Reduced voltage sensitivity in a K+-channel voltage sensor by electric field remodeling

Abstract

Propagation of the nerve impulse relies on the extreme voltage sensitivity of Na(+) and K(+) channels. The transmembrane movement of four arginine residues, located at the fourth transmembrane segment (S4), in each of their four voltage-sensing domains is mostly responsible for the translocation of 12 to 13 e(o) across the transmembrane electric field. Inserting additional positively charged residues between the voltage-sensing arginines in S4 would, in principle, increase voltage sensitivity. Here we show that either positively or negatively charged residues added between the two most external sensing arginines of S4 decreased voltage sensitivity of a Shaker voltage-gated K(+)-channel by up to approximately 50%. The replacement of Val363 with a charged residue displaced inwardly the external boundaries of the electric field by at least 6 A, leaving the most external arginine of S4 constitutively exposed to the extracellular space and permanently excluded from the electric field. Both the physical trajectory of S4 and its electromechanical coupling to open the pore gate seemed unchanged. We propose that the separation between the first two sensing charges at resting is comparable to the thickness of the low dielectric transmembrane barrier they must cross. Thus, at most a single sensing arginine side chain could be found within the field. The conserved hydrophobic nature of the residues located between the voltage-sensing arginines in S4 may shape the electric field geometry for optimal voltage sensitivity in voltage-gated ion channels.

Fig. 1.

Macroscopic currents of Shaker mutants carrying a charged side chain at position 363. (A) Topological representation of a single Shaker subunit. The S4 sequence is expanded at the right, where R1–R4 are underlined and 363 is enclosed by the green box. The proximity of R1 to 419, instead of 241, makes this drawing more closely represent the activated state. (B) Detail of segment S4 of the Kv1.2-Kv2.1 paddle chimera (PDB:2R9R) (10). R1, R2, and R3 are shown in ball-and-stick representation, Leu294 (equivalent to Shaker's Val363) is in solvent-accessible surface (yellow), and lipids are in wireframe representation (PGW501 and PGW503). Transmembrane S1 and S2 were removed for clarity. Molecular graphics made by VMD (http://www.ks.uiuc.edu/Research/vmd/). (C–E) TEVC currents evoked by voltage pulses incremented by 10 mV from a holding potential of −90 mV. (F) Normalized conductances vs. voltage (G-V relationship). We used the expression G = I_K/(Vm − E_K) to calculate conductance, G, at each voltage, where I_K is the ionic K-current, Vm is the voltage pulse, and E_K, the reversal potential for K+, is assumed to be −90 mV. Results are means ± SE. Data points were fit to a simple Boltzmann distribution function with parameters V_1/2 = 1.9 mV, z = 1.53 e_o for V363R (n = 6); V_1/2 = −26.6, z = 1.53 e_o for V363D (n = 4); and V_1/2 = −9.5 mV, z = 1.76 e_o for wt (n = 5).

Fig. 2.

Charged side chain at 363 decreases the valence of opening. (A) One hundred superimposed macroscopic current traces evoked by the protocols schematized at top. Each column corresponds to the channel construct specified at top. Inset: Nonstationary fluctuation analysis of the tail currents used to obtain the number of channels (N) (23). Continuous lines were drawn according to σ²(I) = C + (iI − I²/N), where i is the unitary channel current. (B) Representative segments of 5-min records from the same patch shown in A. Opening are downward deflections. Voltage increments of 2.5 mV are shown at the right of each trace. (C) Three distributions of the open-duration events detected during 5 min at three different applied voltages. Voltages shown span 1 order of magnitude in Po. Solid line at the top of each distribution is the best maximum-likelihood single-exponential fit to logarithmically binned data. (D) Absolute Po (NPo/N) vs. V. Data points were fitted to the function Po(V) = exp (z_limV/kT), where z_lim is the effective valence of opening. (E) Summary plot of z_lim. Results are means ± SD, and asterisks indicate significant differences from wt according to a two-tailed t test.

Fig. 3.

Hypotheses involving reshaping of the electric field. (Top) In the Native channel, R1 (encircled number 1) is only accessible to modification by MTSET in the Activated conformation, and all sensing arginines (R1–R4) move across the electric field. (Middle) Hypothesis i: the charged side chain at 363 shifts the boundaries of the electric field outward. Then, R1 becomes inaccessible to MTSET at any activation state. All sensing arginines (R1–R4) move in the electric field but scan a smaller fraction of it, resulting in decreased voltage sensitivity. (Bottom) Hypothesis ii: the charged side chain at 363 shifts the boundaries of the electric field inward. Then, R1 becomes accessible to MTSET at any activation state. Some sensing arginines (R1–R4) do not move in the electric field, resulting in decreased voltage sensitivity.

Fig. 4.

Accessibility of R1C to extracellular MTSET becomes high and state-independent. (A and B) Representative K-current traces elicited in TEVC by short-pulse (A) and long-pulse (B) protocols differing in channel open duration by 10-fold. Arrows indicate the beginning of the external MTSET perfusion. (C) Time course plots of cysteine modification using short- or long-pulse protocols. Modification time course drawn by plotting current amplitudes at the times marked with the discontinuous vertical lines shown in A and B. Modification time courses were fitted to a single exponential function: I(t) = I₀ + Ae^−t/τ, with τ value shown. (D) Time constants of modification using short- and long-pulse protocols. Results are means ± SD, and asterisks indicate significant differences (P < 0.05) between protocols according to a two-tailed t test. From these τ values the apparent second-order modification rate constants in the closed and open state, k_c and k_o respectively, were calculated in mM⁻¹s⁻¹ according to a procedure described previously (31). k_c = 0.32 and k_o = 14.2 for R1C; k_c/o = 1.3 for R1C/V363R; k_c/o= 3.2 for R1C/V363D; k_c = 0.046 and k_o = 1.8 for R2C; k_c = 0.053 and k_o = 3.4 for R2C/V363R; and k_c = 0.041 and k_o = 0.7 for R2C/V363D.

Fig. 5.

Charged side chain at 363 influences the electrostatic field sensed by the voltage sensor. (A) Oppositely charged side chains affect the voltage profile across the membrane in opposite fashion. The transmembrane electric field (thick dashed line) is locally affected by nearby extracellular charges [represented by (+) and (−) “lollipops”]. According to the sign of the charge, the external border is made more positive or more negative (thick solid lines at right and left, respectively). The net difference between these opposite electrostatic effects corresponds to ΔV_1/2. At lower ionic strength, the produced electrostatic field should be stronger, increasing ΔV_1/2. (B and C) Voltage dependence of activation obtained at high and low ionic strength. G-V relationships were obtained in TEVC. Currents were first recorded in a normal-ionic-strength solutions (B; I ≈ 0.11 M); then at low ionic strength (C; I ≈ 0.022 M). Finally, a G-V curve was obtained at normal ionic strength to compare with those taken at the beginning. Only variations of V_1/2 <5 mV between the first and third recording were used. Data points were fitted to a simple Boltzmann distribution with parameters V_1/2 = 3.5 mV, z = 1.5 e_o for V363R in high ionic strength (n = 5); V_1/2 = −26.5, z = 1.62 e_o for V363D in high ionic strength (n = 6); V_1/2 = −38.8 mV, z = 1.26 e_o for V363R in low ionic strength; and V_1/2 = −78.6 mV, z = 1.74 e_o for V363D in low ionic strength.