Wu D et al. (2010), State-dependent electrostatic interactions of S...

XB-ART-42026

J Gen Physiol 2010 Jun 01;1356:595-606. doi: 10.1085/jgp.201010408.

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State-dependent electrostatic interactions of S4 arginines with E1 in S2 during Kv7.1 activation.

Wu D , Delaloye K , Zaydman MA , Nekouzadeh A , Rudy Y , Cui J .

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The voltage-sensing domain of voltage-gated channels is comprised of four transmembrane helices (S1-S4), with conserved positively charged residues in S4 moving across the membrane in response to changes in transmembrane voltage. Although it has been shown that positive charges in S4 interact with negative countercharges in S2 and S3 to facilitate protein maturation, how these electrostatic interactions participate in channel gating remains unclear. We studied a mutation in Kv7.1 (also known as KCNQ1 or KvLQT1) channels associated with long QT syndrome (E1K in S2) and found that reversal of the charge at E1 eliminates macroscopic current without inhibiting protein trafficking to the membrane. Pairing E1R with individual charge reversal mutations of arginines in S4 (R1-R4) can restore current, demonstrating that R1-R4 interact with E1. After mutating E1 to cysteine, we probed E1C with charged methanethiosulfonate (MTS) reagents. MTS reagents could not modify E1C in the absence of KCNE1. With KCNE1, (2-sulfonatoethyl) MTS (MTSES)(-) could modify E1C, but [2-(trimethylammonium)ethyl] MTS (MTSET)(+) could not, confirming the presence of a positively charged environment around E1C that allows approach by MTSES(-) but repels MTSET(+). We could change the local electrostatic environment of E1C by making charge reversal and/or neutralization mutations of R1 and R4, such that MTSET(+) modified these constructs depending on activation states of the voltage sensor. Our results confirm the interaction between E1 and the fourth arginine in S4 (R4) predicted from open-state crystal structures of Kv channels and reveal an E1-R1 interaction in the resting state. Thus, E1 engages in electrostatic interactions with arginines in S4 sequentially during the gating movement of S4. These electrostatic interactions contribute energetically to voltage-dependent gating and are important in setting the limits for S4 movement.

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Species referenced: Xenopus
Genes referenced: arfgap1 kcne1 kcnq1 ogdh

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	Figure 1. Sequence alignment of S2 and S4 from various voltage-dependent ion channels and proteins. (A) Conserved negatively charged residues in S2 and positively charged residues in S4 are in bold. (B) Currents generated from WT, E1K, and endogenous channels. Oocytes were held at −80 mV, depolarized from −80 to +60 mV for 5 s, and repolarized at −40 mV for 3 s. Scale, 4 µA for all except Kv7.1+KCNE1 (20 µA); 2 s for all currents in this and subsequent figures. (C) Western blot probing for Kv7.1 and Gβ in the whole cell lysate and biotinlyated membrane fraction from oocytes. Gβ is a cytoplasmic protein. Black lines indicate that intervening lanes have been spliced out.
	Figure 2. Currents from various E1 mutations to negative or neutral residues in Kv7.1 obtained using the protocol in Fig. 1 B. (A) Scale, 4 µA. (B) G-V relationship from mutations in A. Gray line represents WT Kv7.1. Error bars represent standard error of the means.
	Figure 3. S4 mutations to glutamate restore E1R current. (A) Currents were recorded from double mutations shown using the voltage protocol as in Fig. 1 B. Scale, 6 µA. (B) Peak current amplitudes in A were averaged for each mutation. Error bars represent standard error of the means. (C) Current from E1R paired with S4 residues mutated to glutamate coexpressed with KCNE1. Scale, 20 µA. (D) Peak current amplitudes in C were averaged for each mutant. Error bars represent standard error of the means.
	Figure 4. I-V relationships of various mutants generating constitutive current (left). Protocol same as Fig. 1 B. (Right) Block of current by KCNQ1 pore blocker chromanol 293B (100 µM) while pulsing to +40 mV for 5 s, repolarizing at −40 mV for 3 s, and holding at −80 mV for 32 s.
	Figure 5. E1C currents after superfusion of MTSES− or MTSET+. (A; left) Oocytes were repeatedly held at −80 mV for 32 s, depolarized at +40 mV for 5 s, and repolarized at −40 mV for 3 s. Scale, 2 µA. (Middle) Peak current amplitudes at +40 mV plotted against time. (Right) Normalized peak current amplitude after various MTS treatments. Error bars represent standard error of the means. (B; left) E1C+KCNE1 currents after superfusion of MTSES−, MTSET+, or MTSACE. Same protocol used as in A. Scale, 3 µA. (Middle) peak current amplitudes at +40 mV plotted against time. (Right) Normalized peak current amplitude after various MTS treatments. Error bars represent standard error of the means.
	Figure 6. E1C/R4E+KCNE1 currents after superfusion of MTSES− or MTSET+. (A, left) The same pulse protocol was used as in Fig. 5 A. Scale, 5 µA. (Middle) Peak current amplitudes plotted against time. (Right) Normalized peak current amplitude after various MTS treatments. Error bars represent standard error of the means. (B; left) E1C/R1Q+KCNE1 currents after superfusion of MTSES− or MTSET+. The same pulse protocol was used as in A. Scale: top, 5 µA; bottom, 2 µA. (Middle) Peak current amplitudes plotted against time. (Right) Normalized peak current amplitude after various MTS treatments. Error bars represent standard error of the means.
	Figure 7. Currents from E1C/R1E and E1C/R1E+KCNE1. (A, left) Same protocol used as in Fig. 1 B. (Right) E1C/R1E+KCNE1 currents after superfusion of MTSES− or MTSET+. (B; left) Currents from E1C/R1E/Q3R and E1C/R1E/Q3R+KCNE1. Same protocol as in A. (Right) G-V relationships from E1C/R1E/Q3R with or without KCNE1 were plotted with G-V relationships of WT Kv7.1 (left gray line) or WT Kv7.1+KCNE1 (right gray line). Error bars represent standard error of the means. Scale: E1C/R1E, 0.7 µA; E1C/R1E+KCNE1, 12 µA; E1C/R1E/Q3R, 1 µA; E1C/R1E/Q3R+KCNE1, 5 µA. (C; left) E1C/R1E/Q3R+KCNE1 currents after superfusion of MTSES− or MTSET+. Same protocol as in Fig. 5 A. Scale: top, 5 µA; bottom, 2 µA. (Middle) Peak current amplitudes plotted against time. Error bars represent standard error of the means. (Right; top) Normalized peak current amplitude after various MTS treatments. (Right; bottom) G-V relationships before and after MTS treatment. Red line indicates the G-V before MTS treatment. Error bars represent standard error of the means.
	Figure 8. E1C/R1E/Q3R+KCNE1 currents after superfusion MTSET+. (A; left) Oocytes were held at either −80 or +40 mV, with perfusion of MTSET+ as indicated, followed by resumption of test pulses. Scale, 0.7 µA. (Right) Peak current amplitudes plotted against time. (B) Voltage dependence of MTSET+ modification. (Left) Pulse protocol used. The holding potential was varied. (Middle) Time course of MTSET+ modification at different holding potentials. (Right) Rates of MTSET+ modification plotted against holding potential. The rates were fit with a double Boltzmann function (see Materials and methods) with V1/2,a of −75 mV and slopea of 5 mV, and V1/2,b of 8 mV and slopeb of 8 mV. Error bars represent standard error of the means. G-V before MTS modification was also plotted. (C) Structures of the deep resting, intermediate resting, and activated states of Kv7.1 generated from molecular dynamics simulations and Poisson-Boltzmann continuum electrostatic calculations. Red, E1; magenta, R1; cyan, R2; green, R3; blue, R4.
	Figure 9. E1C/R4E+KCNE1 currents. (A) Oocytes were repeatedly depolarized to 40 mV for 5 s, repolarized at 40 mV for 3 s, and held at −80 mV for 32 s. (B) Time course of instantaneous and peak current after repeated pulses.

References [+] :

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed, Xenbase