recording-gap-junction-current-from-xenopus-oocytes

Recording Gap Junction Current from <em>Xenopus</em> Oocytes

Heterologous expression of connexins and innexins in Xenopus oocytes is a powerful approach for studying the biophysical properties of gap junctions ...

University of Connecticut School of Medicine

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and the BK channel are enriched at the presynaptic nerve terminal, where CaMKII associates with synaptic vesicles whereas the BK channel colocalizes with voltage-sensitive Ca(2+) channels in the plasma membrane. Mounting evidence suggests that these two proteins play important roles in controlling neurotransmitter release. Presynaptic BK channels primarily serve as a negative regulator of neurotransmitter release. In contrast, presynaptic CaMKII either enhances or inhibits neurotransmitter release and synaptic plasticity depending on experimental or physiological conditions and properties of specific synapses. The different functions of presynaptic CaMKII appear to be mediated by distinct downstream proteins, including the BK channel.

Regulation of synaptic transmission by presynaptic CaMKII and BK channels.

The neuromuscular junction (NMJ) of Caenorhabditis elegans has proved to be a very useful model synapse for investigating molecular mechanisms of synaptic transmission. Intriguingly, miniature postsynaptic currents (minis) at this synapse occur at an unusually high frequency (50-90 Hz in wild-type worms) and show large variation in quantal size (from <10 pA to >200 pA). It is important to understand the cellular and molecular bases for these properties of minis in order to interpret electrophysiological data from this synapse properly. Existing data suggest that several factors may contribute to the high frequency and quantal size variation, including 1) the establishment of multiple NMJs with each body-wall muscle cell, 2) diversity of postsynaptic receptors (two acetylcholine receptors and one GABA receptor), 3) association of one presynaptic site with several body-wall muscle cells, 4) effects of Ca(2+) at the presynaptic site, and 5) a possibly elevated (less negative) resting membrane potential in motoneurons. Neither the frequency nor the quantal size of minis is affected by electrical coupling of body-wall muscle cells. Furthermore, quantal size variation is not due to synchronized multivesicular release. Analyses of the C. elegans NMJ may lead to a better understanding of the mechanisms controlling the frequency and quantal size of minis of other synapses as well.

Origin of quantal size variation and high-frequency miniature postsynaptic currents at the Caenorhabditis elegans neuromuscular junction.

The sinusoidal locomotion of Caenorhabditis elegans requires synchronous activities of neighboring body wall muscle cells. However, it is unknown whether the synchrony results from muscle electrical coupling or neural inputs. We analyzed the effects of mutating gap junction proteins and blocking neuromuscular transmission on the synchrony of action potentials (APs) and Ca2+ transients among neighboring body wall muscle cells. In wild-type worms, the percentage of synchronous APs between two neighboring cells varied depending on the anatomical relationship and junctional conductance (Gj) between them, and Ca2+ transients were synchronous among neighboring muscle cells. Compared with the wild type, knock-out of the gap junction gene unc-9 resulted in greatly reduced coupling coefficient and asynchronous APs and Ca2+ transients. Inhibition of unc-9 expression specifically in muscle by RNAi also reduced the synchrony of APs and Ca2+ transients, whereas expression of wild-type UNC-9 specifically in muscle rescued the synchrony defect. Loss of the stomatin-like protein UNC-1, which is a regulator of UNC-9-based gap junctions, similarly impaired muscle synchrony as unc-9 mutant did. The blockade of muscle ionotropic acetylcholine receptors by (+)-tubocurarine decreased the frequencies of APs and Ca2+ transients, whereas blockade of muscle GABAA receptors by gabazine had opposite effects. However, both APs and Ca2+ transients remained synchronous after the application of (+)-tubocurarine and/or gabazine. These observations suggest that gap junctions in C. elegans body wall muscle cells are responsible for synchronizing muscle APs and Ca2+ transients.

Gap junctions synchronize action potentials and Ca2+ transients in Caenorhabditis elegans body wall muscle.

Pituitary tumors are frequently associated with mutations in the AIP gene and are sometimes associated with hypersecretion of growth hormone. It is unclear whether other factors besides an enlarged pituitary contribute to the hypersecretion. In a genetic screen for suppressors of reduced neurotransmitter release, we identified a mutation in Caenorhabditis elegans AIPR-1 (AIP-related-1), which causes profound increases in evoked and spontaneous neurotransmitter release, a high frequency of spontaneous calcium transients in motor neurons and an enlarged readily releasable pool of vesicles. Calcium bursts and hypersecretion are reversed by mutations in the ryanodine receptor but not in the voltage-gated calcium channel, indicating that these phenotypes are caused by a leaky ryanodine receptor. AIPR-1 is physically associated with the ryanodine receptor at synapses. Finally, the phenotypes in aipr-1 mutants can be rescued by presynaptic expression of mouse AIP, demonstrating that a conserved function of AIP proteins is to inhibit calcium release from ryanodine receptors.

AIP limits neurotransmitter release by inhibiting calcium bursts from the ryanodine receptor.

Rectifying electrical synapses (RESs) exist across animal species, but their rectification mechanism is largely unknown. We investigated why RESs between AVA premotor interneurons and A-type cholinergic motoneurons (A-MNs) in  escape circuit conduct junctional currents ( ) only in the antidromic direction. These RESs consist of UNC-7 innexin in AVA and UNC-9 innexin in A-MNs. UNC-7 has multiple isoforms differing in the length and sequence of the amino terminus. In a heterologous expression system, only one UNC-7 isoform, UNC-7b, can form heterotypic gap junctions (GJs) with UNC-9 that strongly favor   in the UNC-9 to UNC-7 direction. Knockout of  alone almost eliminated the  , whereas AVA-specific expression of UNC-7b substantially rescued the coupling defect of  mutant. Neutralizing charged residues in UNC-7b amino terminus abolished the rectification property of UNC-7b/UNC-9 GJs. Our results suggest that the rectification property results from electrostatic interactions between charged residues in UNC-7b amino terminus.

Molecular basis of junctional current rectification at an electrical synapse.

Melatonin (Mel) promotes sleep through G protein-coupled receptors. However, the downstream molecular target(s) is unknown. We identified the  BK channel SLO-1 as a molecular target of the Mel receptor PCDR-1-. Knockout of , , or  (a gene required for Mel synthesis) causes substantially increased neurotransmitter release and shortened sleep duration, and these effects are nonadditive in double knockouts. Exogenous Mel inhibits neurotransmitter release and promotes sleep in wild-type (WT) but not  and  mutants. In a heterologous expression system, Mel activates the human BK channel (hSlo1) in a membrane-delimited manner in the presence of the Mel receptor MT but not MT A peptide acting to release free Gβγ also activates hSlo1 in a MT-dependent and membrane-delimited manner, whereas a Gβλ inhibitor abolishes the stimulating effect of Mel. Our results suggest that Mel promotes sleep by activating the BK channel through a specific Mel receptor and Gβλ.

Melatonin promotes sleep by activating the BK channel in .

Proper threat-reward decision-making is critical to animal survival. Emerging evidence indicates that the motor system may participate in decision-making but the neural circuit and molecular bases for these functions are little known. We found in C. elegans that GABAergic motor neurons (D-MNs) bias toward the reward behavior in threat-reward decision-making by retrogradely inhibiting a pair of premotor command interneurons, AVA, that control cholinergic motor neurons in the avoidance neural circuit. This function of D-MNs is mediated by a specific ionotropic GABA receptor (UNC-49) in AVA, and depends on electrical coupling between the two AVA interneurons. Our results suggest that AVA are hub neurons where sensory inputs from threat and reward sensory modalities and motor information from D-MNs are integrated. This study demonstrates at single-neuron resolution how motor neurons may help shape threat-reward choice behaviors through interacting with other neurons.

Zhao-Wen Wang

Department of Neuroscience

University of Connecticut School of Medicine

Recording Gap Junction Current from Xenopus Oocytes