Dissection of K+ currents in Caenorhabditis elegans muscle cells by genetics and RNA interference

Abstract

GFP-promoter experiments have previously shown that at least nine genes encoding potassium channel subunits are expressed in Caenorhabditis elegans muscle. By using genetic, RNA interference, and physiological techniques we revealed the molecular identity of the major components of the outward K+ currents in body wall muscle cells in culture. We found that under physiological conditions, outward current is dominated by the products of only two genes, Shaker (Kv1) and Shal (Kv4), both expressing voltage-dependent potassium channels. Other channels may be held in reserve to respond to particular circumstances. Because GFP-promoter experiments indicated that slo-2 expression is prominent, we created a deletion mutant to identify the SLO-2 current in vivo. In both whole-cell and single-channel modes, in vivo SLO-2 channels were active only when intracellular Ca2+ and Cl- were raised above normal physiological conditions, as occurs during hypoxia. Under such conditions, SLO-2 is the largest outward current, contributing up to 87% of the total current. Other channels are present in muscle, but our results suggest that they are unlikely to contribute a large outward component under physiological conditions. However, they, too, may contribute currents conditional on other factors. Hence, the picture that emerges is of a complex membrane with a small number of household conductances functioning under normal circumstances, but with additional conductances that are activated during unusual circumstances.

Fig. 1.

The voltage-dependent outward component in WT and slo-2 mutant cells. Comparison of whole-cell currents from a WT cell in low Ca²⁺, low Cl^- intracellular concentrations (10 nM and 4 mM, respectively) (a) and a slo-2 mutant cell (b). Currents were elicited from a -70 mV holding potential, in 10-mV steps from -70 to +60 mV. (c) G-V data plotted for WT cells (▪; n = 5), in low intracellular Ca²⁺/Cl^- solution; and slo-2 mutant cells (•; n = 7). G-V data were plotted as described in Materials and Methods, using a value of -70 mV for K⁺ reversal potential (E_k), which is close to the expected K⁺ equilibrium potential of ≈-80 mV. (d) Prepulse inactivation data for WT cells (▪, n = 6) and slo-2 mutants (•, n = 5). Curves were obtained as described in Materials and Methods and fitted with the sum of two Boltzmann functions (1 and 2). (e) Each of the two components was plotted separately for both WT and slo-2 cells. The parameter values for each component were comparable between WT and slo-2 cells, respectively: V_0.5(1) = -6.9 mV, k₍₁₎ = 8.5; V_0.5(2) = -30.9 mV, k₍₂₎ = 7.6 and V_0.5(1) = -15.6, k₍₁₎ = 6.33; V_0.5(2) = -39.3 mV, k₍₂₎ = 8.4.

Fig. 2.

Dissection of voltage-dependent components by RNAi. The slo-2 mutant was used to obtain these data so that there would be no chance of contamination from the SLO-2 current. (ai) Family of K⁺ currents obtained from a slo-2 mutant muscle cell showing only the voltage-dependent components. (aii) Family of K⁺ current traces for SHAL currents (slo-2 mutant cell treated with Shaker RNAi). (aiii) SHAKER currents (a slo-2 mutant cell treated with Shal RNAi). Cells were held at -70 mV and stepped from -70 to +60 mV in 10-mV increments. (b) G-V plots and prepulse inactivation curves for SHAL currents (○) and SHAKER currents (•). The activation parameter values were V_0.5a = 11.2 ± 1.5 mV, k_a = 14.1 ± 1.04 (n = 5); V_0.5a = 20.4 ± 2, k_a = 7.7 ± 1.1 (n = 3) for SHAL and SHAKER currents, respectively. SHAL currents inactivated with values of V_0.5i =-33.1 mV ± 1.2 and k_i = 8.3 ± 0.7 (n = 6), whereas SHAKER inactivation parameter values were V_0.5i =-6.95 ± 1.7, k_i = 5.8 ± 0.5 (n = 2). Internal concentrations of Cl^- and Ca²⁺ were, respectively, 120 mM and 10 nM.

Fig. 3.

SLO-2 is a major reserve component of the delayed outward current in C. elegans. Whole-cell currents are shown from body wall muscle cells in culture, demonstrating that SLO-2 is the major outward current in these cells. Note that the scale is changed from that in Fig. 1. so that the small amplitude of the voltage-dependent components (ai and aii) can be seen relative to the SLO-2 component (aiii). (ai) Current traces from a WT cell using low Ca²⁺ and Cl^- (10 nM and 4 mM, respectively). (aii) Current traces from a slo-2 (nf100) mutant cell using high Ca²⁺ and Cl^- internal solution (200 μM and 128 mM, respectively). (aiii) Current traces from a WT cell using high Ca²⁺ and Cl^- internal solution. (aiv) Current-voltage relationships of the currents shown in ai-aiii.(b) Whole-cell currents recorded in situ from body wall muscle of adult animals. (bi) Current-traces from a WT cell using 4 mM Cl^- internal solution. (bii) Current traces from a slo-2 nf100 mutant cell using 128 mM Cl^- internal solution. (biii) Current traces from a WT cell using 128 mM Cl^- internal solution. Free internal Ca²⁺ was 10 nM in these experiments. (biv) Comparison of current-voltage relationships of currents shown in bi-biii.

Fig. 4.

Separation of the voltage-dependent inactivating currents from the SLO-2 current in a WT cell. (ai) Current traces of test pulses at +50 mV. Top trace is before, and bottom trace is after a 1-s conditioning prepulse at +5 mV; the holding potential was -70 mV. (aii) The difference between the two currents in ai shows the inactivating component alone. (aiii) Current trace of a slo-2 nf100 mutant muscle cell in culture at +50 mV (-70 mV holding potential). For comparison, the current amplitude was scaled to the same amplitude as the trace shown in aii.

Fig. 5.

(a) Single-channel SLO-2 currents from an inside-out patch from a WT muscle cell in culture. The intracellular surface of the membrane was perfused with and concentrations as indicated while the (intracellular) membrane potential was held at +40 mv. Perfusion with 100 μM and 100 mM produced periods of high activity followed by periods of inactivity. Records shown are from periods of higher activity in each condition. (b) Analysis of open probability and plots of an all-points histogram for 20-s intervals. Each plot is shown directly below the current traces for each condition. A 20-s interval was chosen for analysis of each condition because it was much longer than the longest inactive state. An asterisk indicates the peak of the single-channel level in the all-points histograms. Similar channels were not seen in patches from slo-2 nf100 mutant cells (n = 7). (c) Voltage ramps (-80 to +80 mV) of an inside-out patch perfused with and concentrations as indicated. The duration of the ramp was 1,600 ms. The pipette (external) solution contained 120 mM K⁺-gluconate, 20 mM KOH, 2 mM MgCl₂, 4 mM Mg²⁺-gluconate₂, 5 mM Tris, 0.25 mM CaCl₂, 36 mM sucrose, 5 mM EGTA, and 4 mM Na₂ATP. The bath (internal) solutions contained 100 mM KCl or 100 mM K⁺-gluconate, 59 mM K⁺-gluconate, 10 mM Hepes, 1 mM KOH, and 0.1 mM Ca²⁺-gluconate₂, pH 7.2, with KOH. The Ca²⁺-free solutions contained 100 mM KCl or 100 mM K⁺-gluconate, 30 mM K⁺-gluconate, 10 mM Hepes, 30 mM KOH, and 11 mM EGTA, pH 7.2, with KOH.