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1.
Figure 5.

Figure 5. From: Gating Pore Currents in DIIS4 Mutations of NaV1.4 Associated with Periodic Paralysis: Saturation of Ion Flux and Implications for Disease Pathogenesis.

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 Fig. 2 B) for different blocking affinities. High-affinity block ([B]/KD ≥ 10) predicts a ∼33% decline in current amplitude, which is inconsistent with the experimental data (see Fig. S1). The lack of a significant change in amplitude in Fig. 2 B would be consistent with a low-affinity block ([B]/KD ≤ 10), but low-affinity block is not sufficient to account for the broad voltage range of current saturation.

Arie F. Struyk, et al. J Gen Physiol. 2008 October;132(4):447-464.
2.
Figure 9.

Figure 9. From: Gating Pore Currents in DIIS4 Mutations of NaV1.4 Associated with Periodic Paralysis: Saturation of Ion Flux and Implications for Disease Pathogenesis.

Permeation for a single-well two-barrier model. Steady-state ion flux and occupancy of the binding site were computed for the case of two permeant ions present at physiological concentrations on either side of the gating pore (in mM [K]e = 3, [K]i = 103, [Na]e = 112, [Na]i = 7). The “open-channel” current–voltage relation that is in absence of gating is compared in A for the assumption of independence (Scheme 1, dashed line) or competition by Na+ and K+ for the single binding site (Scheme 3, solid line). When voltage-dependent gated accessibility of the pore is included (B), the current–voltage relation has strong inward rectification and the distinction between Schemes 1 and 3 is no longer discernable.

Arie F. Struyk, et al. J Gen Physiol. 2008 October;132(4):447-464.
3.
Figure 2.

Figure 2. From: Gating Pore Currents in DIIS4 Mutations of NaV1.4 Associated with Periodic Paralysis: Saturation of Ion Flux and Implications for Disease Pathogenesis.

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 charge in the corresponding oocyte. The mean I-V relationship of these normalized steady-state currents is shown in A. A nonlinear current component was not detected for WT-expressing oocytes in K+-containing bath solutions (n = 5). Inward gating pore current is seen when R666G channels are recorded in K+-containing bath solution (n = 6), whereas the gating pore current is abolished by NMDG substitution in the bath (n = 4; symbol key in figure inset). In B, R666G gating pore K+ currents were recorded in the presence of different divalent cations in the bath. No difference in the amplitude or voltage-dependent plateau of the R666G gating pore current is seen despite substitution with either 6 mM Ca2+ (n = 4), Ba2+ (n = 4), or Zn2+ (n = 3; symbol legend in the figure inset).

Arie F. Struyk, et al. J Gen Physiol. 2008 October;132(4):447-464.
4.
Figure 8.

Figure 8. From: Gating Pore Currents in DIIS4 Mutations of NaV1.4 Associated with Periodic Paralysis: Saturation of Ion Flux and Implications for Disease Pathogenesis.

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 leak correction and normalization to the corresponding maximal gating charge displacement, are shown for recordings made in bath and internal solutions approximating the normal mammalian physiological cation gradient (A) or in bath and internal solutions containing NMDG (B). Scale bars for all traces are shown in the inset to A. The mean I-V relationships of gating pore currents seen in each mutant under these conditions is shown in C. For each panel, the filled circles represent currents recorded in the physiological cation gradient, whereas open circles represent currents recorded when NMDG was present in both internal and external compartments. Number of samples recorded for each condition is denoted in parenthesis in the inset legend for each figure. Gating pore currents presumably carried by monovalent cations and abolished by NMDG substitution are seen in R666S and R666C mutant channels. In contrast, inward gating pore currents of similar magnitude are seen in R666H channels in both ionic conditions, consistent with the notion that the charge carriers of this gating pore current are protons rather than larger monovalent cations.

Arie F. Struyk, et al. J Gen Physiol. 2008 October;132(4):447-464.
5.
Figure 6.

Figure 6. From: Gating Pore Currents in DIIS4 Mutations of NaV1.4 Associated with Periodic Paralysis: Saturation of Ion Flux and Implications for Disease Pathogenesis.

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 charge movement, the gating pore is accessible for cation binding and permeation, whereas outward movement of gating charges favored by membrane depolarization occludes the permeation pathway. The permeation pathway was modeled as a free-energy barrier profile calculated using Eyring rate theory. Profiles at 0 mV (top panel) and −100 mV (bottom panel) membrane potential of the proposed ion permeation pathway through the R666G gating pore are depicted in B. The black and red lines represent the proposed barrier profiles encountered by K+ and Na+, respectively. The shallow voltage dependence of the I-V relation at negative potentials (Fig. 2 A) is accounted for by a barrier model with a cation binding site that is very near the external face of the electrical field (δ = 0.03). Proposed transition rate constants for K+ and Na+ over the external and internal energy barriers at 0 mV are listed in Table I.

Arie F. Struyk, et al. J Gen Physiol. 2008 October;132(4):447-464.
6.
Figure 7.

Figure 7. From: Gating Pore Currents in DIIS4 Mutations of NaV1.4 Associated with Periodic Paralysis: Saturation of Ion Flux and Implications for Disease Pathogenesis.

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 open probability of the R666G gating pore derived from the best fit parameters of the model in Fig. 6. The solid black line represents the least-squares fit of a single Boltzmann function to the experimental QOn-V data, yielding the following parameters: V1/2 = −48.4 ± 1.0 mV, k = 13.8 ± 0.3 mV, n = 9. In comparison, the gray line represents the single Boltzmann function describing the voltage dependence of relative open probability of the R666G gating pore permeation pathway. The best fit parameters from this function are as follows: V1/2 = −50 mV, k = 11.3 mV. The inverse relationship between R666G gating pore accessibility (note the inverted scale to the right) and the voltage dependence of gating charge movement supports the notion that cation access to the permeation pathway is controlled by S4 translocation (presumably the DIIS4 voltage sensor). In B, the I-V relationship of normalized cation currents flowing through the R666G gating pore in oocytes exposed to bath and internal solutions approximating the normal mammalian physiological Na+/K+ gradient is shown (n = 5). The solid line represents the behavior of the R666G gating pore current under these conditions, as predicted by the model (using parameters listed in Table I).

Arie F. Struyk, et al. J Gen Physiol. 2008 October;132(4):447-464.
7.
Figure 1.

Figure 1. From: Gating Pore Currents in DIIS4 Mutations of NaV1.4 Associated with Periodic Paralysis: Saturation of Ion Flux and Implications for Disease Pathogenesis.

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 at top of figure). For comparison, currents recorded from a mock-injected oocyte in K+-containing bath solution are also shown. Representative raw gating current traces are shown in A. Gating charge displacement was assessed by recording the nonlinear transient current responses elicited by 15-ms voltage steps from a prepulse potential of −160 mV (see voltage protocol in inset). Membrane potential commands (in mV) eliciting charge movement are denoted to the left of each trace. Scale bars for the current records in A are shown at the bottom right. In B, representative steady-state background current recordings are shown for the same oocytes whose gating currents are depicted in A. Steady-state currents were elicited by a 300-ms voltage command from a holding potential of −100 mV (see voltage protocol in inset). Scale bars for the current records in B are shown at the bottom left. The mean I-V relationships of the steady-state background currents from the oocyte recordings depicted in B are shown in C. The symbol legend is to the right. In K+-containing bathing solutions, oocytes expressing R666G mutant channels exhibit robust inward currents at voltages <+20 mV that are not seen in oocytes expressing comparable levels of WT channels under the same conditions (open circles are not fully visible beneath filled squares). Steady-state currents in R666G-expressing oocytes recorded in NMDG bath solution, however, are indistinguishable from the nonspecific membrane leak seen in mock-injected oocytes and oocytes expressing WT rNav1.4.

Arie F. Struyk, et al. J Gen Physiol. 2008 October;132(4):447-464.
8.
Figure 4.

Figure 4. From: Gating Pore Currents in DIIS4 Mutations of NaV1.4 Associated with Periodic Paralysis: Saturation of Ion Flux and Implications for Disease Pathogenesis.

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 Fig. 3 A) was transformed to a G-V relationship in A by assuming a reversal potential of 0 mV and therefore a linear open-state current through the origin. These conductance values were then normalized to the peak conductance (filled black circles). Normalized peak tail current amplitudes (after leak correction) are plotted against the voltage of the preceding pulse at which the normalized conductance values were obtained (filled green circles, right axis). No change in the amplitudes of these tail currents (all measured at −100 mV) was observed at voltages <−80 mV in a region where the normalized conductance declines. (B) A comparison of individual raw current traces extracted from Fig. 1 A confirms that the tail currents exhibit an anomalous increase in amplitude after more depolarizing voltage pulses during which the gating pore is occluded (red trace) and are absent after hyperpolarized pulses during which the gating pore remains open (black trace). The slow activation kinetics of the tail currents are shown C, in which tail currents were recorded at −100 mV after depolarizing pulse to 0 mV of different durations (see inset at top of figure). Current traces are arranged along the abscissa in proportion to conditioning pulse duration, and the peak current is fit with a single exponential function (dashed blue line), revealing an activation time constant of 1.2 s at 0 mV.

Arie F. Struyk, et al. J Gen Physiol. 2008 October;132(4):447-464.
9.
Figure 3.

Figure 3. From: Gating Pore Currents in DIIS4 Mutations of NaV1.4 Associated with Periodic Paralysis: Saturation of Ion Flux and Implications for Disease Pathogenesis.

R666G K+ and Na+ steady-state gating pore currents. The permeability of the R666G gating pore for different monovalent cations was tested. The transmembrane cation gradient was manipulated as described in the insets, such that either cation was present exclusively in the internal or external compartments, or was distributed nearly symmetrically in both compartments. The normalized, leak-corrected I-V relationships of resultant K+ (A and B) and Na+ (C) gating pore currents are shown. The different colors in A and C represent current recordings made in different ionic gradients, with the legend in each figure inset. The number of samples recorded for each condition is also denoted in parenthesis. Very small outward currents were seen when either Na+ or K+ were present exclusively on the inside, despite significant outward driving force for both cations. In external solutions consisting of 10 mM K+ and 105 mM NMDG, low-amplitude K+ currents were observed (note the normalized current scale in B), indicating that NMDG does not block the pore from outside and therefore cannot account for the lack of outward currents. Furthermore, inward current amplitudes were nearly equivalent whenever K+ or Na+ was present on the outside, despite significant changes to the overall driving force due to differences in the internal cation concentration. Overlying the data are curves from the permeation model depicted in Fig. 6 and described (see Results), representing the best parameter fits listed in Table I. Solid lines represent currents from the full model, which includes voltage-dependent accessibility of the R666G gating pore due to movement of the DIIS4 voltage sensor, whereas the dotted lines demonstrate the predicted open-channel voltage dependence of currents flowing through an R666G gating pore in the absence of gating.

Arie F. Struyk, et al. J Gen Physiol. 2008 October;132(4):447-464.

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