SLO-2 potassium channel is an important regulator of neurotransmitter release in Caenorhabditis elegans

Abstract

Slo2 channels are prominent K(+) channels in mammalian neurons but their physiological functions are not well understood. Here we investigate physiological functions and regulation of the Caenorhabditis elegans homologue SLO-2 in motor neurons through electrophysiological analyses of wild-type and mutant worms. We find that SLO-2 is the primary K(+) channel conducting delayed outward current in cholinergic motor neurons, and one of two K(+) channels with this function in GABAergic motor neurons. Loss-of-function mutation of slo-2 increases the duration and charge transfer rate of spontaneous postsynaptic current bursts at the neuromuscular junction, which are physiological signals used by motor neurons to control muscle cells, without altering postsynaptic receptor sensitivity. SLO-2 activity in motor neurons depends on Ca(2+) entry through EGL-19, an L-type voltage-gated Ca(2+) channel (CaV1), but not on other proteins implicated in either Ca(2+) entry or intracellular Ca(2+) release. Thus, SLO-2 is functionally coupled with CaV1 and regulates neurotransmitter release.

Figure 1

SLO-2 is an important contributor to delayed outward current in cholinergic and GABAergic motor neurons. A. Diagram showing the locations of the recorded motor neurons VD5, VA5 and VB6 (based on published anatomical data). B. Sample whole-cell current traces in response to voltage steps (−60 to +70 mV at 10-mV intervals) from a holding voltage of −60 mV (left) and current-voltage relationships (right) from VA5, VB6 and VD5 of wild type (WT), slo-2(nf101), slo-2 rescue, shk-1(ok1581), slo-2(nf101);shk-1(ok1581), shl-1(ok1168), and slo-1(md1745). In VA5 and VB6, delayed outward current was greatly decreased in slo-2(nf101) compared with WT but not further decreased in the slo-2;shk-1 double mutant. In VD5, delayed outward current was greatly decreased in both slo-2(nf101) and shk-1(ok1581), and was essentially absent in the slo-2;shk-1 double mutant. Data are shown as mean ± SE. The asterisk (*) indicates a significant difference compared with WT whereas the pound sign (#) indicates a significant difference compared with the single mutants of slo-2 and shk-1 (p < 0.01, two-way mixed model ANOVA with Tukey’s post hoc tests). The recordings were performed with extracellular solution I and pipette solution I.

Figure 2

Loss-of-function mutation of slo-2 reduced the amplitude of a spontaneous up-state in motor neurons. A. Samples traces of spontaneous membrane voltage changes of VA5 from wild type (WT), slo-2(nf101), and slo-2(nf101) rescued by expressing wild-type SLO-2b under the control of Prab-3. B. Comparisons of the resting membrane potential (RP), and up-state (US) frequency, duration, amplitude, and potential. Both the averaged value of each experiment (filled circle) and the mean ± SE of the group (line and bar) are shown. The asterisk (*) indicates a statistically significant difference compared with WT (p < 0.05, one-way ANOVA with Tukey’s post hoc tests). The sample size (n) was 15 for WT, 12 for slo-2, and 8 for Rescue. Samples without obvious up-states (4 WT and 2 slo-2 mutant) were not included in the up-state quantifications. The recordings were performed with extracellular solution I and pipette solution I.

Figure 3

SLO-2 deficiency augmented PSC bursts recorded from body-wall muscle. A. Representative traces of spontaneous PSC. The horizontal gray line marks PSC bursts. B. Comparison of PSC burst properties. C. Comparisons of extra- and intra-burst PSC events. The sample sizes (n) were 17 for wild type (WT), 15 for slo-2(nf101), and 10 for slo-2(nf101) expressing Prab-3::SLO-2::GFP (Rescue). Data are shown as mean ± SE. The asterisk (*) indicates a statistically significant difference compared with WT (p < 0.05, one-way ANOVA with Tukey’s post hoc tests). The recordings were performed with extracellular solution I and pipette solution II.

Figure 4

Postsynaptic receptor sensitivities and ePSCs were normal in slo-2(nf101). A. Comparison of muscle cell response to pressure-ejected acetylcholine (ACh, 100 μM) or GABA (100 μM) between wild type (WT) and slo-2(nf101). B. Comparison of ePSC between WT and slo-2(nf101) in the presence of two different extracellular Ca²⁺ concentrations (5 mM and 0.5 mM). Data are shown as mean ± SE. No significant difference was detected (unpaired t-test). The holding voltage was −60 mV in all the experiments. The numbers inside the columns indicate the sample sizes (n). All the recordings were performed with pipette solution I. The recordings of ePSC at 0.5 mM [Ca²⁺]_o were performed with extracellular solution II whereas the remaining recordings were recorded with extracellular solution I.

Figure 5

Single-channel conductance of motor neuron SLO-2. A. Sample current traces showing SLO-2 single-channel activities in inside-out patches of wild type and the absence of such activities in slo-2(nf101). B. Sample SLO-2 single-channel current traces at various membrane voltages from inside-out patches, and the relationship between single-channel current amplitude and holding potential. Data are shown as mean ± SE. SLO-2 single-channel conductance (G) and sample size (n) are shown in the graphs. For display purpose, the sample traces were chosen from regions where only one channel was open. Arrows mark the baseline. The experiments were performed in symmetrical K⁺ (pipette solution III and bath solution as specified in Methods).

Figure 6

SLO-2 activity in motor neurons depends on cytosolic [Cl⁻] and entry of extracellular Ca²⁺. A. Whole-cell current traces and relationships between delayed outward current and voltage in representative motor neurons (VA5, VB6 and VD5) of wild type (WT), slo-2(nf101), WT with low [Cl⁻] in pipette solution (WT + low [Cl⁻]_i), and slo-2(nf101) with low [Cl⁻] in pipette solution (slo-2 + low [Cl⁻]_i) showing that reducing [Cl⁻]_i from the control level (128.5 mM) to a low level (15.3 mM) caused a great reduction of delayed outward current in WT but no change in slo-2(nf101). The asterisk (*) on the right side of a current-voltage relationship indicates a statistically significant (p < 0.01) difference compared with WT (two-way mixed model ANOVA with Tukey’s posthoc test). All the recordings were performed with extracellular solution I and pipette solution I except for the low [Cl⁻] experiments, in which pipette solution II was used instead. B. Effects of varying [Ca²⁺]_o on SLO-2 single channel activity in outside-out patches at a holding voltage of +30 mV. The open probability (NP_o) of SLO-2 was normalized to that of the first 5 mM [Ca²⁺]_o period. The asterisk (*) indicates a statistically significant difference compared with the first 5 mM [Ca²⁺]_o period (p < 0.01, one-way ANOVA with Tukey’s posthoc test, n = 5). The recordings were performed with pipette solution I and either extracellular solution I (5 mM Ca²⁺) or extracellular solution III (0 Ca²⁺). In both A and B, data are shown as mean ± SE.

Figure 7

Mutations of egl-19 altered delayed outward current of motor neurons in a SLO-2-dependent manner. Whole-cell delayed outward current were recorded from VA5, VB6 and VD5 of wild type (WT), egl-19(ad695) (gain-of-function), egl-19(n582) (hypomorph), slo-2(nf101), slo-2(nf101);egl-19(ad695); and slo-2(nf101);egl-19(n582). Compared with WT, egl-19(ad695) and egl-19(n582) augmented and inhibited delayed outward current, respectively, which were not observed in the presence of slo-2(nf101). Left. Sample current traces in response to voltage steps (−60 to +70 mV at 10-mV intervals) from a holding potential of −60 mV. Right. Current-voltage relationships. Data are shown as mean ± SE. The asterisk (*) indicates a statistically significant difference compared with WT (p < 0.05, two-way mixed model ANOVA and Tukey’s post hoc tests). The recordings were performed with extracellular solution I and pipette solution I.

Figure 8

Delayed outward current was normal in mutants of other Ca²⁺ channels. Delayed outward currents were unchanged in unc-2(e55), cca-1(ad1650), trp-1(ok323), clhm-1(ok3617), unc-68(r1162), and the double mutant nca-1(gk9);nca-2(gk5) compared with wild type (WT) (two-way mixed model ANOVA). Left. Sample current traces in response to voltage steps (−60 to +70 mV at 10-mV intervals) from a holding potential of −60 mV. Right. Current-voltage relationships. Data are shown as mean ± SE. The recordings were performed with extracellular solution I and pipette solution I.