Zarrabi T , Cervenka R , Sandtner W , Lukacs P , Koenig X , Hilber K , Mille M , Lipkind GM , Fozzard HA , Todt H .

1R01 HL096476-01 NHLBI NIH HHS , R01 HL096476 NHLBI NIH HHS

	FIGURE 1. The mutation K1237E produces an inactivated state from which recovery is very slow (IUS). A, shown is a graphic representation of the voltage-clamp protocol for assessment of the time course of recovery from IUS. From a holding potential of −120 mV, the channels were inactivated by a 300-s depolarizing step to −20 mV. After return to −120 mV, recovery from inactivation was monitored by repetitive test pulses to −20 mV at 20-s intervals. B, shown are inward currents through wild type and K1237E channels elicited by test pulses after the holding potential was returned to −120 mV. Here, to allow for better resolution of the current traces, test-pulse duration was 100 ms. In both constructs the first test pulse failed to elicit inward current. With wild type channels the second test pulse resulted in almost full recovery, whereas in K1237E channels full recovery was not attained before ∼300 s had elapsed. C, shown is the effect of charge-altering mutations of K1237E and of residues at homologous positions in DI, II, and IV. Oocytes expressing currents of the constructs D400A, E755A, and A1529D were subjected to the protocol shown in A. Currents are normalized to the value after full recovery and plotted as a function of time after the conditioning prepulse. Only the mutation K1237E results in substantial ultra-slow recovery.
	FIGURE 2. Previously published molecular model of the voltage gated Na+ channel (2). The P loops are represented as green ribbons, S6 helices are presented as pink ribbons. A, amino acids Lys-1237 of the selectivity filter and Ile-1575 and Phe-1579 of DIV-S6 are shown by space-filling models. Amino acid numbering corresponds to the rNaV1.4 channel. Amino acid Lys-1237 of the DIII P-loop is in close spatial relationship to amino acid Ile-1575 of the DIV-S6 segment. Phe-1579 is located one helix turn internal to Ile-1575 and lines the permeation pathway in the inner vestibule. B, shown is a helical wheel representation of the DIV-S6 segment. Only amino acids downstream of Ile-1575 are shown. Amino acids in bold are considered to face the permeation pathway.
	FIGURE 3. IUS is modulated by mutations in the DIV-S6 segment. Time course of recovery from a 300-s conditioning prepulse to −20 mV. Experimental protocol was as in Fig. 1. Maximum inward currents during recovery were normalized to the level after full recovery (n = 6–8). Data points were fitted with Equation 1 (lines). Fitting parameters are presented in Table 1. A, shown is a comparison of the time course of recovery of K1237E channels and the combination of K1237E with the DIV-S6 mutants I1575A, F1579A, and Y1586A. These amino acids in DIV-S6 are considered to face the pore (Fig. 2B) and are, thus, likely candidates for interaction with site 1237 in the DIII P-loop. Whereas the addition of F1579A and Y1586A enhanced recovery from IUS in K1237E background, the addition of I1575A protected K1237E channels from entry into the IUS state. B, shown is a comparison of the time course of recovery of wild type channels and the DIV-S6 mutants I1575A, F1579A, and Y1586A. F1579A and Y1586A show enhanced ultra-slow recovery, whereas the time course of recovery in I1575A is comparable with wild type channels.
	FIGURE 4. Schematic representation of the proposed molecular mechanism underlying the IUS state. Shown are the P-loops and the S6 segments of DII and DIV. A, the non-inactivated state is shown. Lys-1237 of DIII P-loop is located close to Ile-1575 of the DIV-S6 segment. B, mutation K1237E results in electrostatic repulsion between Glu-1237 and other negatively charged amino acids in the selectivity filter (Asp-400, Glu-755). This electrostatic repulsion gives rise to a swinging out of the DIII P-loop, resulting in an interaction between the Glu-1237 and the side chain of Ile-1575 (upper arrow). This interaction favors a conformational change of the DIV-S6 segment which gives rise to the IUS state (lower arrow).
	FIGURE 5. Effect of cysteine scanning mutagenesis of the DIV-S6 segment on IUS. Shown is the result of double exponential fits (Equation 1) to the time course of recovery from a prolonged depolarization as shown in Fig. 1A. Because of the low amplitude of recovery from IUS in these mutants, the parameter τ2 was fixed at 120 s during the fitting procedure. The asterisks indicate significant differences from wild type (**, p < 0.01; ***, p < 0.001; n = 5–7). With the exception of I1575C, all mutants significantly enhanced recovery from IUS, indicating a role of the DIV-S6 segment in the generation of the IUS state. Mutants I1576C and N1584C did not express sufficient current to be studied.
	FIGURE 6. Effect of addition of serial DIV-S6 cysteine mutations on IUS produced by K1237E. Experiments were performed as in Fig. 1. Shown is the normalized time course of recovery from inactivation as in Fig. 3. To allow better discrimination between the graphs, a log scale was chosen for the time axis. The time course of recovery in K1237E/I1575C was significantly faster than in all other double mutants. n = 3–7.
	FIGURE 7. Modulating effect of serial cysteine mutagenesis of the DIV-S6 segment on IUS produced by the mutation K1237E. Shown are the results of fitting Equation 1 to the data points in Fig. 6. Asterisks denote significant difference from K1237E (*, p < 0.05; **, p < 0.01; ***, p < 0.001, n = 3–7). Most S6 mutations increased both the time constant of recovery from IUS and the fraction of channels recovering from IUS. Only in K1237E/I1575C both time constants and amplitude were reduced.
	FIGURE 8. Passage of large organic cations through the pore increases the likelihood of entry into IUS. Presumably, the passage of large organic cations through the selectivity filter is associated with an increase in diameter of the pore. If IUS results from a widening of the pore, then the permeation of large cations should increase the likelihood of entry into IUS. A, recovery from the IUS state was assessed in K1237E. The voltage clamp protocol and data analysis were the same as shown in Figs. 1 and 3. The following charge carriers were assessed (90 mm, n = 3–6): Na+, K+, choline, methylammonium (MA), and diethylammonium (DEA). When Na+ was the permeating ion, IUS recovered with a time constant of 155 ± 12 s. This time constant increased during permeation with K+ (175 ± 19 s), MA (208 ± 25 s), choline (208 ± 58 s), and DEA (351 ± 51 s). The amplitudes of IUS were only slightly increased during permeation with K+. Permeation with large organic cations resulted in a substantial increase in the amplitude of IUS (p < 0.05, see panel B for data). B, correlation between the size of permeating ions and the increase in amplitude of IUS is shown. As the parameter of molecular size, we used the diameter calculated from the Pauling ionic radii for Na+ and K+. For the organic cations we calculated the minimum and maximum diameter by using the functions Minimum projection radius and maximum projection radius of the software marvinSketch. Both minimum and maximum diameter were significantly correlated with the amplitude of IUS (p < 0.01).
	FIGURE 9. Mutations I1575A and I1575C increase selectivity for Na+ ions in K1237E. The permeability of the channels for the large organic cation choline was assessed from the shift in reversal potential produced by replacing 90 mm Na+ of the bath solution with 90 mm choline. Current-voltage relationships were fit with the function Equation 2 to derive the reversal potential. Permeability ratios were then calculated from the shift in reversal potential with Equation 3. In K1237E/I1575A and K1237E/I1575C permeability to choline was significantly smaller than in K1237E alone or in the double mutant K1237E/F1579A, indicating that the mutations at site 1575 reduce the diameter of the selectivity filter (n = 6 for each construct).
	FIGURE 10. Molecular model of the conformational changes inside the selectivity filter associated with IUS. Shown are the amino acid residues at the internal turn of the P-loops (green ribbons) forming the selectivity filter: DIAsp-400, DII-Glu-755, DIII-Lys-1237, DIV-Ala-1529. The adjacent S6 segments are represented by pink ribbons. A, wild type rNaV1.4 is shown. Lys-1237 is in close proximity to Glu-755 and Ile-1575 of DIV-S6. B, mutation K1237E gives rise to an electrostatic repulsion between Glu-755 and Glu-1237, resulting in a lateral displacement of Glu-1237, thereby establishing an interaction with Ile-1575. C, the additional mutation I1575A produces a further displacement of the side chain of Glu-1237 in the direction of Ala-1575. Glu-755 and Glu-1237 are now separated by a bulky water molecule (not shown) with a high dielectric constant that removes the strong electrostatic repulsion between these side chains.
	FIGURE 11. Ile-402 in KV1. 2 channel is homologous to Ile-470 in Shaker. Amino acid sequence alignment of inner helices between KcsA, Shaker, KV1.2, and DIV-S6 of rNaV1.4 is shown. Underlines, residues homologous to Ile-1575 in rNaV1.4. Box, glycine hinge residues. Light Gray, residues homologous to Phe-103 in the KcsA channel.
	FIGURE 12. Close spatial relationship between gating-sensitive amino acids in the outer and the inner vestibule of K+ channel crystal structures. Shown are two of the four subunits of the published structures of KcsA (bacterial, non-voltage gated; Protein Data Bank code 1BL8 (1)) and mammalian, voltage gated (KV1.2, Protein Data Bank Protein Data Bank code 2A79 (61)). For clarity, protein backbone ribbons are colored differently for each subunit. Key amino acid side chains are shown as van der Waals spheres (blue, amino acid at position equivalent to site 1575 in rNaV1.4; rose, amino acid at the inner turn of the P-loop, possible equivalent to Lys-1237 in rNaV1.4; green, amino acid for which a major interaction with the P-loop is proposed to account for C-type inactivation in KcsA (9). A, KcsA; Thr-74 of one subunit (P-loop; yellow ribbon) is in close relationship to Met-96 (TM2) of the adjacent subunit (white ribbon) and with Phe-103 (TM2) of the same subunit. B, KV1.2; Thr-373 of one subunit (yellow ribbon) is close to Ala-395 of the adjacent subunit (white ribbon) and to Ile-402 of the same subunit.

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