We characterized gating pore currents in the rat NaV1.4 isoform (rNaV1.4) expressing missense mutations at position R666 (ortholog of the human R672 position, which is the second arginine from the outermost one in DIIS4). Xenopus
oocytes were injected with RNA encoding either mutant or WT rNaV1.4 (Trimmer et al., 1989
), along with the β1 subunit (McClatchey et al., 1993
), and membrane currents were studied under voltage clamp in the cut-open configuration. Expression of rNaV1.4 mutant constructs at the plasma membrane was confirmed by recording current transients arising from nonlinear charge displacement (gating current). Corresponding steady-state currents, consisting of a combination of nonspecific oocyte membrane leak and rNaV1.4-specific gating pore current (if present), were subsequently assessed at higher amplifier gain.
Representative current recordings from WT and R666G mutants are shown in . Translocation of rNaV1.4 gating charge was detectable in oocytes expressing both WT and R666G channels and absent in water-injected control oocytes. Significant differences in the steady-state background currents, however, could be discerned between WT and R666G-expressing oocytes, despite similar amounts of gating charge movement. In bath solution containing K+ as the predominant extracellular cation, oocytes expressing WT rNav1.4 exhibited a small amplitude membrane leak current with linear voltage dependence, indistinguishable from the nonspecific leak currents recorded from mock-injected oocytes. However, oocytes expressing R666G mutant channels revealed larger amplitude inward currents with nonlinear voltage dependence, consistent with ionic current flowing through an accessory gating pore created by the S4 missense mutation. This current was abolished when NMDG was substituted for K+ (), suggesting that the R666G gating pore is permeable for K+ but not NMDG. Examination of the steady-state I-V relationships reveals that at voltages >+20 mV, currents recorded from R666G-expressing oocytes are identical to the nonspecific membrane leak currents recorded in mock-injected oocytes and in oocytes expressing comparable levels of WT channels. An inward current appears at more negative voltages, however, in R666G-expressing oocytes. This inward current has steep voltage dependence in a midrange of membrane potentials and transitions to reduced voltage dependence (shallower slope conductance) at more hyperpolarized potentials ().
Figure 1. Charge movement and gating pore currents in rNaV1.4-R666G channels. Oocytes expressing WT and R666G mutant channels were recorded using different bath solutions in which either K+ or NMDG was substituted as the predominant external cation (denoted (more ...)
To segregate the R666G-specific steady-state current from the nonspecific oocyte membrane leak, the contribution of nonspecific leakage current at hyperpolarized voltages was extrapolated from a linear fit to the current responses to command potentials between +30 and +50 mV (a voltage range in which the R666G-specific conductance appears to be closed). The amplitudes of inward currents that remained after this leak correction were proportional to the maximal gating charge displacement in each oocyte (unpublished data), consistent with the notion that the R666G-specific inward current is the consequence of a gating pore conductance created by the mutation. For subsequent analyses, gating pore currents were normalized to the maximal gating charge displacement for comparison between oocytes with different levels of rNaV1.4 expression. Leak-corrected currents normalized in this manner are compared in for WT oocytes and R666G-expressing oocytes recorded in K+ and NMDG bath solutions.
Figure 2. Normalized leak-corrected R666G gating pore currents are not altered by divalent substitution in the bath. Gating pore currents were segregated from the nonspecific oocyte membrane leak as described (see Results) and scaled to the maximal rNaV1.4 gating (more ...)
Several disparities in the current amplitude, selectivity, and voltage dependence were evident between the R666G gating pore currents we recorded and those reported in Sokolov et al. (2007)
. Because the recording solutions we used were slightly different, it was important to rule out the possibility that these differences in ionic composition might account for these discrepancies. Specifically, in addition to Ca2+
, our external bath contained Ba2+
, which we found was critical to suppress the background leak conductance of the oocyte membrane sufficiently to discern the low-amplitude gating pore current (note the ratio of nonspecific leak to gating pore current in ). When oocytes were bathed in solutions identical to those used by Sokolov et al. (2007)
, high-amplitude background leak currents were observed roughly 100-fold higher than the typical oocyte leak current recorded in our standard bath solutions. Because the R666G-specific gating pore current was a minor component of the total current under these low-divalent conditions, we instead sought to test whether bath solutions with different divalent cation compositions affected the R666G gating pore current. Sokolov et al. (2007)
reported that the addition of 6 mM Zn2+
fully blocked the R666G gating pore current, whereas 6 mM Ba2+
partially blocked the current (~50%). In no instance did they report current saturation in the hyperpolarized voltage range, even in bathing solutions characterized by high divalent cation concentrations.
Various divalent cations were substituted in K+
bath solution (). K+
currents flowing through the R666G gating pore recorded using our standard divalent cation concentrations (2.5 mM Ba2+
, 1.5 mM Ca2+
) were not discernibly different from K+
currents recorded when these divalent cations were fully substituted with 6 mM Ca2+
, 6 mM Ba2+
, or 6 mM Zn2+
. These data are consistent with two alternative interpretations: either all the divalent cations that were tested exert similar blocking effects on the R666G gating pore, or the divalent composition of the bath has no significant influence over the gating pore current. These alternative possibilities are considered below. These results do indicate, however, that the differences between our data and those reported in Sokolov et al. (2007)
are probably not due to differences in bath divalent cation composition.
The ionic selectivity of the R666G gating pore was explored in greater detail through substitution of monovalent cations in the internal and external solutions, and the R666G conductance was found to exhibit several further unexpected features. When the ionic driving force for K+ was biased outwardly by raising the internal K+ to 110 mM and using NMDG as the predominant external cation, very low amplitude outward currents were observed, despite the strong outward driving force for K+ at all voltages (, blue symbols). The possibility that such small outward currents were due to external block by NMDG was excluded by recording inward K+ currents in bath solutions composed of 10 mM K+ and 105 mM NMDG (). K+ currents under these conditions are proportionately reduced in amplitude compared with the 105-mM K+ bath (notice the change in current scale for ). When nearly equal concentrations of K+ were present on both sides of the membrane (), thereby reducing the inward electrochemical K+ driving force at hyperpolarized voltages (EK ~ +1 mV), the gating pore current amplitude was nearly identical to the currents recorded when K+ was limited to the extracellular compartment (e.g., EK→∞). When Na+ was substituted for K+, nonlinear currents were also observed that exhibited the same voltage dependence as the K+ currents (). However, the normalized amplitude of the Na+ currents was reduced, suggesting that Na+ was less permeable through the R666G gating pore. Like the K+ currents, the amplitude of inward Na+ currents at hyperpolarized potentials was largely independent of changes to the inward Na+ driving force, and only very small outward currents were observed under conditions strongly favoring outward flow of Na+ ions at hyperpolarized voltages.
We sought to understand whether the unusual current saturation at hyperpolarized voltages was the consequence of changes in the open probability, POPEN, voltage-dependent block, or whether this was an intrinsic feature of the permeation pathway in the R666G gating pore. The relative open probability of a putative R666G gating pore exhibiting Ohmic conductance properties can be estimated by transforming the I-V relationship recorded in symmetric K+ into a normalized G-V relationship, assuming a EREV ≈ 0. This transformation is demonstrated in (black circles). As anticipated, the gating pore permeation pathway is closed by membrane depolarization favoring outward movement of the DIIS4 voltage sensor, but this model also requires an ~40% decline from peak in normalized POPEN to account for the saturation of the I-V curve at hyperpolarized voltages. As another index of POPEN, we examined whether the amplitudes of the tail currents observed upon membrane repolarization to the holding potential of −100 mV () also support the notion that POPEN declines at potentials <−80 mV. Instantaneous tail current amplitudes were estimated by extrapolating a single exponential fit to the tail current decay after leak correction and normalization to gating charge. No change in normalized tail current amplitude was observed at voltages <−80 mV (, filled green circles), and thus these data fail to provide support for the notion that a reduction in POPEN at increasingly hyperpolarized potentials accounts for the current saturation at voltages <−80 mV. Furthermore, the aggregate behavior of the R666G gating pore currents, including the low amplitude of the outward currents, and the relative independence of inward currents from the ionic driving force cannot be explained parsimoniously with the assumption of a simple Ohmic conduction pathway.
Figure 4. Anomalous R666G tail currents do not support a change in POPEN to explain the current saturation at hyperpolarized voltages. The I-V relationship of the steady-state currents in a symmetrical K+ gradient (from ) was transformed to a G-V (more ...)
The unexpected, anomalous behavior of the tail currents, which exhibited increased amplitudes after more depolarizing voltage pulses that close the gating pore conductance (; note larger transient tail current amplitudes are indicated by symbols nearer to the bottom edge of the plot in A), suggested that more than one open state is traversed during the recovery from nonconducting state(s) at depolarized potentials. In addition, the slow relaxation of the tail currents indicated that this recovery occurred over several hundred milliseconds, much slower than the fast transitions anticipated due to rapid movement of a single voltage sensor. The slow decay of the tail current suggested that entry to closed state(s) at depolarized potentials (from which recovery results in the tails) might also occur on a slow time scale. To test this hypothesis, we measured the time course for the development of the transient tail currents after progressively longer conditioning pulses to 0 mV (). Inward tail currents are small after short depolarizations (<100 ms), whereas longer depolarizations elicit progressively larger inward tail current transients, which approach a maximal saturating amplitude with a time constant of ~1.2 s. The possible significance of these tail currents is discussed below, but the pertinent issue at hand is that these anomalous instantaneous tails do not reflect the POPEN of the R666G gating pore for the antecedent conditioning pulse.
The gating pore current saturation (shallow slope conductance) at hyperpolarized voltages suggests the presence of an impediment to current flow with low-voltage sensitivity in this range. This might either be the consequence of voltage-dependent ionic block at a shallow electrical distance from the outside, or intrinsic saturability of the permeation pathway. In either circumstance, the steep portion in the midrange of the I-V relationship is likely due to voltage-dependent changes to the accessibility of the permeation pathway due to translocation of the mutant DIIS4 segment. The possibility of ionic block was considered in a model with a two-state gating transition to account for the steep decrease in current amplitude as membrane voltage becomes depolarized. The open channel I-V was modeled using the GHK current equation to account for the effects of the asymmetrical permeant ion gradients. Bath divalent cations were considered the most likely voltage-dependent blocking candidates (see Materials and methods, Online supplemental material). Divalent block occurs as the result of binding at a site within the gating pore located at electrical distance δ from the external side. The complete model, assuming K+
as the permeant ion, is thus given by:
is the K+
permeability of the gating pore, and [B]/KD
is the ratio of external divalent cation concentration to blocking affinity. First, we fit this equation to the special case of symmetrical K+
to determine PK
, and δ (the gating parameters V1/2
were already known). Parameter estimation was somewhat limited because for high-affinity block ([B]/KD
> 1), the current is scaled by the ratio of PK
), and so separate determination of PK
is difficult. Nevertheless, it was clear that high-affinity block is required for an adequate fit; otherwise, the predicted I-V was too curvilinear in the −80 to −140 mV range. The best fit, obtained with [B]/KD
= 30 and δ = 0.1, is shown in Fig. S1. As the [B]/KD
ratio was further increased, the fit did not improve. This configuration was sufficient to account for the strong rectification of the permeation pathway in which outward current flow was impeded (only a small fraction of the block is relieved by depolarization before the gating pore becomes occluded by outward S4 movement). The model fails, however, to simulate the effect of increasing [B] by 50%, which was done experimentally when we increased the divalent from 4 to 6 mM (). The model predicts that steady-state K+
currents should decline by ~33% under these conditions if the block is of very high affinity ([B]/KD
≥ 10). The relationship between the expected current reduction due to a 50% increase in the concentration of a putative divalent blocker, starting from different [B]/KD
baseline values, is shown in . If the block is high affinity, such that [B]/KD
is any value >1, the prediction is a reduction in gating pore current amplitude by ~30%. This magnitude block would have easily been detected but was not observed for our measurements in 6 versus 4 mM divalent (). Alternatively, if a low-affinity scenario is proposed, [B]/KD
< 1, to explain the failure to observe divalent block, the voltage dependence of the I-V would be too curvilinear and cannot simulate the saturation observed for V <−80 mV. Therefore, we reject the hypothesis that R666G gating pore current saturation at negative potentials is a consequence of voltage-dependent block by an external cation.
Figure 5. High-affinity ionic block predicts a reduced current after a 50% increase in blocker concentration The model described by Eq. 5 was used to predict the effect on current amplitude of changing the divalent cation concentration from 4 to 6 mM (as is (more ...)
Our preferred model, which parsimoniously accounts for the saturation at negative voltages and the apparent independence from permeant ion driving force, is based on a single binding site scheme for ion permeation. As in the ionic block model, accessibility of the permeation pathway is gated by a two-state voltage-dependent rearrangement of the mutant voltage–sensing domain. The components of this model are displayed schematically in . At hyperpolarized voltages favoring inward movement of the S4 segment, the position of the R666G mutant residue renders the gating pore permissive for cation permeation, whereas membrane depolarization (outward S4 movement) abolishes the permeation pathway (). This scheme accounts for most of the steep voltage dependence of the R666G current between −80 and −20 mV and explains why no gating pore current is observed at depolarized voltages (>0 mV) under any ionic conditions. The exposed permeation pathway is modeled as a pore with a single cation binding site, flanked by two energy barriers (). The pronounced eccentric location of the binding site at a very shallow electrical distance from the external surface (δ = 0.03) accounts for the reduced voltage dependence exhibited by R666G gating pore currents. The maximal ion flux at hyperpolarized voltages is determined predominantly by the relatively voltage-independent rate of cation transitions over the external barrier. For the model in Scheme 1
, as the membrane potential becomes very negative, the net current is approximately equal to
which has a very shallow voltage dependence because δ is very small. The electrical distances of the binding sites of the two different monovalent cations can be estimated from fitting the above equation to the saturating portions of individual I-V curves at voltages <−100 mV. This yields δ = 0.07 ± 0.04 (n
= 6) for K+
and 0.02 ± 0.02 (n
= 5) for Na+
(statistically insignificant), suggesting that the two cations probably bind to the same site.
Figure 6. A barrier model of R666G cation permeation. In the proposed model, accessibility of the R666G gating pore is due to voltage-dependent movement of the DIIS4 voltage sensor as depicted schematically in A. At hyperpolarized voltages favoring inward gating (more ...)
In contrast to both the Ohmic and divalent block models, the barrier model of the R666G gating pore is able to reproduce almost all of the fundamental features of the steady-state R666G currents (, solid lines). The dashing lines in show the “open channel” I-V relationship (POPEN fixed at 1.0) for the single binding site model of permeation. Notice that the saturation of gating pore currents at negative potentials is accounted for entirely by permeation features of the external barrier model. When permeant ions are present intracellularly, steep outward rectification is noted only when the membrane potential approaches 0 mV and then becomes positive; however, in these instances the voltage-dependent movement of the DIIS4 segment occludes the gating pore (, solid lines), and so these changes to the gating pore current cannot be detected. Similarly, for inward currents the ionic concentration effect on driving force (dashed lines; V > −20 mV) is masked by voltage-dependent gated access to the permeation pathway. Finally, because voltage-dependent block is not a component of this model, it is also consistent with the independence of external divalent concentration, as shown in .
In our model, the occlusion and availability of the gating pore are simulated as a two-state transition with voltage-dependent rates, described by a single Boltzmann function. The relation between the voltage dependence of gating pore accessibility and measured charge displacement is shown in . The solid gray curve shows the voltage dependence for POPEN of the gating pore, as determined from the fits to the steady-state I-V data in . The black circles and dashed curve show the measured gating charge displacement of R666G channels (S4 translocation). These curves exhibit a close, inverse relationship, supporting our contention that accessibility of the R666G permeation pathway is coupled to the resting conformation of the DIIS4 segment and, conversely, that outward movement of gating charge favored by membrane depolarization occludes the R666G gating pore.
Figure 7. Comparison of the model POPEN to the QOn-V relationship and steady-state ionic currents in a physiologically relevant cation gradient. In A, the QOn-V relationship of R666G gating charge displacement is compared with the voltage dependence of the relative (more ...)
Because the permeation pathway is open only at hyperpolarized voltages, the bulk of the current carried by the R666G gating pore under normal physiological conditions is likely to be inward Na+ flow. To test this, we recorded R666G gating pore currents under conditions approximating the physiological monovalent cation gradient (scaled to account for differences in the osmolarity of solutions used to record from oocytes). These experimentally determined currents were compared with the model predictions () using parameters for Na+ and K+ permeation derived independently. Under these approximate physiological conditions, the bulk of the R666G gating pore current is indeed directed inwardly and is of similar magnitude as currents recorded when Na+ was the sole external monovalent cation (compare the normalized current amplitudes in to Na+ currents in ). The model predictions (solid line) lie in close agreement with the experimental data. These results suggest that in the presence of a normal physiological ionic gradient, the R666G gating pore conductance is likely to introduce a small Na+-predominant leak to the sarcolemma.
One notable deviation is the amplitude inflection (hump) between −90 and −60 mV where the inward current amplitude exceeds the model curve. This hump was prominent in recordings with asymmetrical ionic solutions wherein Na+ was the major extracellular cation, and the predominant internal cation was either K+ or an impermeant species (see also and ). Similarly, the amplitude of the anomalous tail current transients was larger in Na+-containing bath solutions (for instance, ). Both of these observations can be reconciled with a modification to our model wherein a second open state can be elicited in the R666G gating pore at intermediate depolarizations (to account for the hump in steady-state behavior) or during recovery after prolonged depolarizations (to account for the anomalous tails). In this scenario, both the permeability and ionic selectivity may differ from the primary open state (e.g., higher Na+ permeability). This possibility is discussed below.
Figure 8. Gating pore currents from other HypoPP mutations at site R666. Steady-state gating pore currents were recorded from R666S, -C, and -H mutant channels and compared with currents recorded in WT channels (denoted at top). Representative current traces, after (more ...)
We tested whether other HypoPP mutations at the R666 position might create gating pores with similar currents. Representative traces of steady-state currents are shown in for WT and rNaV1.4 channels expressing cysteine, serine, and histidine substitutions at the R666 position, after leak correction and normalization to maximal gating charge displacement. Currents were recorded in either a physiological cation gradient, as in , or in external and intracellular solutions in which the predominant cation was NMDG. As expected, no gating pore currents could be detected in WT channels. However, inward currents were observed in R666C, -S, and –H mutants, which exhibited the same sigmoidal voltage dependence with saturation of the current amplitude at hyperpolarized potentials, as with the R666G gating pore current. The magnitudes of normalized inward currents were each slightly different from the normalized amplitude of the R666G inward current, ranging between smaller currents (R666C) to currents that were nearly twice as large (R666H). These differences suggest that specific mutations expose pathways with different permeation characteristics. In R666C and –S channels, both steady-state gating pore and transient tail currents were abolished in recording solutions containing NMDG, consistent with the notion that these mutant gating pores predominantly conduct small monovalent cations. In contrast, when NMDG was substituted as the predominant cation in R666H channel recordings, the steady-state inward current persisted (, rightmost column). Although these results could be interpreted as demonstrating equivalent permeability for NMDG and Na+
, it is more likely that the steady-state R666H gating pore current is carried by protons and not these larger monovalent cations. This view is consistent with previous findings that gating pores created by histidine substitutions in Shaker K+
channels (Starace et al., 1997
; Starace and Bezanilla, 2001
), and at the more exterior R663 position in rNaV1.4 (Struyk and Cannon, 2007
) are selective for protons. Inward gating pore currents with similar amplitude and voltage dependence were recorded from R666H channels bathed in solutions predominantly consisting of Tris-HEPES buffer as described in Struyk and Cannon, (2007)
, which does not contain Na+
or NMDG (unpublished data), supporting the notion that protons are the charge carriers of this current. However, the tail currents recorded from R666H channels in each of the two conditions exhibit marked differences in amplitude and suggest that the tail current permeation pathway (the putative second open state) has a different ionic selectivity compared with the steady-state permeation pathway (as mentioned above to account for the hump in the I-V relation in the voltage range of −60 to −80 mV). Possible interpretations of this observation are discussed below, but it does suggest that the R666H gating pore is capable of conducting cations other than protons under the appropriate conditions.