Cervenka R , Lukacs P , Gawali VS , Ke S , Koenig X , Rubi L , Zarrabi T , Hilber K , Sandtner W , Stary-Weinzinger A , Todt H .

W 1232 Austrian Science Fund FWF

	Figure 1. Gating perturbations by serial cysteine mutagenesis of DIV-S6. (A) Mutation-induced changes in the voltage-dependence of fast inactivation at site 1575 in DIV-S6. The normalized currents were fit by a Boltzman equation (Eq. 1). The estimated values for V1/2 were −43.7 ± 0.5 mV and −51.8 ± 1.1 mV for wild type and I1575C channels, respectively (P < 0.01. n = 7–8). The respective values for the slope factors were 10.1 ± 0.3 mV, and 11.5 ± 0.3 mV for wild type and I1575 channels, respectively (P = n.s.). The mutation in the selectivity filter of DIII. K1237E also caused a hyperpolarizing shift in the V1/2 relative to wild type (−51.1 ± 1.2 mV, n = 5, P < 0.001 vs. wild type). The combination of I1575 with K1237E resulted in a further negative shift of V1/2 (−59.6 ± 1.4 mV, n = 5, P < 0.001 vs. wild type, P = 0.01 vs. K1237E). The slope factors were unaffected by K1237E (10.8 ± 0.5 mV) and K1237E/I1575C (11.3 ± 0.4 mV). (B) Effect of cysteine-scanning mutagenesis on the V1/2 of fast inactivation. Bars indicate the mutation-induced change in V1/2 with respect to wild type (n = 5–9, *P < 0.05, **P < 0.01). (C) K1237E gives rise to a hyperpolarizing shift in V1/2. Shown are the shifts in V1/2 produced by adding the mutation K1237E to the indicated mutations in DIV-S6 (n = 5–9; *P < 0.05, **P < 0.01). The constructs K1237E/I1576C and K1237E/N1584C did not express current. (D) Effect of background on gating-perturbation by cysteine-scanning mutagenesis in DIV-S6. Shown are the shifts in V1/2 produced by cysteine substitutions in DIV-S6 in the background of wild type (abscissa) and in the background of the mutation K1237E (ordinate). There is a clear correlation between the examined gating perturbations with the notable exception of position 1581, which is an obvious outlier and. therefore. was omitted from the regression analysis (R = 0.94, P < 0.0001).
	Figure 2. Time course of recovery from inactivation in wild type and K1237E channels. (A) Short prepulse duration: Ooytes were subjected to a 100 ms depolarizing prepulse to −20 mV. The normalized inward currents elicited by the test pulses were fit with the sum of three exponentials (Eq. 3). A1, A2 and A3 were 0.50 ± 0.02. 0.25 ± 0.06, and 0.25 ± 0.06 for wild type and 0.33 ± 0.03, 0.11 ± 0.03 and 0.56 ± 0.01 for K1237E, respectively (P < 0.01, n = 3). τ1, τ2 and τ3 were 0.8 ± 0.05 ms, 231.8 ± 62.2 ms, 1667.8 ± 512.2 ms for wild type and 8.3 ± 1.4 ms, 295.8 ± 100.4 ms and 3443.8 ± 114.7 ms for K1237E, respectively (P < 0.01, n = 3). (B) Long prepulse duration: clamp protocols was as in described in A. with the exception that the prepulse duration was 1 s. The data points were fit with the sum of two exponentials (Eq. 2). A1 and A2 were 0.70 ± 0.05 and 0.30 ± 0.05 for wild type and 0.45 ± 0.05 and 0.55 ± 0.05 for K1237E, respectively (P < 0.01, n = 6–9). τ1 and τ2 were 486.5 ± 41.9 ms and 2588.1 ± 403.9 ms for wild type and 1804.1 ± 181.6 ms and 8140.6 ± 566.7 ms for K1237E, respectively.
	Figure 3. The observed shifts in V1/2 of inactivation are independent from shifts in activation. Each data point is labelled with the respective mutation. The voltage-dependence of activation was determined as described in Methods and the normalized currents were fit with Eq. 4 in order to derive the V1/2 for activation (R = −0.22, P = 0.54).
	Figure 4. The selectivity filter does not couple directly to DIV-S6 during inactivation. Histogram of the coupling energies between site 1237 in the selectivity filter and the indicated positions in DIV-S6. Coupling energies were calculated from the values of V1/2 and k which were derived from the fits of a Boltzmann equation (Eq. 1) to the normalized data of steady state inactivation. as described in “Methods” and Fig. 1.
	Figure 5. Gating perturbations by amino acid substitutions of I1581. (A) Hydrophobic substitutions at site 1581 give rise to large shifts in V1/2 of inactivation. I1581W significantly shifted the V1/2 to more positive values compared to wild type (**P < 0.01. n = 5–9). For comparison the data for I1581C are reproduced from Fig. 1B. (B,C) A histidine engineered to site 1581 allows for modulation of inactivation by pH. V1/2 values were for wild type −66.5 ± 1.2 mV, −71.1 ± 2.5 mV, −69.1 ± 1.0 mV for pH values of 6.6, 7.4 and 8.2, respectively (n = 7–9). V1/2 values were for I1581H −66.4 ± 1.7 mV, −65.8 ± 1.5 mV, and −75.3 ± 1.3 mV for pH values of 6.6, 7.4, and 8.2, respectively (n = 4–6). The only significant difference was with I1581 for V1/2 at pH 7.4 and pH 8.8. (D) The mutation I1581V recapitulates isoform differences in gating. I1581V was transfected into mammalian tsA201 cells and examined using the patch-clamp technique. The holding potential was -140 mV. The values for V1/2 were −66.2 ± 0.6 mV for wild type and -53.8 ± 0.6 mV for I1581V (n = 5–9. P < 0.05).
	Figure 6. Potential gating mechanism of I1581 and biological significance. (A) Site 1581 is homologous to a pivot point for activation in NavMs. Sequence alignment between DIV-S6 of rNav 1.4., the recently crystallized eukaryotic NavPaS33, and the prokaryotic VGSC structures of NavMs30 and of NavAb28. The alignment is based upon figure S1 in Shen et al.33, Fig. 1 in McCusker et al.30, Supplementary Figure 7 in Payandeh et al.28. I1581 aligns with V1401 of NavPaS, with T209 of NavMs and with V208 of NavAb (shaded area). (B) Crystal structures of DIV S6 of NavPaS (left, PDB#5x0 M) and an alignment of the pre-open NavAb28 (PDB#3RVZ)) and the presumably open state of NavMs30 (right, (PDB#4F4)). In all stuctures the S6 segment has a bend starting with the amino acids homologous to rNav1.4 I1581 (see A., amino acid side chain indicated). For better identification of the bend the long axis-centers of the proximal and distal parts of the S6 segments are indicated by lines. Right panel: The structures of NavAb of NavMs are aligned with reference to the selectivity filter. The NavMs structure does not contain voltage-sensors, therefore no S4-S5 linker is available. All four S6 segments of the tetrameric NavMs are shown (red ribbons) These S6 segments are clearly displaced with respect to the pre-open NavAb structure (white ribbon), albeit to slightly different degrees. This displacement is generated by a rotation around the backbone angle of T209 (side chain indicated), which swings that helix away from the central pore opening up the bottom of the structure30.
	Figure 7. Spacial relationship between the S4-S5 linker of one domain/subunit and the S6 segment of an adjacent subunit/domain in recently published VGSC structures. The backbone of one subunit/domain is indicated by a ribbon of a single color. Shown are only side chains of the amino acids homologous to rNav1.4 L1146 (S4-S5 linker) and I1581 (DIV S6) which are in close proximity in all structures. “P” denotes P-loop. (A) NavPaS (eukaryotic)33} (PDB#5x0 M), DIII and DIV. (B) NavAb (prokaryotic, closed)32} (PDB#5XB2) chains C and D. (C) NavMs (prokaryotic, open)31} (PDB#5HVD). Unlike NavAb this structure has not been crystallized as tetramer thus two identical chains are shown (1, 2).
	Figure 8. Spacial relationship of the inactivation gate (“IFM” motif) and the amino acids homologous to rNav I1581 and L1146 in the structure of EeNav1.434 (PDB#5xsy). Shown are the backbones of S5, P-loop (“P”) and S6 of DIII (white) and DIV (red), the S4-S5 linker of DIII (white), and the intracellular linker between DIII and DIV (green) containing the IFM motif (here LFM) represented as spheres. Also represented as spheres are the amino acids homologous to rNav1.4 I1581 and L1146, V1557 and L1122, respectively. (A) Side view as with the structures in Fig. 7. (B) View from the intracellular side. Obviously, residues V1557 and L1122 are in close proximity to the inactivation gate (LFM).

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