Conductance stability and Na+ interaction with Shab K+ channels under low K+ conditions

Figure 1.. GK drop in hypokalemic solutions. (a) Left panel, Control IK evoked by a 0 mV/30 ms pulse with the cell bathed in 30Ko/Nai solutions. HP = −80 mV. Right panel, IK left after exposing the cell to 0 K+ (Nao/Nai) solutions across the membrane for 5 min (see Text), while Vm was kept constant at the HP. (b) I(t)/I(0) vs. Time in 0K+, I(t) is IK left after exposing the cell for t-min to 0 K+, Io is the initial, control, IK, as in A. The line is the fit of the points with a single exponential function I(t)/I(0) = exp(-t/Ʈ), with Ʈ = 1.4 min. (c) GK stability assessed as I(t)/I(0) vs. Time of whole-cell recording. IK was measured applying 0 mV/30 ms activating pulses at the indicated times, Vm = −80 mV between pulses. Cells were bathed in either Nao/35Ki (closed circles) or 30Ko/Nai (open circles) solutions. The solid line is the fit of the points (Nao/35Ki) with a single exponential function with Ʈ = 5.7 min, as in B. A&B were taken from Gomez-Lagunas, 2007 [7]

Figure 2.. GK stability vs. [Ki]. (a) Remaining GK (I(t)/Io) vs Time of recording, as in Figure 1c. Cells were bathed in Na+-containing internal solutions containing the indicated [Ki] (with XKi+XNai = 1, see text). External solution was Nao. Note that the data clusters into two sets. (b) Initial slope vs. [Ki]. Initial slope was assessed as the slope of the straight line that connects the first two points in A (I(t = 0) with the next I(t ≈ 2 min) point. The dashed lines have no physical meaning, they serve to best point out the data division into two sets. (c) GK stability of channels bathed in Nao/35Ki solutions at the HP of either −80 or −120 mV, as indicated

Figure 3.. Inhibition of GK drop by externally added monovalent cations. (a) Representative IK recorded in 30Ko/Nai solutions, before (left traces) and after (right traces) bathing the cells for 5 min in Nao solution containing the indicated test cation ([test cation] = 5 mM). Vm was kept constant at −80 mV during cells exposure to test solutions. (b) Remaining IK (IK(t = 5 min)/Io) after a 5-min exposure to the indicated solutions. Effects of Rb+ (0.9 ± 0.05) and K+ (0.9 ± 0.04) are not statistically different

Figure 4.. TEAo block and inhibition of GK drop. (a) Left panel, control IK evoked by a 0 mV/30 ms pulse applied from −80 mV in Nao/Ki solutions. Right panel, IK recorded upon bathing the same cell with 145 mM TEAo solution (see Methods), ~80% of IK was blocked. (b) Fractional TEAo block vs. [TEAo]. Fraction blocked = 1-(I([TEA])/Io), where I([TEA]) is IK amplitude in the presence of the indicated [TEA], Io is the corresponding control IK. Closed circles: test pulse = 0 mV; open circles: test pulse = +60 mV. The lines are the fit of the points with a Michaelis-Menten equation with Kd(0 mV) = 10.8 mM, or Kd(+60 mV) = 14.5 mM. Inset: Kd vs. Vm. Kds are the best fit parameters of the corresponding Michaelis-Menten curves. The line is the fit of the points with a Woodhull equation: Kd(Vm) = Kd(0)*exp[zd(F/RT)*Vm]; with parameters d = 0.12, Kd(0 mV) = 10.5 mM, z = +1; F,R,T have their usual meaning. (c) TEAo inhibition of GK collapse in 0 K+. Left panel: Control IK evoked by a 0 mV/30 ms pulse, applied from −80 mV, in 30Ko/Nai solutions (labeled 1). Right panel: IK left after immersing the cell in, 0 K+, TEAo solution (TEAo/Nai) for 5 min, with Vm constant at −80 mV (trace labeled 2, see…

Figure 5.. Selectivity and stability in hypokalemic conditions. (a) IK recorded in standard Nao/Ki solutions. Current was evoked by a 0 mV/30 ms pulse applied from −80 mV; repolarization potential was −170 mV. There is no inward current at −170 mV (b) Superposed currents recorded in Nao/35Ki solutions in a different cell. Currents were evoked as in A, at pulse end Vm was stepped to the potentials indicated in the inset, which shows tail currents in an expanded scale, HP was −120 mV. Note the small but conspicuous inward Na+-carried current. (c) Currents recorded in 4Ko/35Ki solutions in the same cell as in B. Tail currents Vm are indicated. Notice the shift in current reversal potential as compared to that in B. HP was −120 mV. (d) Instantaneous I–V relationship of the experiments in B &C (see Text). (e) Conductance stability in 4Ko/35Ki solutions. Note that 4 mM Ko affords GK stability. Stability in 4Ko/35Ki solutions turned out to be similar to that in standard Nao/Ki solutions, HP was −80 mV in both cases

Figure 6.. Na+ permeation and block in low K+ conditions. (a) Left panel: Superposed currents (Nao/20Ki solutions) evoked by 0 mV/30 ms pulses, at pulse end Vm was repolarized to the indicated potentials. Note the inward Na+ current. Right panel: currents recorded in the same cell in A illustrating internal Na+ block, and the voltage-driven unblock of the pore (see Tex). (b) Instantaneous I–V relationship of the traces in A. (c) Fractional Nai block vs. Vm. Fractional block was assessed as: fb = 1-(Io/Ie), were Ie is the expected current in the absence of Nai block, and Io is the actual, observed, current (see Text). Ie was evaluated from the least-squares line (solid straight line through the points in B) that fits the observed current (Io) between −80 and −40 mV, where Io varies linearly with Vm (r = 0.999): Ie = 1.98+(0.019)Vm. The vertical dashed line helps to visually separate fb at negative vs. positive Vm. (d) Fractional Na+ block restricted to the voltage range where fb does not decrease with Vm, data from C. The line is the fit of the points with a Woodhull equation: Kd(0)*exp[zd(F/RT)*Vm], with z = +1, d = 0.98, Kd(0) = 170 mM

Figure 7.. Na+ permeation & block with physiological [Ko+]. (a) Average instantaneous I–V relationships recorded either in Nao/20Ki or 5Ko/20Ki conditions obtained from experiments as in Figure 6A. To allow comparison, currents were normalized to its local maximal value (see Text). (b) Fractional Nai block vs. Vm. Nai+ block was assessed with the least-squares lines that fit the linear regions of the points at negative Vm, as in Figure 6, namely: 0Ko(−90 to −50 mV): Ie = 1.52 + 0.016*Vm, r = 0.998; 5Ko(−70 to −30): Ie = 1.24 + 0.037*Vm, r = 0.999. The vertical dashed line serves to visually separate negative from positive voltages. (c) Comparison of 0 voltage affinity, Kd(0 mV), in 0 vs 5 Ko conditions; affinities are statistically different (P = 0.0634). (d) Comparison of block electrical distance, d, in 0 vs. 5Ko conditions; values are not statistically different (P = 0.911). The parameters Kd(0 mV) & d were obtained by fitting the corresponding fractional block vs Vm relationships with a Woodhull equation, as in Figure 6. (e) PNa/PK vs. [Ki]. PNa/PK was evaluated with the Goldman equation (see Text) applied to the corresponding average current reversal-potentials, from experiments as in…

Figure 8.. Summary of Shab K+ channel behavior as a function of [K+] across the membrane. Upper left image (state 1): Under saturating [K+] the selectivity filter has 2 K+ ions at a time (blue spheres, Doyle et al [9]), Gk is stable & selective, irrespective of Vm. Upper right panel (state 2): Upon K+ replacement by equimolar amounts of Na+ ions the channel visits a “transition”, meaning unstable, state (1->2, right panel), where Gk falls with a rate that depends on XK & Vm (K+ molar fraction) and Vm. In this unstable state the pore conformation allows Na+ permeation from the open, non-inactivated, state (a Na+ ion is indicated in the pore by the small red sphere), depending on Vm. Negative HP or K+ addition restore stability (indicated by the left pointing arrow), although: the already lost G is not recovered (i.e., arrows connecting states 1 & 2 do not indicate a truly reversible transition). Lower panel (state 3): With 0 K+ across the membrane (1->3) the channel readily falls into the stable (i.e., irreversible) not conducting state 3; alternatively, under hypokalemic (non-zero K+ conditions) the channels sink although more slowly into state 3 (2->3) depending on XK+. In both cases (1->3…