Voltage-induced calcium release in Caenorhabditis elegans body muscles.

Type 1 voltage-activated calcium channels (CaV1) in the plasma membrane trigger calcium release from the sarcoplasmic reticulum (SR) by two mechanisms. In voltage-induced calcium release (VICR), CaV1 voltage sensing domains are directly coupled to ryanodine receptors (RYRs), an SR calcium channel. In calcium-induced calcium release (CICR), calcium ions flowing through activated CaV1 channels bind and activate RYR channels. VICR is thought to occur exclusively in vertebrate skeletal muscle while CICR occurs in all other muscles (including all invertebrate muscles). Here, we use calcium-activated SLO-2 potassium channels to analyze CaV1-SR coupling in Caenorhabditis elegans body muscles. SLO-2 channels were activated by both VICR and external calcium. VICR-mediated SLO-2 activation requires two SR calcium channels (RYRs and IP3 Receptors), JPH-1/Junctophilin, a PDZ (PSD95, Dlg1, ZO-1 domain) binding domain (PBD) at EGL-19/CaV1's carboxy-terminus, and SHN-1/Shank (a scaffolding protein that binds EGL-19's PBD). Thus, VICR occurs in invertebrate muscles.

Post-synaptic GABAA receptors potentiate transmission by recruiting CaV2 channels to their inputs.

We describe a retrograde synaptic signal at the C. elegans GABAergic neuromuscular junction. At this synapse, GABA release is controlled by two voltage-activated calcium channels (UNC-2/CaV2 and EGL-19/CaV1), and muscle responses are mediated by a single GABA receptor (UNC-49/GABAA). Mutations inactivating UNC-49 or those preventing UNC-49 synaptic clustering cause retrograde defects in GABAergic motor neurons, whereby UNC-2/CaV2 levels at active zones, UNC-2 current, and pre-synaptic GABA release are decreased. Inactivating post-synaptic GABAA receptors has no effect on GABA neuron EGL-19/CaV1 levels nor on several other pre-synaptic markers. The effect of GABAA receptors on pre-synaptic strength is not a consequence of decreased GABA transmission and is input selective. Finally, pre-synaptic UNC-2/CaV2 levels are increased when post-synaptic GABAA receptors are increased but are unaffected by increased extra-synaptic receptors. Collectively, these results suggest that clustered post-synaptic GABAA receptors adjust the strength of their inputs by recruiting CaV2 to contacting active zones.

Shank promotes action potential repolarization by recruiting BK channels to calcium microdomains.

Mutations altering the scaffolding protein Shank are linked to several psychiatric disorders, and to synaptic and behavioral defects in mice. Among its many binding partners, Shank directly binds CaV1 voltage activated calcium channels. Here, we show that the Caenorhabditis elegans SHN-1/Shank promotes CaV1 coupling to calcium activated potassium channels. Mutations inactivating SHN-1, and those preventing SHN-1 binding to EGL-19/CaV1 all increase action potential durations in body muscles. Action potential repolarization is mediated by two classes of potassium channels: SHK-1/KCNA and SLO-1 and SLO-2 BK channels. BK channels are calcium-dependent, and their activation requires tight coupling to EGL-19/CaV1 channels. SHN-1's effects on AP duration are mediated by changes in BK channels. In shn-1 mutants, SLO-2 currents and channel clustering are significantly decreased in both body muscles and neurons. Finally, increased and decreased shn-1 gene copy number produce similar changes in AP width and SLO-2 current. Collectively, these results suggest that an important function of Shank is to promote microdomain coupling of BK with CaV1.

The industrial solvent 1,4-dioxane causes hyperalgesia by targeting capsaicin receptor TRPV1.

BACKGROUND: The synthetic chemical 1,4-dioxane is used as industrial solvent, food, and care product additive. 1,4-Dioxane has been noted to influence the nervous system in long-term animal experiments and in humans, but the molecular mechanisms underlying its effects on animals were not previously known. RESULTS: Here, we report that 1,4-dioxane potentiates the capsaicin-sensitive transient receptor potential (TRP) channel TRPV1, thereby causing hyperalgesia in mouse model. This effect was abolished by CRISPR/Cas9-mediated genetic deletion of TRPV1 in sensory neurons, but enhanced under inflammatory conditions. 1,4-Dioxane lowered the temperature threshold for TRPV1 thermal activation and potentiated the channel sensitivity to agonistic stimuli. 1,3-dioxane and tetrahydrofuran which are structurally related to 1,4-dioxane also potentiated TRPV1 activation. The residue M572 in the S4-S5 linker region of TRPV1 was found to be crucial for direct activation of the channel by 1,4-dioxane and its analogs. A single residue mutation M572V abrogated the 1,4-dioxane-evoked currents while largely preserving the capsaicin responses. Our results further demonstrate that this residue exerts a gating effect through hydrophobic interactions and support the existence of discrete domains for multimodal gating of TRPV1 channel. CONCLUSIONS: Our results suggest TRPV1 is a co-receptor for 1,4-dioxane and that this accounts for its ability to dysregulate body nociceptive sensation.

Directed Differentiation of Zebrafish Pluripotent Embryonic Cells to Functional Cardiomyocytes.

A cardiomyocyte differentiation in vitro system from zebrafish embryos remains to be established. Here, we have determined pluripotency window of zebrafish embryos by analyzing their gene-expression patterns of pluripotency factors together with markers of three germ layers, and have found that zebrafish undergoes a very narrow period of pluripotency maintenance from zygotic genome activation to a brief moment after oblong stage. Based on the pluripotency and a combination of appropriate conditions, we established a rapid and efficient method for cardiomyocyte generation in vitro from primary embryonic cells. The induced cardiomyocytes differentiated into functional and specific cardiomyocyte subtypes. Notably, these in vitro generated cardiomyocytes exhibited typical contractile kinetics and electrophysiological features. The system provides a new paradigm of cardiomyocyte differentiation from primary embryonic cells in zebrafish. The technology provides a new platform for the study of heart development and regeneration, in addition to drug discovery, disease modeling, and assessment of cardiotoxic agents.

Selective potentiation of 2-APB-induced activation of TRPV1-3 channels by acid.

Temperature-sensitive TRP channels are important for responses to pain and inflammation, to both of which tissue acidosis is a major contributing factor. However, except for TRPV1, acid-sensing by other ThermoTRP channels remains mysterious. We show here that unique among TRPV1-3 channels, TRPV3 is directly activated by protons from cytoplasmic side. This effect is very weak and involves key cytoplasmic residues L508, D512, S518, or A520. However, mutations of these residues did not affect a strong proton induced potentiation of TRPV3 currents elicited by the TRPV1-3 common agonist, 2-aminoethoxydiphenyl borate (2-APB), no matter if the ligand was applied from extracellular or cytoplasmic side. The acid potentiation was common among TRPV1-3 and only seen with 2-APB-related ligands. Using (1)H-nuclear magnetic resonance to examine the solution structures of 2-APB and its analogs, we observed striking structural differences of the boron-containing compounds at neutral/basic as compared to acidic pH, suggesting that a pH-dependent configuration switch of 2-APB-based drugs may underlie their functionality. Supporting this notion, protons also enhanced the inhibitory action of 2-APB on TRPM8. Collectively, our findings reveal novel insights into 2-APB action on TRP channels, which should facilitate the design of new drugs for these channels.

A novel donor splice-site mutation of major intrinsic protein gene associated with congenital cataract in a Chinese family.

PURPOSE: To identify the disease-causing gene in a Chinese family with autosomal dominant congenital cataract. METHODS: Clinical and ophthalmologic examinations were performed on all members of a Chinese family with congenital cataract. Nine genes associated with congenital cataract were screened using direct DNA sequencing. Mutations were confirmed using restriction fragment length polymorphism (RFLP) analysis. The mutated multi-intronic plasmid (MIP) minigene, which carries the disease-causing splice-site mutation, and the wild-type (WT) MIP minigene were constructed using the pcDNA3.1 expression vector. Wild-type and mutant MIP minigene constructs were transiently transfected into HeLa cells. After 48 h of incubation at 37 °C, total RNA isolation and reverse transcription (RT)-PCR analysis were performed, and PCR products were separated and confirmed with sequencing. RESULTS: Direct DNA sequence analysis identified a novel splice-site mutation in intron 3 (c.606+1 G>A) of the MIP gene. To investigate the manner in which the splice donor mutation could affect mRNA splicing, WT and mutant MIP minigenes were inserted in the pcDNA3.1 (+) vector. Constructs were transfected into HeLa cells. RT-PCR analysis showed that the donor splice site mutation led to deletion of exon 3 in the mRNA encoded by the MIP gene. CONCLUSIONS: The present study identified a novel donor splice-site mutation (c.606+1G>A) in the MIP gene in a Chinese family with congenital cataract. In vitro RT-PCR analysis showed that this splice-site mutation resulted in the deletion of exon 3 from mRNA encoded by the MIP gene. This is the first report to show that donor splice-site mutation in MIP genes can cause autosomal dominant congenital cataract.

Gain-of-function mutations in SCN11A cause familial episodic pain.

Many ion channel genes have been associated with human genetic pain disorders. Here we report two large Chinese families with autosomal-dominant episodic pain. We performed a genome-wide linkage scan with microsatellite markers after excluding mutations in three known genes (SCN9A, SCN10A, and TRPA1) that cause similar pain syndrome to our findings, and we mapped the genetic locus to a 7.81 Mb region on chromosome 3p22.3-p21.32. By using whole-exome sequencing followed by conventional Sanger sequencing, we identified two missense mutations in the gene encoding voltage-gated sodium channel Nav1.9 (SCN11A): c.673C>T (p.Arg225Cys) and c.2423C>G (p.Ala808Gly) (one in each family). Each mutation showed a perfect cosegregation with the pain phenotype in the corresponding family, and neither of them was detected in 1,021 normal individuals. Both missense mutations were predicted to change a highly conserved amino acid residue of the human Nav1.9 channel. We expressed the two SCN11A mutants in mouse dorsal root ganglion (DRG) neurons and showed that both mutations enhanced the channel's electrical activities and induced hyperexcitablity of DRG neurons. Taken together, our results suggest that gain-of-function mutations in SCN11A can be causative of an autosomal-dominant episodic pain disorder.