Characterization of K(+) currents using an in situ patch clamp technique in body wall muscle cells from Caenorhabditis elegans

Abstract

The properties of K(+) channels in body wall muscle cells acutely dissected from the nematode Caenorhabditis elegans were investigated at the macroscopic and unitary level using an in situ patch clamp technique. In the whole-cell configuration, depolarizations to potentials positive to -40 mV gave rise to outward currents resulting from the activation of two kinetically distinct voltage-dependent K(+) currents: a fast activating and inactivating 4-aminopyridine-sensitive component and a slowly activating and maintained tetraethylammonium-sensitive component. In cell-attached patches, voltage-dependent K(+) channels, with unitary conductances of 34 and 80 pS in the presence of 5 and 140 mM external K(+), respectively, activated at membrane potentials positive to -40 mV. Excision revealed that these channels corresponded to Ca(2+)-activated K(+) channels exhibiting an unusual sensitivity to internal Cl(-) and whose activity progressively decreased in inside-out conditions. After complete run-down of these channels, one third of inside-out patches displayed activity of another Ca(2+)-activated K(+) channel of smaller unitary conductance (6 pS at 0 mV in the presence of 5 mM external K(+)). In providing a detailed description of native K(+) currents in body wall muscle cells of C. elegans, this work lays the basis for further comparisons with mutants to assess the function of K(+) channels in this model organism that is highly amenable to molecular and classical genetics.

Figure 1. Whole-cell currents elicited by positive and negative voltage pulses

In A, whole-cell outward currents were elicited by applying voltage pulses of 200 ms duration in 10 mV increments from a holding potential of −70 mV. The two sets of current traces were obtained from two different cells and are representative of two typical outward current waveforms. In B, from a holding potential of −80 mV, a two-pulse protocol was applied: depolarizing pulses of 10 mV positive increments followed by hyperpolarizing pulses of 10 mV negative increments, as illustrated by the voltage protocol below the current traces.

Figure 2. Tail currents and tail current-voltage relationships of the depolarization-activated outward currents

A, after depolarization to +20 mV from a holding potential of −70 mV, tail currents were evoked during membrane hyperpolarization to potentials between 0 and −140 mV (left panel) or between −20 and −130 mV (right panel). Hyperpolarizing voltage steps were presented 5 ms (left panel) or 200 ms (right panel) after the onset of the depolarization to +20 mV. The voltage protocol is illustrated below the current records. B, mean current-voltage relationships were established for tail currents recorded after a short (left panel) or a long (right panel) depolarizing pulse. For tail currents recorded after a short depolarizing pulse, measurements were only performed above −60 mV because of the low resolution of the rapidly deactivating inward current tails at hyperpolarized potentials.

Figure 3. Pharmacological separation of the outward currents and current-voltage relationships

A, from a holding potential of −70 mV, voltage pulses of 200 ms duration in 10 mV increments were applied from −60 to +40 mV in control conditions (left panel), in the presence of 3 mm 4-AP (middle panel) and in the presence of 3 mm 4-AP plus 20 mm TEA (right panel). B, the 4-AP-sensitive component (left panel) was obtained by subtracting records in the presence of 4-AP from those in control conditions. The TEA-sensitive component was obtained by subtracting records in the presence of both drugs from those in the presence of 4-AP alone (right panel). C, current-voltage relationships for the control outward currents (▪), the 4-AP-sensitive component (▴), the TEA-sensitive component (•) and the 4-AP- and TEA-insensitive component (♦). In each case, measurements were performed 5 ms (left panel) or 200 ms (right panel) after the onset of depolarization. For control outward currents, the curves were fitted using eqn (1) with values for V_rev of −47.3 mV (left panel) and −69.7 mV (right panel).

Figure 4. Single-channel currents recorded in cell-attached patches

A, single-channel currents were recorded in cell-attached configuration at different membrane potentials indicated next to each current trace in the presence of Na⁺-rich solution in the pipette and K⁺-rich solution in the bath. B, mean current-voltage relationships were obtained in the presence of 5 mm K⁺ (•) or 140 mm K⁺ (○) in the pipette. Slopes indicate conductances of 34 and 80 pS in the presence of 5 and 140 mm K⁺, respectively. C, mean relationship between NP_o and membrane potential obtained in the presence of Na⁺-rich solution in the pipette. In each patch, values of NP_o were normalized to the value obtained at +50 mV.

Figure 5. Voltage- and Cl⁻-sensitive Ca²⁺-activated K⁺ currents in inside-out patches

In A, left panel, the arrow indicates excision of a cell-attached patch held at 0 mV in K⁺-rich solution with 0.1 mm CaCl₂. Na⁺-rich solution was present in the pipette. The right panel illustrates channel activity in the same patch 45 s after excision; bars indicate the periods during which solution containing EGTA and no added Ca²⁺ (0 Ca) and solution in which [Cl⁻] was reduced from 150 to 10 mm (10 Cl) were applied to the internal face of the patch. In B, traces correspond to segments of single-channel currents recorded in an inside-out patch at the different membrane potentials indicated next to each current trace. In C, the upper panel shows the current-voltage relationship. The slope indicates a conductance of 33.9 pS. The lower panel shows the relationship between NP_o and membrane potential; in each patch, values of NP_o were normalized to the value obtained at +50 mV. The curve was drawn by eye.

Figure 6. Small conductance Ca²⁺-activated K⁺ channels in inside-out patches

In A, channel activity was recorded at +30 mV in the presence of K⁺-rich solution with 0.1 mm CaCl₂ at the internal face of the patch and Na⁺-rich solution in the pipette, 5.5 min after excision. Bars indicate the period during which 0 Ca and 10 Cl solutions (see legend to Fig. 5) were applied to the internal face of the patch. B, single-channel currents were recorded at the different membrane potentials indicated next to each current trace 3 min after excision of the patch. C, current-voltage relationship. The curve was drawn by eye.