Characterization of a voltage-gated K+ channel that accelerates the rod response to dim light

Abstract

In this study a K+ current, IKx, in isolated salamander rod photoreceptors was characterized and its role in shaping small photovoltages was examined. IKx is a standing outward current of about 40 pA at -30 mV that deactivates slowly when the cell is hyperpolarized (tau max = 0.25 s). The voltage and time dependence of IKx are similar to that of M-current, but IKx can be distinguished from M-current because it is not suppressed by acetylcholine and is "blocked" by external Ba2+ in a surprising manner: the activation range of IKx is shifted strongly in the positive direction. Using current-clamp recordings and a computer simulation of the photo-response, we show that IKx figures prominently in setting the dark resting potential and accelerates the voltage response to small photocurrents.

Figure 1. Separation of I_Kx from I_h (Using Cs⁺) and from I_Ca and I_K(Ca) (Using Cd²⁺) in a Rod

(A) Voltage-clamped whole-cell current in a rod bathed in Ringer solution. Voltage command steps (500 ms; drawn below the current records) were applied every 3 s to a cell held at –40 mV. (B) Superimposed current responses to a two-step hyperpolarizing protocol showed that 2 mM Cs⁺ blocked only the inward current elicited by steps to –90 mV. Voltage steps (500 ms) to –60 and –90 mV, separated by 500 ms, were applied every 3 s. The current record in the presence of Cs⁺ is plotted in dots. The two records shown are from the 2d and the 32d sweeps of the experiment in (C). (C) Block of I_h by Cs⁺. Current measurements made at the holding potential of –40 mV and at the end of the steps to –60 and –90 mV from the same experiment as in (B). Application of 0.2 mM Cs⁺ reduced the inward current at –90 mV. The 0.2 mM Cs⁺ was washed out, and 2 mM Cs⁺ was applied, resulting in more block of inward current. (D) Block of I_K(Ca) by Cd²⁺ without affecting I_Kx. Voltage steps (1 s) were applied every 5 s to a rod held at –30 mV. Currents elicited by test steps to –10 and –50 mV are shown. Upper traces, before application of Cd²⁺; lower traces, after application.

Figure 2. I_Kx Is a Standing Outward Current That Turns Off with Hyperpolarization

Recordings were made from cells bathed in Ringer solution containing 5 mM Cs⁺ and 0.1 mM Cd²⁺. (A) Current elicited by 1 s voltage steps applied every 5 s from a holding potential of –30 mV. (B) Averaged currents (n = 3) elicited by steps to –60 mV from a holding potential of –30 mV: (a) brief (20 ms) –4 mV steps were applied during the –60 mV step; (b) –4 mV steps were not applied. The difference current (a–b) shows that the –4 mV steps gradually resulted in less current as time progressed at –60 mV. The superimposed broken line has the same time constant as an exponential fitted to the decaying current in (b), showing a correlation between the rate of conductance decrease and current decay.

Figure 3. Time and Voltage Dependence of I_Kx

Recordings were made from ceils bathed in Ringer solution containing 5 mM Cs⁺ and 0.1 mM Cd²⁺. (A) Voltage dependence of activation for a typical experiment. Peak tail currents at –30 mV (the same protocol as shown in Figure 2A) were converted to conductance and plotted against conditioning potential. The reversal potential for I_Kx was taken as –74 mV (see Figure 4), zero conductance corresponds to the minimum value of g_Kx, and the smooth curve is the Boltzmann equation fitted using a least-squares method. (B) Deactivation of I_Kx described by a single exponential. Hyperpolarizing voltage steps (1 s) were applied from a holding potential of –30 mV, and exponentials were fitted to the current decay using a nonlinear least-squares method. The currents (circles) and exponentials (continuous lines) are shown normalized and on a semilogarithmic scale for two test potentials. (C) Time constants (τ) of exponentials fitted to the decay of I_Kx (means ± SD, n = 5–8) plotted against test potential. The smooth curve is 1/(α + β), in which α and β are rate constants for transitions between the closed and open states of the channels. Mean values for α and β (n = 6) were calculated from α = A_∞/τ and β = (1 – A_∞)/τ (Hodgkin and Huxley, 1952), in which A_∞ is the normalized steady-state conductance, and τ is the time constant (s) of the fitted exponential. α and β were fitted by eye with the functions: α = 0.23(E + 45)/(1 – exp(– (E + 45)/7)); β = 1.2(E + 55)/(exp((E + 55)/5.5) – 1), in which E is the membrane potential (mV).

Figure 4. Reversal Potential for I_Kx

Recordings were made from cells bathed in Ringer solution containing 5 mM Cs⁺ and 0.1 mM Cd²⁺. Hyperpolarizing voltage steps (not shown) wereapplied from a holding potential of –30 mV. Current (shown) was measured at the beginning and the end of the hyperpolarizing steps: the difference (beginning minus end) was then plotted against test voltage, and the reversal potential was estimated (data not shown). Recordings were first made in 2.5 mM external K⁺ and then the external K⁺ concentration was (A) raised to 8 mM or (B) lowered to 1 mM. (C) Reversal potentials plotted against the logarithm of the external K⁺ concentration. The points are means (±SD) measured in 1 (n = 3), 2.5 (n = 8), 8 (n = 4), and 17.5 (n = 1) mM K⁺. The broken line shows the Nernst relation for a pure K⁺ electrode, and the continuous line is the Goldman-Hodgkin-Katz voltage equation fitted by eye (P_Na/P_K = 0.024).

Figure 5. Modification of I_Kx by External Ba²⁺

Recordings were made from cells bathed in Ringer solution containing 5 mM Cs⁺ and 0.1 mM Cd²⁺. (A) Whole-cell current in response to voltage steps from –30 mV before (left) and after (right) 5 mM Ba²⁺ was added to the Cs⁺- and Cd²⁺-containing Ringer solution (note that more depolarizing steps are shown after Ba²⁺ application). (B) Time-dependent current in 5 mM Ba²⁺ had kinetics similar to those of the control I_Kx. The points are mean time constants, τ (±SD, n = 3–6), of single exponentials fitted to the time-dependent current component elicited by test steps from –30 mV in 5 mM Ba²⁺. The continuous smooth curve describes 1/(α + β) (derived as in Figure 3C) and was estimated for data from six experiments in 5 mM BaCl₂. The broken curve is the control repeated from Figure 3C. (C) Activation curves, from tail currents measured at –30 mV, for I_Kx in 0 (closed circles), 1 (open circles), 5 (closed squares), and 25 (open squares) mM added Ba²⁺. The smooth curves are the Boltzmann equation fitted using a least-squares method. The midpoint of the activation curve in 5 mM Ba²⁺ was –6.9 ± 1.7 mV, with a slope factor of 11.2 ± 0.9 mV (means ± SD, n = 7). (D) Concentration dependence of the Ba²⁺-induced shifts in the midpoint of the activation curve for I_Kx. The points are means (±SD) for cells in 0 (n = 7), 1 (n = 4), 5 (n = 7), or 25 (n = 3) mM added Ba²⁺.

Figure 6. Effects of I_Kx and I_h on the Voltage Response to Current Injection

Current components were identified in the voltage-clamp mode, and voltage responses to square pulses of injected current were assessed in the same cell. In each condition, cells were made to “rest” at about –38 mV by injection of a constant holding current (Axopatch 1-C). (A) To reveal I_h, recordings were made in the absence of Cs⁺ but in the presence of Cd²⁺. With both I_h (dominant at –95 mV) and I_Kx (dominant at –55 mV) present, a time-dependent decrease in both small and large voltage signals occurred. (B) Recordings in both (B) and (C) were made from cells bathed in Ringer solution containing 5 mM Cs⁺ and 0.1 mM Cd²⁺. Bath application of 5 mM Cs⁺ blocked I_h, but left I_Kx. The small voltage signal was similar to the control but the large signal became sustained. (C) Bath-applied 5 mM Ba²⁺ inhibited I_Kx in this voltage range (see Figure 5). No time- or voltage-dependent currents (other than capacity currents) were evident, and the response to the small hyperpolarizing current injection was much larger and did not decrease with time (the larger hyperpolarizing current step was not applied).

Figure 7. Comparrson of the Voltage Ranges over Which I_Kx and I_h Gate and the Simulated Effect of I_Kx on the Photovoltage

Recordings were made from cells bathed in Ringer solution containing 5 mM Cs⁺ and 0.1 mM Cd²⁺. (A) Activation and τ curves for I_Kx and I_h as used in our computer reconstruction of the light response. The reversal potentials for I_Kx and I_h were –74 and –32 mV, respectively, and the rod's dark resting potential is marked by the vertical broken line. The data for salamander rod I_h come from Hestrin (1987). (B, top) The photocurrent in response to a dim flash measured from published data for salamander rods (Baylor et al., 1984, their Figure 2A; response to 0.98 photons mm^–2) with the points (closed circles) fitted by eye using the Independence equatron (I = (15.5(exp(–1.6t)))(1 – exp(–1.6t))³) from Baylor et al. (1974), in which I is the photocurrent (pA) and t is time (s). Zero on the vertical axis corresponds to an inward dark current of 40 pA. This current trajectory was applied to a computer model circuit with the following current components: g_Kxn_Kx(E – E_Kx) and g_L(E – E_L), in which E is membrane potential, E_Kx and E_L are reversal potentials with values equal to –74 and –77 mV, respectively, g_Kx is the maximum conductance underlying I_Kx (1.04 ± 0.23 nS, mean ± SD, n = 12), and n_Kx is a value between 0 and 1 that represents the proportion of channels in the open state. A voltage-independent leak conductance, g_L, of 0.35 nS and a membrane capacitance of 21 pF (Baylor et al., 1984) were assumed. (B, middle) Two calculated voltage responses to the photocurrent are shown: (a) the continuous line, when n_Kx was allowed to vary according to the differential equation: dn_Kx/dt = α(1 – n_Kx) – β(n_Kx) (α and β from Figure 3C); (b) the broken line, when the n_Kx term was held constant at the resting value of 0.719 (i.e., I_Kx with no voltage dependence). Zero on the vertical axis corresponds to a resting potential of –38.2 mV. The vertical arrows point to the peaks of the voltage responses. (B, bottom) Cating of the channels underlying I_Kx (described by n_Kx) during the photocurrent, correspondrng to the two voltage responses described above.