Bojun Chen

Presynaptic Ryanodine Receptors Are Required for Normal Quantal Size at the Caenorhabditis elegans Neuromuscular Junction

Analyses of the effect of ryanodine in vertebrate brain slices have led to the conclusion that presynaptic ryanodine receptors (RYRs) may have several functions in synaptic release, including causing large-amplitude miniature postsynaptic currents (mPSCs) by promoting concerted multivesicular release. However, the role of RYRs in synaptic release is controversial. To better understand the role of RYRs in synaptic release, we analyzed the effect of RYR mutation on mPSCs and evoked postsynaptic currents (ePSCs) at the Caenorhabditis elegans neuromuscular junction (NMJ). Amplitudes of mPSCs varied greatly at the C. elegans NMJ. Loss-of-function mutations of the RYR gene unc-68 (uncoordinated 68) essentially abolished large-amplitude mPSCs. The amplitude of ePSCs was also greatly suppressed. These defects were completely rescued by expressing wild-type UNC-68 specifically in neurons but not in muscle cells, suggesting that RYRs acted presynaptically. A combination of removing extracellular Ca2+ and UNC-68 function eliminated mPSCs, suggesting that influx and RYR-mediated release are likely the exclusive sources of Ca2+ for synaptic release. Large-amplitude mPSCs did not appear to be caused by multivesicular release, as has been suggested to occur at vertebrate central synapses, because the rise time of mPSCs was constant regardless of the amplitude but distinctive from that of ePSCs, and because large-amplitude mPSCs persisted under conditions that inhibit synchronized synaptic release, including elimination of extracellular Ca2+, and mutations of syntaxin and SNAP25 (soluble N-ethylmaleimide-sensitive factor attachment protein 25). These observations suggest that RYRs are essential to normal quantal size and are potential regulators of quantal size.

Presynaptic Ca2+/Calmodulin-Dependent Protein Kinase II Modulates Neurotransmitter Release by Activating BK Channels at Caenorhabditis elegans Neuromuscular Junction

Although Ca2+/calmodulin-dependent protein kinase II (CaMKII) is enriched at the presynaptic nerve terminal, its role in neurotransmitter release is poorly defined. We assessed the function of presynaptic CaMKII in neurotransmitter release and tested the hypothesis that BK channel is a mediator of presynaptic CaMKII function by analyzing miniature and evoked postsynaptic currents at the Caenorhabditis elegans neuromuscular junction. Both loss-of-function (lf) and gain-of-function (gf) of unc-43, the gene encoding CaMKII, inhibited neurotransmitter release. The inhibitory effect of unc-43(gf) was reversed by mutation or blockade of the BK channel SLO-1. SLO-1 expressed in Xenopus oocytes could be activated by recombinant rat α-CaMKII, and this effect of CaMKII was abolished by mutating a threonine residue (T425) at a consensus CaMKII phosphorylation site in the first RCK (regulator of conductance for K+) domain of the channel. Expression of slo-1(T425A) in neurons antagonized the inhibitory effect of unc-43(gf) on neurotransmitter release as slo-1(lf) did. The inhibitory effect of unc-43(gf) was not reversed by unc-103(lf), dgk-1(lf), or eat-16(lf), which reportedly suppress behavioral phenotypes of unc-43(gf). These observations suggest that presynaptic CaMKII is a bidirectional modulator of neurotransmitter release, presumably by phosphorylating different molecular targets, and that its negative modulatory effect on the release is mainly mediated by SLO-1 activation.

Low conductance gap junctions mediate specific electrical coupling in body-wall muscle cells of Caenorhabditis elegans.

Invertebrate innexins and their mammalian homologues, the pannexins, are gap junction proteins. Although a large number of such proteins have been identified, few of the gap junctions that they form have been characterized to provide combined information of biophysical properties, coupling pattern, and molecular compositions. We adapted the dual whole cell voltage clamp technique to in situ analysis of electrical coupling in Caenorhabditis elegans body-wall muscle. We found that body-wall muscle cells were electrically coupled in a highly organized and specific pattern. The coupling was characterized by small (350 pS or less) junctional conductance (Gj), which showed a bell-shaped relationship with junctional potential (Vj) but was independent of membrane potential (Vm). Injection of currents comparable to the junctional current (Ij) into body-wall muscle cells caused significant depolarization, suggesting important functional relevance. The innexin UNC-9 appeared to be a key component of the gap junctions. Both Myc- and green fluorescent protein-tagged UNC-9 was localized to muscle intercellular junctions. Gj was greatly inhibited in unc-9(fc16), a putative null mutant. Specific inhibition of UNC-9 function in muscle cells reduced locomotion velocity. Despite UNC-9 expression in both motor neurons and body-wall muscle cells, analyses of miniature and evoked postsynaptic currents in the unc-9 mutant showed normal neuromuscular transmission. These analyses provide a relatively detailed description of innexin-based gap junctions in a native tissue and suggest that innexin-based small conductance gap junctions can play an important role in processes such as locomotion.

UNC-1 regulates gap junctions important to locomotion in C. elegans

In C. elegans, loss-of-function (lf) mutations of the stomatin-like protein (SLP) UNC-1 and the innexin UNC-9 inhibit locomotion [1, 2] and modulate sensitivity to volatile anesthetics [3, 4]. It was unknown why unc-1(lf) and unc-9(lf) mutants have similar phenotypes. We tested the hypothesis that UNC-1 is a regulator of gap junctions formed by UNC-9. Analyses of junctional currents between body-wall muscle cells showed that electrical coupling was inhibited to a similar degree in unc-1(lf), unc-9(lf), and unc-1(lf);unc-9(lf) double mutants, suggesting that UNC-1 and UNC-9 function together. Expression of Punc-1::DsRED2 and Punc-9::GFP transcriptional fusions suggests that unc-1 and unc-9 are coexpressed in neurons and body-wall muscle cells. Immunohistochemistry showed that UNC-1 and UNC-9 colocalized at intercellular junctions and that unc-1(lf) did not alter UNC-9 expression or subcellular localization. Bimolecular fluorescence complementation (BiFC) assays suggest that UNC-1 and UNC-9 are physically very close at intercellular junctions. Targeted rescue experiments suggest that UNC-9 and UNC-1 function predominantly in neurons to control locomotion. Thus, in addition to the recently reported function of regulating mechanosensitive ion channels [5, 6], SLPs might have a novel function of regulating gap junctions.

Genetic dissection of ion currents underlying all-or-none action potentials in C. elegans body-wall muscle cells

Mammalian skeletal muscle contractions are initiated by all‐or‐none action potentials (APs) triggered by motoneurons. In C. elegans locomotory muscle, however, the characteristics of APs, the underlying ion channels, and their role in Ca2+ release are poorly understood. Here we show that C. elegans locomotory muscle fires spontaneous, all‐or‐none APs, which appear to be modulated by motoneuron activity. The upstroke of muscle APs requires Ca2+ entry through a voltage‐gated Ca2+ channel (EGL‐19), whereas the downstroke of the APs relies on a voltage‐gated potassium channel as well as a Ca2+‐ and Cl−‐activated potassium channel. AP‐elicited elevations of intracellular Ca2+ concentration require both EGL‐19 in the plasma membrane and the ryanodine receptor in the sarcoplasmic reticulum membrane. The discovery of all‐or‐none action potentials in C. elegans body‐wall muscle brings the physiology of C. elegans much closer to that of other metazoans, and strengthens its utility as a model organism.

A Novel Auxiliary Subunit Critical to BK Channel Function in Caenorhabditis elegans

The BK channel is a Ca2+- and voltage-gated potassium channel with many important physiological functions. To identify proteins important to its function in vivo, we screened for Caenorhabditis elegans mutants that suppressed a lethargic phenotype caused by expressing a gain-of-function (gf) isoform of the BK channel α-subunit SLO-1. BKIP-1 (for BK channel interacting protein), a small peptide with no significant homology to any previously characterized molecules, was thus identified. BKIP-1 and SLO-1 showed similar expression and subcellular localization patterns and appeared to interact physically through discrete domains. bkip-1 loss-of-function (lf) mutants phenocopied slo-1(lf) mutants in behavior and synaptic transmission and suppressed the lethargy, egg-laying defect, and deficient neurotransmitter release caused by SLO-1(gf). In heterologous expression systems, BKIP-1 decreased the activation rate and shifted the conductance–voltage relationship of SLO-1 in a Ca2+-dependent manner and increased SLO-1 surface expression. Thus, BKIP-1 is a novel auxiliary subunit critical to SLO-1 function in vivo.

α‐Catulin CTN‐1 is required for BK channel subcellular localization in C. elegans body‐wall muscle cells

The BK channel, a voltage‐ and Ca2+‐gated large‐conductance potassium channel with many important functions, is often localized at specific subcellular domains. Although proper subcellular localization is likely a prerequisite for the channel to perform its physiological functions, little is known about the molecular basis of localization. Here, we show that CTN‐1, a homologue of mammalian α‐catulin, is required for subcellular localization of SLO‐1, the Caenorhabditis elegans BK channel α‐subunit, in body‐wall muscle cells. CTN‐1 was identified in a genetic screen for mutants that suppressed a lethargic phenotype caused by expressing a gain‐of‐function (gf) isoform of SLO‐1. In body‐wall muscle cells, CTN‐1 coclusters with SLO‐1 at regions of dense bodies, which are Z‐disk analogs of mammalian skeletal muscle. In ctn‐1 loss‐of‐function (lf) mutants, SLO‐1 was mislocalized in body‐wall muscle but its transcription and protein level were unchanged. Targeted rescue of ctn‐1(lf) in muscle was sufficient to reinstate the lethargic phenotype in slo‐1(gf);ctn‐1(lf). These results suggest that CTN‐1 plays an important role in BK channel function by mediating channel subcellular localization.

Postsynaptic current bursts instruct action potential firing at a graded synapse

Nematode neurons generally produce graded potentials instead of action potentials (APs). It is unclear how the graded potentials control postsynaptic cells under physiological conditions. Here we show that postsynaptic currents (PSCs) frequently occur in bursts at the neuromuscular junction of C. elegans. Cholinergic bursts concur with facilitated AP firing, elevated cytosolic [Ca2+], and contraction of the muscle whereas GABA ergic bursts suppress AP firing. The bursts, distinct from artificially evoked responses, are characterized by a persistent current (the primary component of burst-associated charge transfer)and increased frequency and mean amplitude of PSC events. The persistent current of cholinergic PSC bursts is mostly mediated by levamisole-sensitive acetylcholine receptors, which correlates well with locomotory phenotypes of receptor mutants. Eliminating command interneurons abolishes the bursts whereas mutating SLO-1 K+ channel, a potent presynaptic inhibitor of exocytosis, greatly increases the mean burst duration. These observations suggest that motoneurons control muscle by producing PSC bursts.

Dystrobrevin controls neurotransmitter release and muscle Ca(2+) transients by localizing BK channels in Caenorhabditis elegans.

Dystrobrevin is a major component of a dystrophin-associated protein complex. It is widely expressed in mammalian tissues, including the nervous system, in which it is localized to the presynaptic nerve terminal with unknown function. In a genetic screen for suppressors of a lethargic phenotype caused by a gain-of-function isoform of SLO-1 in Caenorhabditis elegans, we isolated multiple loss-of-function (lf) mutants of the dystrobrevin gene dyb-1. dyb-1(lf) phenocopied slo-1(lf), causing increased neurotransmitter release at the neuromuscular junction, increased frequency of Ca2+ transients in body-wall muscle, and abnormal locomotion behavior. Neuron- and muscle-specific rescue experiments suggest that DYB-1 is required for SLO-1 function in both neurons and muscle cells. DYB-1 colocalized with SLO-1 at presynaptic sites in neurons and dense body regions in muscle cells, and dyb-1(lf) caused SLO-1 mislocalization in both types of cells without altering SLO-1 protein level. The neuronal phenotypes of dyb-1(lf) were partially rescued by mouse α-dystrobrevin-1. These observations revealed novel functions of the BK channel in regulating muscle Ca2+ transients and of dystrobrevin in controlling neurotransmitter release and muscle Ca2+ transients by localizing the BK channel.

Stomatin inhibits pannexin-1-mediated whole-cell currents by interacting with its carboxyl terminal.

The pannexin-1 (Panx1) channel (often referred to as the Panx1 hemichannel) is a large-conductance channel in the plasma membrane of many mammalian cells. While opening of the channel is potentially detrimental to the cell, little is known about how it is regulated under physiological conditions. Here we show that stomatin inhibited Panx1 channel activity. In transfected HEK-293 cells, stomatin reduced Panx1-mediated whole-cell currents without altering either the total or membrane surface Panx1 protein expression. Stomatin coimmunoprecipitated with full-length Panx1 as well as a Panx1 fragment containing the fourth membrane-spanning domain and the cytosolic carboxyl terminal. The inhibitory effect of stomatin on Panx1-mediated whole-cell currents was abolished by truncating Panx1 at a site in the cytosolic carboxyl terminal. In primary culture of mouse astrocytes, inhibition of endogenous stomatin expression by small interfering RNA enhanced Panx1-mediated outward whole-cell currents. These observations suggest that stomatin may play important roles in astrocytes and other cells by interacting with Panx1 carboxyl terminal to limit channel opening.