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

Liu P, Chen B, Wang ZW - Nat Commun (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.

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.

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SLO-2 activity in motor neurons depends on cytosolic[Cl−] and entry of extracellularCa2+. A. Whole-cell current traces and relationshipsbetween delayed outward current and voltage in representative motor neurons (VA5, VB6 andVD5) 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 nochange in slo-2(nf101). The asterisk (*) on the right side of acurrent-voltage relationship indicates a statistically significant (p< 0.01) difference compared with WT (two-way mixed model ANOVA withTukey’s posthoc test). All the recordings were performed with extracellularsolution I and pipette solution I except for the low[Cl−] experiments, in which pipette solution II wasused instead. B. Effects of varying[Ca2+]o on SLO-2 single channel activityin outside-out patches at a holding voltage of +30 mV. The open probability(NPo) 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 eitherextracellular solution I (5 mM Ca2+) or extracellular solution III (0Ca2+). In both A and B, data are shown asmean ± SE.
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Figure 6: SLO-2 activity in motor neurons depends on cytosolic[Cl−] and entry of extracellularCa2+. A. Whole-cell current traces and relationshipsbetween delayed outward current and voltage in representative motor neurons (VA5, VB6 andVD5) 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 nochange in slo-2(nf101). The asterisk (*) on the right side of acurrent-voltage relationship indicates a statistically significant (p< 0.01) difference compared with WT (two-way mixed model ANOVA withTukey’s posthoc test). All the recordings were performed with extracellularsolution I and pipette solution I except for the low[Cl−] experiments, in which pipette solution II wasused instead. B. Effects of varying[Ca2+]o on SLO-2 single channel activityin outside-out patches at a holding voltage of +30 mV. The open probability(NPo) 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 eitherextracellular solution I (5 mM Ca2+) or extracellular solution III (0Ca2+). In both A and B, data are shown asmean ± SE.

Mentions: Analyses with inside-out patches from Xenopus oocytes show thatSLO-2 has a mean single channel conductance of 110 pS in symmetricalK+, and is activated synergistically by Ca2+ andCl− on the intracellular side23. However, single-channel conductance of SLO-2 in neurons has not beenreported, the concentration of free Ca2+ required for significant SLO-2activity23 is much higher than whatcould possibly be reached in the bulk cytoplasm under physiological conditions, and thereported Cl− sensitivity of SLO-2 was rebutted by a recentstudy38. We therefore performedexperiments to assess the single-channel conductance of SLO-2 in motor neurons and todetermine whether SLO-2 in motor neurons is sensitive to Ca2+ andCl−. First, we recorded SLO-2 single channel activities in inside-outpatches held at a series of voltages in the presence of symmetrical K+solutions to determine SLO-2 single channel conductance. In wild type, each patchtypically showed activities of a few channels, which were apparently of the same type(Figure 5A). In contrast, similar single-channelopening events were never observed in slo-2(lf) (Figure 5A). We determined SLO-2 single channel conductance fromthe slope of a linear fit to SLO-2 single channel current amplitudes at the variousvoltages, and found it to be 117 ± 2 pS in VA5, 120 ± 2 pS in VB6 and 111± 2 pS in VD5 (Figure 5B). These values aresimilar to those of SLO-2 expressed in Xenopus oocytes23 and in cultured body-wall musclecells34. Next, we analyzed theeffect of intracellular Cl− on SLO-2 by reducing[Cl−] from 128.5 mM to 15.3 mM in the standardpipette solution. This treatment essentially abolished SLO-2 current in wild-type worms,as suggested by the similar outward current between WT with low[Cl−]i and slo-2(lf) withhigh [Cl−]i, and the lack of an effect ofthe low [Cl−]i on outward current inslo-2(lf) (Figure 6A). Finally, weanalyzed SLO-2 Ca2+ dependence. In recording the whole-cell current,the concentration of free Ca2+ in the pipette solution was ~50 nM,which was not expected to cause significant SLO-2 activation23. We hypothesized that extracellularCa2+ plays a role in SLO-2 activation by establishingCa2+ microdomains at the inner openings of Ca2+permeable channels, as is the case for mammalian Slo2 activation by extracellularNa+4, 5. We therefore examined the effects of varying[Ca2+]o between 5 mM and zero on SLO-2single channel activity in outside-out patches with little free Ca2+(~50 nM) in the pipette solution. Indeed, SLO-2 activity was high when[Ca2+]o was 5 mM but greatly decreasedafter [Ca2+]o was changed to zero (Figure 6B). Taken together, our observations suggest thatSLO-2 in motor neurons depends on both Cl− and Ca2+for activation, and that extracellular Ca2+ plays an important role inSLO-2 activation.


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

Liu P, Chen B, Wang ZW - Nat Commun (2014)

SLO-2 activity in motor neurons depends on cytosolic[Cl−] and entry of extracellularCa2+. A. Whole-cell current traces and relationshipsbetween delayed outward current and voltage in representative motor neurons (VA5, VB6 andVD5) 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 nochange in slo-2(nf101). The asterisk (*) on the right side of acurrent-voltage relationship indicates a statistically significant (p< 0.01) difference compared with WT (two-way mixed model ANOVA withTukey’s posthoc test). All the recordings were performed with extracellularsolution I and pipette solution I except for the low[Cl−] experiments, in which pipette solution II wasused instead. B. Effects of varying[Ca2+]o on SLO-2 single channel activityin outside-out patches at a holding voltage of +30 mV. The open probability(NPo) 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 eitherextracellular solution I (5 mM Ca2+) or extracellular solution III (0Ca2+). In both A and B, data are shown asmean ± SE.
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Figure 6: SLO-2 activity in motor neurons depends on cytosolic[Cl−] and entry of extracellularCa2+. A. Whole-cell current traces and relationshipsbetween delayed outward current and voltage in representative motor neurons (VA5, VB6 andVD5) 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 nochange in slo-2(nf101). The asterisk (*) on the right side of acurrent-voltage relationship indicates a statistically significant (p< 0.01) difference compared with WT (two-way mixed model ANOVA withTukey’s posthoc test). All the recordings were performed with extracellularsolution I and pipette solution I except for the low[Cl−] experiments, in which pipette solution II wasused instead. B. Effects of varying[Ca2+]o on SLO-2 single channel activityin outside-out patches at a holding voltage of +30 mV. The open probability(NPo) 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 eitherextracellular solution I (5 mM Ca2+) or extracellular solution III (0Ca2+). In both A and B, data are shown asmean ± SE.
Mentions: Analyses with inside-out patches from Xenopus oocytes show thatSLO-2 has a mean single channel conductance of 110 pS in symmetricalK+, and is activated synergistically by Ca2+ andCl− on the intracellular side23. However, single-channel conductance of SLO-2 in neurons has not beenreported, the concentration of free Ca2+ required for significant SLO-2activity23 is much higher than whatcould possibly be reached in the bulk cytoplasm under physiological conditions, and thereported Cl− sensitivity of SLO-2 was rebutted by a recentstudy38. We therefore performedexperiments to assess the single-channel conductance of SLO-2 in motor neurons and todetermine whether SLO-2 in motor neurons is sensitive to Ca2+ andCl−. First, we recorded SLO-2 single channel activities in inside-outpatches held at a series of voltages in the presence of symmetrical K+solutions to determine SLO-2 single channel conductance. In wild type, each patchtypically showed activities of a few channels, which were apparently of the same type(Figure 5A). In contrast, similar single-channelopening events were never observed in slo-2(lf) (Figure 5A). We determined SLO-2 single channel conductance fromthe slope of a linear fit to SLO-2 single channel current amplitudes at the variousvoltages, and found it to be 117 ± 2 pS in VA5, 120 ± 2 pS in VB6 and 111± 2 pS in VD5 (Figure 5B). These values aresimilar to those of SLO-2 expressed in Xenopus oocytes23 and in cultured body-wall musclecells34. Next, we analyzed theeffect of intracellular Cl− on SLO-2 by reducing[Cl−] from 128.5 mM to 15.3 mM in the standardpipette solution. This treatment essentially abolished SLO-2 current in wild-type worms,as suggested by the similar outward current between WT with low[Cl−]i and slo-2(lf) withhigh [Cl−]i, and the lack of an effect ofthe low [Cl−]i on outward current inslo-2(lf) (Figure 6A). Finally, weanalyzed SLO-2 Ca2+ dependence. In recording the whole-cell current,the concentration of free Ca2+ in the pipette solution was ~50 nM,which was not expected to cause significant SLO-2 activation23. We hypothesized that extracellularCa2+ plays a role in SLO-2 activation by establishingCa2+ microdomains at the inner openings of Ca2+permeable channels, as is the case for mammalian Slo2 activation by extracellularNa+4, 5. We therefore examined the effects of varying[Ca2+]o between 5 mM and zero on SLO-2single channel activity in outside-out patches with little free Ca2+(~50 nM) in the pipette solution. Indeed, SLO-2 activity was high when[Ca2+]o was 5 mM but greatly decreasedafter [Ca2+]o was changed to zero (Figure 6B). Taken together, our observations suggest thatSLO-2 in motor neurons depends on both Cl− and Ca2+for activation, and that extracellular Ca2+ plays an important role inSLO-2 activation.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.

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.

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