Stability of the Shab K+ channel conductance in 0 K+ solutions: the role of the membrane potential

Abstract

Shab channels are fairly stable with K(+) present on only one side of the membrane. However, on exposure to 0 K(+) solutions on both sides of the membrane, the Shab K(+) conductance (G(K)) irreversibly drops while the channels are maintained undisturbed at the holding potential. Herein it is reported that the drop of G(K) follows first-order kinetics, with a voltage-dependent decay rate r. Hyperpolarized potentials drastically inhibit the drop of G(K). The G(K) drop at negative potentials cannot be explained by a shift in the voltage dependence of activation. At depolarized potentials, where the channels undergo a slow inactivation process, G(K) drops in 0 K(+) with rates slower than those predicted based on the behavior of r at negative potentials, endowing the r-V(m) relationship with a maximum. Regardless of voltage, r is very small compared with the rate of ion permeation. Observations support the hypothesized presence of a stabilizing K(+) site (or sites) located either within the pore itself or in its external vestibule, at an inactivation-sensitive location. It is argued that part of the G(K) stabilization achieved at hyperpolarized potentials could be the result of a conformational change in the pore itself.

FIGURE 1

Collapse of Shab K⁺ conductance in 0 K⁺ solutions. (A) Control I_K evoked by a 0-mV/30-ms test pulse applied from the HP of −80 mV, with the cell in K_o/Na_i (see Methods) solutions (left panel). Thereafter, the cell was immersed 5 min in the 0 K⁺, Na_o solution (Na_o/Na_i), keeping the membrane potential constant at −80 mV, as indicated by the arrow. Finally, the cell was superfused with the K_o solution, and the state of channels was tested with the delivery of a test pulse (right panel). (B) I_K decay as a function of the time spent in 0 K⁺, as in A. I(t)/I_o is the ratio of the I_K left after t minutes in 0 K⁺, I(t), to the control current I_o. Currents were isochronally measured at the end of the test pulse. The line is the fit of the points with the function I(t)/I_o = exp (−rt), with r(−80 mV) = 0.79 min⁻¹. The points are the mean ± SE of at least four experiments at each time.

FIGURE 2

Inhibition of G_K collapse at −140 mV. (A, left panel) control I_K evoked by a −10-mV/30-ms pulse applied from the HP of −80 mV in K_o/Na_i solutions. Thereafter, the HP was shifted to −140 mV, and 15 s later the cell was superfused for 5 min with the Na_o solution (Na_o/Na_i, not shown, indicated by the arrow). Then, the cell was returned to the K_o solution, the HP was shifted back to −80 mV, and 30 s later I_K in the middle panel was recorded, as in the control. There was only a scant decrement of I_K. Next, the cell was immersed again for 5 min in the Na_o solution, this time keeping the HP at −80 mV (indicated by the arrow). Finally, with the cell back in K_o the trace in the right panel was recorded. (B) G_K drop (%) after a 5-min exposure to 0 K⁺ (Na_o/Na_i) solutions with the HP either at −140 (17 ± 4%, n = 5) or at −80 mV (93 ± 2.5%, n = 4), as indicated.

FIGURE 3

Collapse of G_K as a function of the membrane potential during channel exposure to 0 K⁺ solutions. (A) Fraction of conducting channels (I(t)/I_o) as a function of the time spent in 0 K⁺ (Na_o/Na_i) solutions at the indicated HPs. The lines are the fit of the points with exponential functions, as in Fig. 1 B. (B) Decay rate constant r as a function of the membrane potential V; r was obtained from the curves in A. The line is fit to the points with the equation r_n(V) = 0.01 + 86exp(0.06V). Subscript n stands for negative potentials.

FIGURE 4

Conductance-versus-voltage relationship of Shab channels as a function of the K⁺ distribution across the membrane. (A) I_K evoked by 100-ms pulses from −60 to 0 mV applied in 20-mV increments from the HP of −80 mV, in either 100 mM or 2 mM solutions, as indicated (see text). (B) G_K versus pulse potential. G_K was obtained from the relative, isochronal, tail currents at −80 mV I_tail(V)/I_max, where I_tail(V) is the amplitude of the tail current at the end of the pulse potential V, and I_max is the maximal amplitude of the tail currents. V was varied from −70 to +50 mV in 10-mV steps. In the experiments in Na_o/K_i, the tail currents were measured at a repolarization potential of −60 mV. The points are the mean ± SE of at least four experiments at each [K⁺]. The lines are fit to the points with Boltzmann functions, as indicated: 2K_o/Na_i: V_½ = −33.3 mV, z = 2.7; 20K_o/Na_i: V_½ = −31.2 mV, z = 2.4; 100K_o/Na_i: V_½ = −31.2 mV, z = 2.6; Na_o/K_i: V_½ = −29.3, z = 3.3.

FIGURE 5

Drop of G_K with the delivery of activating pulses from the stabilizing HP of −140 mV. (A) Control I_K elicited by a 0-mV/30-ms pulse from the HP of −80 mV in K_o/Na_i (left panel, Before). Thereafter, the HP was shifted to −140 mV, and 15 s later the cell was superfused with the Na_o solution, and 10 0-mV/5-ms pulses were applied at the rate of 1 Hz (indicated by the arrow). Finally, after 1.5 min in Na_o/Na_i, the cell was returned to the control K_o solution, the HP was shifted back to −80 mV, and I_K was recorded as in the control (right panel). (B) G_K drop after a 1.5-min exposure to 0 K⁺ solutions at the HP of −140 mV, obtained either without pulsing (left bar, 5.9 ± 1.5%, n = 8) or with the delivery of 10 0-mV/5-ms pulses (right bar, 9.6 ± 2.8%, n = 7), as in A (see text).

FIGURE 6

G_K drop after a 50-s exposure of the channels to 0 K⁺ at 0 mV. I_K evoked by a −10-mV/30-ms test pulse applied from −80 mV, in K_o/Na_i (Before). Thereafter, the HP was shifted to 0 mV, and 15 s later the cell was superfused for 50 s with the Na_o solution. Finally, the cell was superfused back with K_o, HP was shifted back to −80 mV, and 30 s later the channels were activated by a test pulse (right panel).

FIGURE 7

Channel inactivation reduces the rate of drop of G_K in 0 K⁺. (A) I_k evoked by either a short 30-ms (left panel) or a long 7-s pulse (right panel) to 0 mV from the HP of −80 mV, in Na_o/K_i solutions. (B) Steady-state inactivation (h_∞) as a function of the membrane potential. Inactivation was measured by the standard two-pulse method. The membrane was first depolarized for 20 s to the indicated prepulse potential, each prepulse was followed by a test pulse to 0 mV, with the cell in either Na_o/K_i (solid circles, solid line) or 100 (open circles, slashed line) solutions. I(prepulse)/I_max, is the ratio of I_K evoked by the test pulse that followed the indicated prepulse potential, I(prepulse), to the maximal I_K, I_max, evoked by the test pulse. The points are the mean ± SE of n = 4 experiments in each condition. The lines are the fit of the points with the Boltzmann equation: I(prepulse)/I_max = 1/(1 + exp((zF/RT)(V_m − V_½))), where F, R, and T have their usual meaning. In Na_o/K_i: V_½ = −56.3 mV, z = 4.6; 100K_o/K_i: V_½ = −53 mV, z = 5.0. HP = −80 mV. (C) Collapse of G_K from inactivated channels. G_K drop after either a 1.5- (left bars) or a 5-min (right bars) exposure to 0 K (Na_o/Na_i) solutions at 0 mV, as a function of time (0.25 or 1 min) at which the HP was shifted from −80 to 0 mV before the perfusion of the Na_o solution, as indicated (see text).

FIGURE 8

Collapse of G_K at depolarized membrane potentials (V_m > −80 mV). (A) Fraction of conducting channels (I(t)/I_o) as a function of the time spent in 0 K⁺ (Na_o/Na_i) solutions at the indicated HPs. The lines are the fit of the points with exponential functions, as in Fig. 3 A, with rate constants: r(−50) = 1.78 min⁻¹, r(−30) = 1.36 min⁻¹, and r(0) = 0.9 min⁻¹. (C) r versus HP in the whole range of voltages studied. The dashed line is the plot of r_n(V) (Fig. 3 B). (D) Comparison of the voltage dependence of r (squares) with the activation (G_rel), and the steady-state inactivation (h_∞) gating (solid circles). The values of r were normalized with their maximum experimental value (r(−50)). G_rel and h_∞ are the relationships in 0 solutions (Na_o/K_i) of Figs. 4 B and 7 B, respectively.

FIGURE 9

Voltage dependence of the decay rate constant r in the entire range of voltages studied. The points in the plot are the experimental values of r of Fig. 8 C. The lines are the plot of the equation r(V) = r_n(V) × h_∞ + r(0) × (1 − h_∞), where r(0s) = 0.90 min⁻¹, r_n(V) = 0.01 + 86exp(0.06V) is the variation of r at negative potentials obtained in Fig. 3 B; and h_∞ is the steady-state inactivation relation (see text). The dotted line is the plot of the equation with the parameters of h_∞ in Na_o/K_i of Fig. 7. The solid line is the fit of the points obtained considering the parameters of h_∞ as adjustable parameters (V_½ = −59 mV, z = 3.0).