+ (8). Overall, the results suggested that the up-regulated instantaneous currents we observed in cells expressing HCN2 Y331D had similarities to both If and Iinst.
HCN2 Y331D Is Expressed on the Cell Surface of CHO Cells in Significant Amounts—The large decrease in If in cells expressing Y331D may have reflected a decrease in the total amount of protein expressed in the cell and/or localized to the plasma membrane. Therefore, we examined the localization of mutant channels labeled with an anti-HCN2 primary antibody and a cyanine 3-tagged secondary antibody using confocal microscopy, which would unmask any large reductions in protein expression. There was a very strong pattern of fluorescence along the periphery of the cells consistent with the presence of HCN2 protein on the plasma membrane (Fig. 4). Fluorescent regions were also observed in the interior of the cells, probably representing channel protein present in intracellular compartments such as the endoplasmic reticulum, Golgi apparatus, vesicles mediating transport to or from the plasma membrane, or degradatory compartments. There was relatively little fluorescence in nontransfected cells or in mock-transfected cells (not shown). We have shown previously that the wild-type channel demonstrated a similar pattern of localization and overall intensity of fluorescence, whereas mutants lacking the cyclic nucleotide binding domain and distal C terminus or the complete N terminus did not express If or instantaneous currents significantly larger than GFP-transfected cells and demonstrated a very different pattern of localization that did not include fluorescence on the periphery of CHO cells (12). The pattern of fluorescence observed is consistent with the idea that the large instantaneous currents recorded in these cells were due to HCN2 Y331D and that the large reduction of If in cells expressing HCN2 Y331D was not due to a low level of HCN2 protein in the cell or on the plasma membrane.
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We also found that the total amount of Cs+-sensitive current was larger in cells expressing HCN2 Y331D (If + Cs+-sensitive instantaneous current) than in cells expressing HCN2 (If only) (Fig. 5G). The larger Cs+-sensitive current in cells expressing HCN2 Y331D suggests some effect of the mutation on maximal conductance, since the channels are fully activated at -150 mV (see Fig. 7F). One possibility is that the number of functional mutant channels was larger than the number of functional wild-type channels. If this were true, we would expect Cs+-insensitive Iinst to also be larger, because it is proportional to the amount of HCN protein on the plasma membrane (see Ref. 11 for HCN2 and Fig. 10A for HCN1). We found that Iinst associated with HCN2 Y331D was not significantly larger than Iinst associated with HCN2, which implies a difference in single channel conductance or open probability. However, single channel measurements and assays of HCN protein on the plasma membrane will be required to accurately and directly determine whether the mutation affects expression of the channel on the plasma membrane.
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2 value for the former (see legend to Fig. 6). Together, the data strongly suggest that the block of instantaneous current by 2 mM Cs+ was complete at the more negative voltages and that the unblocked instantaneous current at -150 mV reflected Iinst in cells expressing HCN2 Y331D. According to the fitting, our data suggest that Cs+ ions cross 0.87 of the membrane electrical thickness () before reaching the binding site in HCN2 Y331D channels. The dissociation constant of Cs+ binding (ko) was 4.04 mM. We carried out a similar analysis of Cs+ block of If in CHO cells expressing HCN2 and determined values of 0.82 and 4.02 mM for and ko, respectively (see legend to Fig. 6). The ko and for the mutant and wild-type channel were approximately the same, indicating that the Y331D mutation did not affect the affinity or location of the Cs+ binding site in the pore.
The Y331D Mutation Disrupts Channel Closing and Destabilizes the Closed State—The above experiments support the suggestion that a fraction of If is already activated by the Y331D mutation, thus producing an instantaneous current in addition to Iinst and If. We call this current the If-like instantaneous current to differentiate it from Iinst. In order to estimate the fraction of If activated by the mutation, we determined the ratio of If-like instantaneous current to the total fully activated If. We used a standard leak-subtracting protocol (Fig. 7A) to determine the fully activated If in cells expressing the wild-type channel (Fig. 7B) and Y331D (Fig. 7C). The If-like instantaneous current was determined from the difference in instantaneous currents between cells expressing HCN2 Y331D and HCN2 (Fig. 7C). The fully activated If and the If-like instantaneous current were added to yield the total fully activated If for HCN2 Y331D (Fig. 7C), which crosses the x axis at 9.5 mV. This is very close to the reversal potential of fully activated If determined from cells expressing wild-type HCN2 (10.1 ± 3.4 mV, n = 5 cells) recorded under identical conditions and suggests that Y331D did not affect selectivity of Na+ and K+ (both relations are plotted in Fig. 7B). Using the values in Fig. 7C, we then determined the ratio of the If-like instantaneous current to the total fully activated If as a function of voltage for HCN2 Y331D (Fig. 7D). The fraction of If activated by the mutation is 0.68 on average over voltages ranging from +30 to -150 mV.
We next generated activation curves for If using 1-s test pulses and obtaining tail currents at -30 mV (arrows in Fig. 7E) and plotting normalized tail currents versus test voltage (Fig. 7F). We found that the activation curves determined from cells expressing HCN2 Y331D were shifted to more positive voltages and were less steep than those determined from cells expressing wild-type HCN2. If began to activate in the range of -40 mV in cells expressing HCN2 Y331D unlike If in cells expressing the wild-type HCN2, which began to activate at much more negative voltages using the same protocol. We also found that the rates of If activation/deactivation were shifted to more positive voltages, as would be expected from a positive shift in the activation curve, but we also found that the rates of deactivation were significantly slowed at more positive voltages by the Y331D mutation (Fig. 7H). The slower rates of If deactivation can also be observed by comparing the tail currents at -30 mV in Fig. 7F (arrows). On the other hand, the rates of If activation were not different between HCN2 Y331D and HCN2 at voltages in the fully activated range (-120 and -150 mV). Together, the data are consistent with a disruption of channel closing and destabilization of the closed state by the Y331D mutation.
The Fraction of If Activated by Y331D Is Modified by the Relative Amounts of External K+ and Na+ but Permeation Is Unaffected—When examining the selectivity of the If-like instantaneous current for Na+, we found that the amplitude of If (measured at -150 mV) in cells expressing HCN2 Y331D increased when perfused with solutions containing decreasing amounts of external Na+ while external K+ was kept constant (Fig. 8A). This was unusual and was not like cells expressing wild-type HCN2 in which the amplitude of If (also measured at -150 mV) decreased when perfused with the same solutions. However, the total If (If and If-like instantaneous current) determined from cells expressing HCN2 Y331D did decrease with decreasing amounts of Na+ (Fig. 8, A and B). The changes in the total amount of If flowing through the wild-type and mutated channels due to changes in external Na+ were almost identical for the wild-type and mutant channels. When going from 110 to 5.4 mM Na+, the amplitude of fully activated If in cells expressing wild-type HCN2 decreased from -248.1 to -160.3 pA/pF (to 0.65 times the initial value), whereas the total If in cells expressing HCN2 Y331D decreased from -592.6 to -357.8 pA/pF (to 0.60 times the initial value). These data confirm that the permeation of Na+ was not affected by the Y331D mutation.
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Finally, we generated If activation curves from tail currents in three of the five external concentrations used (the amplitudes of If were too small to generate accurate activation curves in the mutant channels at 5.4 and 15 mM external K+). These indicated that If was fully activated at -150 mV in cells expressing the mutant channel (n = 2 cells, not shown) as it was in the high K+ solution (Fig. 7F). These data and the similarity in the changes in total If amplitude at different concentrations of external Na+ and K+ between cells expressing either the wild-type or mutant channels are consistent with the channels being fully activated at -150 mV.
Because the changes in total If were similar, the above experiments suggested that it was the relative amplitudes of If and the If-like instantaneous current produced by HCN2 Y331D that was affected and furthermore that these changes were related to changes in both external Na+ and K+. We previously showed that the rate of If deactivation increased as the ratio of external K+ versus Na+ concentration ([K+]e/[Na+]e) increased (14), and we suspected a similar relationship between the proportion of If and the ratio of external cations was involved here. A plot of If/(If + If-like instantaneous current) versus [K+]e/[Na+]e showed an increase that was well fit by a single decaying exponential function. The range of greatest change was at ratios of [K+]e/[Na+]e between 0 and 1 (in the "physiological range"), whereas the proportion of If reaches a close-to-maximum level of 0.33 at [K+]e/[Na+]e value of 5.5. This exponential relationship is consistent with our findings in Fig. 7D, where the fraction of If was 0.32 at a [K+]e/[Na+]e value of 25 (135 mM K+/5.4 mM Na+ in the external solution).
The HCN1 Isoform Also Produces Iinst—Because the above analysis suggested that mutations in the S4-S5 linker did not alter Iinst and since our previous work showed that mutations in the selectivity filter or S4 segment also did not alter Iinst, we next took a more global approach to uncover regions involved in generating Iinst. We looked for Iinst in CHO cells expressing HCN1 in order to determine whether structural elements in common between the HCN1 and HCN2 isoforms are involved. Previous results have suggested that other HCN isoforms are associated with the presence of instantaneous currents, although the nature of these currents is not always very clear (5-7, 9, 19). Fig. 9, A and B, shows representative current traces from CHO cells expressing HCN1 or GFP alone recorded in varying external K+ or Na+ solutions, respectively. The density of instantaneous currents measured at -150 mV was significantly larger in cells expressing HCN1 than in cells expressing only GFP (Fig. 9, C and D). The amplitude of instantaneous current in cells expressing HCN1 increased with increasing changes in external K+ or Na+ in a linear fashion, suggesting the current was carried by both cations (Fig. 9, E and F). Interestingly, the amplitude of instantaneous currents in cells expressing only GFP also increased with increasing concentrations of external K+ and Na+, suggesting the presence of an endogenous cation channel. This is consistent with other studies that have demonstrated endogenous mixed cation channels in CHO cells (20, 21).
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Iinst Is Not Due to Excess Levels of HCN Protein on the Plasma Membrane as Compared with Native Cells—The mixed cationic nature of Iinst and the correlation of Iinst with If do not support a nonspecific leakage phenomenon associated with the expression of HCN protein on the plasma membrane of CHO cells. Another possibility is that overexpression of HCN protein on the plasma membrane allowed for the specific entry of Na+ and K+, which would not be observed with presumably lower levels of protein found on the plasma membrane of native cells. In order to examine this issue, we determined the densities of Iinst and fully activated If obtained by expression of HCN1 in CHO cells using "physiological" concentrations of Na+ and K+ (135 and 5.4 mM, respectively, in the extracellular solution and a high K+ solution in the pipette) and compared these with amplitudes of If found in native cells using solutions with similar concentrations of Na+ and K+. Interestingly, we have found that the electrophysiological protocol required for a direct comparison of Iinst and "fully activated" If in the same cell can be carried out most easily and successfully with the HCN1 isoform as compared with HCN2 and HCN4. The densities of Iinst and fully activated If were determined in the same cells at two concentrations of extracellular K+ and Na+ (as described in the legend to Fig. 10B, which shows the pulse protocol used and representative current traces) and plotted against voltage as shown in Fig. 10, C and D. A shift in the reversal potential was found for both If and Iinst in the two concentrations of Na+ and K+, consistent with the mixed cationic nature of these currents. A comparison of If densities collected from studies of native cells (with relatively high levels of If) with the density of If determined from Fig. 10D is shown in Table I. The comparison is not precise, because the measurements were made using similar, but not identical, "physiological" solutions (see legend to Table I). However, the comparison indicates that the density of If determined in native cells may be as high or higher than those determined in our experiments. This suggests that the amount of HCN protein on the plasma membrane of CHO cells in our experiments and HCN protein found on plasma membranes in native cells do not greatly differ and thus that Iinst is not the result of excessive amounts of HCN protein on the plasma membrane of CHO cells relative to native cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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70% of the total If over a range of +30 to -150 mV in a high potassium external solution. In addition, the activation curve of If was shifted to more positive voltages and was less steep, the If activation/deactivation curve was shifted to more positive voltages, and deactivation was significantly slowed at depolarized potentials compared with If recorded from cells expressing wild-type HCN2. On the other hand, the rate of If activation at voltages in the fully activated range was not different between cells expressing HCN2 Y331D or wild-type HCN2. These data are consistent with the idea that the Y331D mutation inhibits the closing of the channel and destabilizes the closed state and does not increase the conductance of a leaky closed state. Our findings are in line with previous studies suggesting a disruption of HCN2 channel closure by mutations of Tyr331 (10). Previous studies suggested a functional and physical uncoupling of the voltage sensor from the activation gate (10, 22), but we think that the Y331D mutation does not physically sever or weaken a link between the voltage-sensing elements and the activation gate. Our reasoning is based partly on an argument made for mutations of the Shaker pore and similarities of this pore to the crystallized pores of KcsA and MthK channels by Yifrach and Mackinnon (23). These authors suggested that because mutations generally tend to destabilize protein packing, shifts toward the open state indicate pore mutations destabilized the closed conformation as opposed to stabilizing the open conformation. The packing of the inner and outer helices of closed Shaker channels were argued to be more optimal based on a comparison of the crystal structures of KcsA and MthK, closed and open channels, respectively. If Shaker channels are intrinsically more stable when they are closed, these authors argued that it follows that the voltage sensors must work to open the pore. If HCN channels have a similar pore structure, which seems plausible based on the conservation of key sequences in the pore including the GYG signature motif and a key glycine "hinge" region (24), then it follows that these channels may also be more stable when they are closed where protein packing would be optimal. Furthermore, the voltage-sensing elements would also have to work to open the pore, although this would be done by hyperpolarization and not by depolarization as in Shaker. This line of reasoning suggests that severing or weakening the link between the voltage-sensing elements and gate would favor the more stable closed state of the channel. Therefore, it seems unlikely that the Y331D mutation reduced or eliminated coupling between voltage-sensing elements and the activation gate but more likely that this mutation disrupted the closing of the channel. The slowing of If deactivation at depolarized potentials, without any effects on the rates of If activation in the fully activated range, and the shift of channel opening to more positive voltages further support a disruption of channel closing and destabilization of the closed state by Y331D rather than an uncoupling of the voltage-sensing elements and the activation gate.
We also suspect that the pore is not directly involved in the effect of Y331D for two reasons. First, permeation is unaffected by Y331D. Second, the disruption of closing by Y331D is additive with the effects of [K+]e/[Na+]e on channel closing, which may be due to the effect of cations in the pore (see below). Because the two effects are additive, it seems likely that they are at least partly independent.
Based on its position, we suspect that the Y331D mutation causes a local allosteric perturbation of the activation gate (S6 segment) and/or of the voltage-sensing elements, which disrupts channel closing. An allosteric effect involving the S6 segment is supported by studies that show that HCN channels with cysteine mutations in the S6 region can be "locked" open by Cd2+ (25). A study using Shaker potassium channels showed that S6 mutations also constitutively activated the channel and shifted the activation curve of time-dependent current to more negative voltages, because of a perturbation of the closed to open equilibrium (26). Exactly how the Tyr331 residue is involved in disrupting gating will require a structural understanding of how HCN channels sense and respond to changes in voltage, and this is presently not completely clear (19, 27, 28).
[K+]e/[Na+]e Ratio Further Modifies HCN2 Y331D Channel Closing—An unusual and interesting finding in the present study was a decrease in the proportion of If-like instantaneous current and an increase in the proportion of If in cells expressing HCN2 Y331D when the [K+]e/[Na+]e ratio increased. The increase in the proportion of If could be accurately described by a decaying single exponential function. The range over which the greatest change occurred was at [K+]e/[Na+]e values between 0 and 1. In CHO cells expressing wild-type HCN2, we have previously shown that a hastening of If deactivation and the amplitude of fully activated If are both related to external K+ and Na+ in a similar way, with the greatest change occurring over the same range of [K+]e/[Na+]e values (14). Therefore, it seems reasonable to suggest that the effects on channel closing and permeation are connected and are due to a competition for the occupancy of site(s) in the pore by Na+ and K+. In HCN2 channels, K+ is the preferred cation, whereas Na+ moves through the pore less efficiently, and the channel itself conducts only when a minimum amount of external K+ is present (6, 14, 29, 30). Based on similarities to the pore and gate of voltage-gated K+ channels (31-33), the slower passage of Na+ suggests a "sodium foot in a potassium channel door" mechanism whereby the presence of Na+ in the pore interferes with the closing of the HCN2 channel. An interesting feature of this mechanism in HCN2 channels is that the effects occur in a range of [K+]e/[Na+]e found in vivo, and thus it is likely to play an important physiological role (14). The effect of [K+]e/[Na+]e also explains the larger proportion of constitutively activated current found previously in Xenopus oocytes expressing HCN2 Y331D, where a value of close to 100% was reported (10). Using the value of [K+]e/[Na+]e in those experiments ([K+]e/[Na+]e = 4 mM/96 mM = 0.04), we would predict a fraction of activated If of greater than 95% using our exponential relationship generated in Fig. 8E.
Iinst Is Also Associated with HCN1, Implicating Regions Conserved among HCN Channels in Generating This Current—We also found that the HCN1 isoform produced an instantaneous current identical to Iinst produced by HCN2 expression in CHO cells restricting the structural elements involved to those common between these isoforms. These include conserved regions in the proximal N and C termini as well as those in the transmembrane segments including the pore. The presence of instantaneous currents in the same tissues as If and HCN channels (34-36) and an apparent up-regulation of instantaneous currents in cells with up-regulated HCN channels or If (37-40) strongly suggest a direct association between them. Whether Iinst is intrinsic to the HCN channel (e.g. through a leaky closed state) or whether it results from a specific association of HCN channels with endogenous subunits or channels remains unknown.
We did find that Iinst was not likely to be an artifact of excess protein on the plasma membrane of CHO cells expressing HCN1, because fully activated If amplitudes were similar between our experiments and studies of If in native cells. It is possible that, in both heterologous expression systems and native cells, HCN proteins may cluster in certain areas of the plasma membrane (e.g. see Refs. 41-44). Molecular crowding of proteins on the cell surface may influence the function of membrane proteins (45). Thus, it is possible that the clustering of HCN protein on the plasma membrane, alone or together with other proteins, leads directly or indirectly to cation-permeable channels underlying the instantaneous current we observed.
It is also possible that Iinst results from the specific up-regulation of an endogenous channel, since CHO cells have a mixed cation instantaneous current. Furthermore, we have yet to find a mutation that modifies Iinst without affecting trafficking, suggesting that the current may not be intrinsic to the channel. On the other hand, if Iinst does result from a leaky closed state, it may be difficult to find a mutation that would disrupt flow through the pore of the closed channel without disrupting the activation gate. Also, we have found that increases in Iinst are directly proportional to the amount of If in the same cell and hence directly proportional to the amount of HCN protein on the plasma membrane in that cell. Therefore, the up-regulation of an endogenous channel or protein and the amplitude of Iinst might not increase in a linear fashion but might be expected to saturate at higher levels HCN expression. Finally, the pore represents a region that is highly conserved between HCN1 and HCN2 and therefore may be responsible for Iinst.
Are closed channels leaky? Recent experiments have shown that closed Shaker channels have a resting conductance and nonzero open probability, although they are very low, at least 100,000-fold lower than fully activated channels (46, 47
value of 0.91.