Goldschen-Ohm MP et al. (2013), Multiple pore conformations driven by asynchron...

XB-ART-47256

Nat Commun 2013 Jan 01;4:1350. doi: 10.1038/ncomms2356.

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Multiple pore conformations driven by asynchronous movements of voltage sensors in a eukaryotic sodium channel.

Goldschen-Ohm MP , Capes DL , Oelstrom KM , Chanda B .

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Voltage-dependent Na(+) channels are crucial for electrical signalling in excitable cells. Membrane depolarization initiates asynchronous movements in four non-identical voltage-sensing domains of the Na(+) channel. It remains unclear to what extent this structural asymmetry influences pore gating as compared with outwardly rectifying K(+) channels, where channel opening results from a final concerted transition of symmetric pore gates. Here we combine single channel recordings, cysteine accessibility and voltage clamp fluorimetry to probe the relationships between voltage sensors and pore conformations in an inactivation deficient Nav1.4 channel. We observe three distinct conductance levels such that DI-III voltage sensor activation is kinetically correlated with formation of a fully open pore, whereas DIV voltage sensor movement underlies formation of a distinct subconducting pore conformation preceding inactivation in wild-type channels. Our experiments reveal that pore gating in sodium channels involves multiple transitions driven by asynchronous movements of voltage sensors. These findings shed new light on the mechanism of coupling between activation and fast inactivation in voltage-gated sodium channels.

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Species referenced: Xenopus
Genes referenced: nav1 scn4a tff3

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	Figure 1. Nav1.4-WCW severely disrupts fast inactivation and eliminates inactivation from closed states.(a) Whole-cell current responses to 20 ms depolarizing voltage steps for rat Nav1.4 (left, only 10 ms shown) and rat Nav1.4-WCW (right) channels at room temperature. Cells were held at −70 mV and hyperpolarized to −120 mV for 20 ms before and following a 20 ms depolarizing pulse from −120 to +50 mV (Nav1.4) or +80 mV (Nav1.4-WCW) in 5–10 mV steps (see inset). (b) Normalized peak conductance–voltage (G–V) relation from recordings as shown in a (mean±s.e.m.). The voltage at which the conductance was half maximal (V1/2) and the effective charge (z) from single Boltzmann fits to the G–V from individual cells were, for Nav1.4 (mean±s.e.m.): V1/2=-32.6±1.9 mV, z=3.8±0.4 e−, n=5; and for Nav1.4-WCW: V1/2=-24.9±0.8 mV, z=4.7±0.2 e−, n=8. Single Boltzmann fits to the mean were for Nav1.4: V1/2=-33.3 mV, z=3.4 e−, and for Nav1.4-WCW: V1/2=-24.4 mV, z=4.2 e−. (c) Summary of the fraction of the peak current remaining after 10 ms for Nav1.4 and Nav1.4-WCW (mean±s.e.m.). ***t-Test, P<0.001. (d) Nav1.4-WCW whole-cell current responses to a steady-state inactivation protocol consisting of a 20 ms test pulse to −20 mV to assay the fraction of available (that is, non-slow inactivated) channels after a 1 s preconditioning pulse from −80 to 0 mV at room temperature (see inset). Holding potential was –120 mV and a 1 ms hyperpolarizing pulse to −140 mV preceded each test pulse. (e) The normalized peak current response during the test pulse is plotted against the voltage during the preconditioning pulse for five cells (open circles, mean±s.e.m., n=5) fit with a single Boltzmann plus an added constant (solid line; V1/2=-29.9 mV, z=4.9 e−, constant=0.27). The G–V relation for Nav1.4-WCW from b is shown inverted for comparison (dashed line).
	Figure 2. Multiple conductance levels during gating of single Nav1.4-WCW channels.(a) Single channel records in response to 200 ms depolarizing pulses from −60 to 0 mV (−120 mV holding) for Nav1.4 (left, only first 100 ms shown) and Nav1.4-WCW (right) channels at 10 °C (filtered at 1 kHz for display). Dashed lines indicate observed conductance levels (C=closed, S1, S2, O; openings are downward). (b) Macroscopic responses obtained by averaging single channel records.
	Figure 3. Voltage dependence of individual conductance levels.(a) Single channel amplitude distributions for Nav1.4 (left) and Nav1.4-WCW (right). Histograms were obtained from all points in a single channel patch after discarding points adjacent to changes in amplitude in the idealized record to remove artifacts due to filtering. Red dashed lines are individual Gaussian fits to each conductance level, and the solid red line is their sum. (b) Current–voltage (I–V) relation for each conductance level (mean±s.e.m.) and its linear fit. The main conductance level for Nav1.4 (squares) nearly overlaps that of Nav1.4-WCW (circles). Data between −120 and −80 mV are from deactivation to the indicated potential (Supplementary Fig. S3). (c) Summary of the conductance (top) and reversal potential (Vrev, bottom) for each conductance level from linear fits to the I–V for individual patches. Conductances were (mean±s.e.m.), for Nav1.4-WCW: S1=6.8±0.9 pS, S2=12.5±1.3 pS, O=17.5±1.4 pS; and for Nav1.4: 17.1±1.4 pS. Extrapolated reversal potentials were (mean±s.e.m.), for Nav1.4-WCW: S1=34.2±9.9 mV, S2=36.3±8.9 mV, O=36.6±6.3 mV; and for Nav1.4: 43.4±5.8 mV.
	Figure 4. Channel activation is not altered in Nav1.4-WCW.(a,b) Cumulative probability of first opening to each individual conductance level during activation from -60 to 0 mV (maximum probability increases with voltage). The reduction of the maximum from unity reflects the probability of observing a null sweep with no channel activity. Note expanded time scale in b. (c) Summary of the weighted time constants from biexponential fits to the cumulative probability of first opening for each individual conductance level and for opening to any level. (d) A comparison of the cumulative probability of first opening for Nav1.4 and Nav1.4-WCW during the first 20 ms of activation at 0 mV. Smooth lines are biexponential fits. (e) Comparison of the average single-channel response scaled to the observed peak open probability during activation at 0 mV for Nav1.4 (solid black) and Nav1.4-WCW (red). The response for Nav1.4 is also shown after normalizing to the peak response for Nav1.4-WCW (dotted black).
	Figure 5. Kinetics of individual conductance levels.(a) Average probability at every time point for each conducting state from idealized records (dots, only first 100 ms shown), and fits to the equation ΣiAi[1−exp(−t/τi)] (lines, see Supplementary Table S2). The first 40 ms period is shown on the right after normalizing the fits to illustrate the relative kinetics of the initial rise in probability for each level. (b) Average conditional probability at every time point after aligning the idealized records so that the first event in each sweep occurs at time zero (dots), and fits to the equation ΣiAi exp(−t/τi)+ constant (lines, see Supplementary Table S2). (c) The probability of transitioning between each conductance level during activation gating at each potential tested (0.5 ms resolution).
	Figure 6. Individual conductance levels are correlated with the movement of specific voltage-sensing domains.(a) Average single channel open probability for Nav1.4 during activation at 0 mV (black) overlaid with the probability for Nav1.4-WCW to be either fully open (PO, blue dots) or in a state associated with the S2 subconductance (PS2, magenta dots). PS2 is shown inverted and scaled to illustrate its similar time course to that of Nav1.4 macroscopic inactivation. (b) PO and PS2 are shown as in a overlaid with fluorescence signals from fluorophores attached to individual voltage sensors from domains I–IV in Nav1.4 (Supplementary Fig. S6). Because fluorescence signals were prohibitively difficult to reliably obtain below room temperature, these signals were uniformly scaled in time to account for the different temperatures at which the single channel (10 °C) and fluorescence (room temperature) were obtained (see methods). (c) Summary of the voltage dependence of the time constants for entry into the S2 and O conductance levels (see Fig. 5), and the fast time constant from exponential fits to the fluorescence responses from individual voltage-sensing domains (Supplementary Fig. S6), after scaling the fluorescence in time as described above.
	Figure 7. The open pore of Nav1.4-WSW is fully accessible to internal MTSET.(a) Current responses to 10 ms voltage steps from −120 to 40 mV (20 mV steps) from a holding potential of −140 mV (see inset) from Nav1.4-WSW channels in inside–out patches excised from Xenopus oocytes. (b) The time course of the reduction in peak current for Nav1.4-WSW and Nav1.4-WSW-F1579C with respect to the cumulative exposure time to internal MTSET at 0 mV (see methods). Current responses for Nav1.4-WSW-F1579C before and after 1.7 s of exposure to internal MTSET at 0 mV are shown. (c) Second-order reaction rates of internal MTSET with Nav1.4-WSW-F1579C in the closed (square) and open (triangle) states. The reported change in closed to open state reaction rates for several residues in the pore of Shaker and BK potassium channels are shown for comparison4142, as well as the reaction rate for Nav1.4-F1579C without (closed circle) and in the presence of the fast inactivation disrupting toxin Anthopleurin B (ApB) (open circle)40.
	Figure 8. Quasi-sequential activation model for sodium channel gating.A simple model depicting the preferential sequence of events during sodium channel activation. The closed to open transition represents all of the steps from resting to activated, and the S2 state represents a distinct conformation of the open pore, which precedes inactivation. In wild-type channels, inactivation occurs sufficiently rapidly from the S2 state so that it is rarely observed. For simplicity, the S1 subconductance is not included here, but this conductance may potentially be represented as a parallel set of states to indicate an independent allosteric conformation of the channel. In this model, inactivation from closed states proceeds via a transition from closed to S2.

References [+] :

Aldrich, A reinterpretation of mammalian sodium channel gating based on single channel recording. , Pubmed