Cx36 makes channels coupling human pancreatic beta-cells, and correlates with insulin expression.

Previous studies have documented that the insulin-producing beta-cells of laboratory rodents are coupled by gap junction channels made solely of the connexin36 (Cx36) protein, and have shown that loss of this protein desynchronizes beta-cells, leading to secretory defects reminiscent of those observed in type 2 diabetes. Since human islets differ in several respects from those of laboratory rodents, we have now screened human pancreas, and islets isolated thereof, for expression of a variety of connexin genes, tested whether the cognate proteins form functional channels for islet cell exchanges, and assessed whether this expression changes with beta-cell function in islets of control and type 2 diabetics. Here, we show that (i) different connexin isoforms are differentially distributed in the exocrine and endocrine parts of the human pancreas; (ii) human islets express at the transcript level different connexin isoforms; (iii) the membrane of beta-cells harbors detectable levels of gap junctions made of Cx36; (iv) this protein is concentrated in lipid raft domains of the beta-cell membrane where it forms gap junctions; (v) Cx36 channels allow for the preferential exchange of cationic molecules between human beta-cells; (vi) the levels of Cx36 mRNA correlated with the expression of the insulin gene in the islets of both control and type 2 diabetics. The data show that Cx36 is a native protein of human pancreatic islets, which mediates the coupling of the insulin-producing beta-cells, and contributes to control beta-cell function by modulating gene expression.

Alpha7 neuronal nicotinic acetylcholine receptors are negatively regulated by tyrosine phosphorylation and Src-family kinases.

Nicotine, a component of tobacco, is highly addictive but possesses beneficial properties such as cognitive improvements and memory maintenance. Involved in these processes is the neuronal nicotinic acetylcholine receptor (nAChR) alpha7, whose activation triggers depolarization, intracellular signaling cascades, and synaptic plasticity underlying addiction and cognition. It is therefore important to investigate intracellular mechanisms by which a cell regulates alpha7 nAChR activity. We have examined the role of phosphorylation by combining molecular biology, biochemistry, and electrophysiology in SH-SY5Y neuroblastoma cells, Xenopus oocytes, rat hippocampal interneurons, and neurons from the supraoptic nucleus, and we found tyrosine phosphorylation of alpha7 nAChRs. Tyrosine kinase inhibition by genistein decreased alpha7 nAChR phosphorylation but strongly increased acetylcholine-evoked currents, whereas tyrosine phosphatase inhibition by pervanadate produced opposite effects. Src-family kinases (SFKs) directly interacted with the cytoplasmic loop of alpha7 nAChRs and phosphorylated the receptors at the plasma membrane. SFK inhibition by PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] or SU6656 (2,3-dihydro-N,N-dimethyl-2-oxo-3-[(4,5,6,7-tetrahydro-1H-indol-2-yl)methylene]-1H-indole-5-sulfonamide) increased alpha7 nAChR-mediated responses, whereas expression of active Src reduced alpha7 nAChR activity. Mutant alpha7 nAChRs lacking cytoplasmic loop tyrosine residues because of alanine replacement of Tyr-386 and Tyr-442 were more active than wild-type receptors and insensitive to kinase or phosphatase inhibition. Because the amount of surface alpha7 receptors was not affected by kinase or phosphatase inhibitors, these data show that functional properties of alpha7 nAChRs depend on the tyrosine phosphorylation status of the receptor and are the result of a balance between SFKs and tyrosine phosphatases. These findings reveal novel regulatory mechanisms that may help to understand nicotinic receptor-dependent plasticity, addiction, and pathology.

Involvement of gap junctional communication in secretion.

Glands were the first type of tissues in which the permissive role of gap junctions in the cell-to-cell transfer of membrane-impermeant molecules was shown. During the 40 years that have followed this seminal finding, gap junctions have been documented in all types of multicellular secretory systems, whether of the exocrine, endocrine or pheromonal nature. Also, compelling evidence now indicates that gap junction-mediated coupling, and/or the connexin proteins per se, play significant regulatory roles in various aspects of gland functions, ranging from the biosynthesis, storage and release of a variety of secretory products, to the control of the growth and differentiation of secretory cells, and to the regulation of gland morphogenesis. This review summarizes this evidence in the light of recent reports.

Connexin36 and pancreatic beta-cell functions.

Most cell types are functionally coupled by connexin (Cx) channels, i.e. exchange cytoplasmic ions and small metabolites through gap junction domains of their membrane. This form of direct cell-to-cell communication occurs in all existing animals, whatever their position in the phylogenetic scale, and up to humans. Pancreatic beta-cells are no exception, and normally cross-talk with their neighbors via channels made of Cx36. These exchanges importantly contribute to coordinate and synchronize the function of individual cells within pancreatic islets, particularly in the context of glucose-induced insulin secretion. Compelling evidence now indicates that Cx36-mediated coupling, and/or the Cx36 protein per se, play significant regulatory roles in various beta-cell functions, ranging from the biosynthesis, storage and release of insulin. Recent preliminary data further suggest that the protein may also be implicated in the balance of beta-cell growth versus necrosis and apoptosis, and in the regulated expression of specific genes. Here, we review this evidence, discuss the possible involvement of Cx36 in the pathophysiology of diabetes, and evaluate the relevance of this connexin in the therapeutic approaches to the disease.

The role of nicotinic acetylcholine receptors in the mechanisms of anesthesia.

Nicotinic acetylcholine receptors are members of the ligand-gated ion channel superfamily, that includes also gamma-amino-butiric-acid(A), glycine, and 5-hydroxytryptamine(3) receptors. Functional nicotinic acetylcholine receptors result from the association of five subunits each contributing to the pore lining. The major neuronal nicotinic acetylcholine receptors are heterologous pentamers of alpha4beta2 subunits (brain), or alpha3beta4 subunits (autonomic ganglia). Another class of neuronal receptors that are found both in the central and peripheral nervous system is the homomeric alpha7 receptor. The muscle receptor subtypes comprise of alphabetadeltagamma (embryonal) or alphabetadeltaepsilon (adult) subunits. Although nicotinic acetylcholine receptors are not directly involved in the hypnotic component of anesthesia, it is possible that modulation of central nicotinic transmission by volatile agents contributes to analgesia. The main effect of anesthetic agents on nicotinic acetylcholine receptors is inhibitory. Volatile anesthetics and ketamine are the most potent inhibitors both at alpha4beta2 and alpha3beta4 receptors with clinically relevant IC(50) values. Neuronal nicotinic acetylcholine receptors are more sensitive to anesthetics than their muscle counterparts, with the exception of the alpha7 receptor. Several intravenous anesthetics such as barbiturates, etomidate, and propofol exert also an inhibitory effect on the nicotinic acetylcholine receptors, but only at concentrations higher than those necessary for anesthesia. Usual clinical concentrations of curare cause competitive inhibition of muscle nicotinic acetylcholine receptors while higher concentrations may induce open channel blockade. Neuronal nAChRs like alpha4beta2 and alpha3beta4 are inhibited by atracurium, a curare derivative, but at low concentrations the alpha4beta2 receptor is activated. Inhibition of sympathetic transmission by clinically relevant concentrations of some anesthetic agents is probably one of the factors involved in arterial hypotension during anesthesia.

A novel human nicotinic receptor subunit, alpha10, that confers functionality to the alpha9-subunit.

We present herein the cloning of the human nicotinic acetylcholine receptor alpha9-ortholog and the identification of a new alpha-like subunit (alpha10) that shares 58% identity with alpha9. Whereas alpha10 fails to produce functional receptors alone, it promoted robust acetylcholine-evoked currents when coinjected with alpha9. The presence of alpha10 modifies the physiological and pharmacological properties of the alpha9 receptor indicating that the two subunits coassemble in a single functional receptor. Fusing the N-terminal domain of alpha9 with the rest of the alpha10-cDNA yielded a functional alpha9:alpha10-chimera that displays the acetylcholine binding properties of alpha9 and ionic pore characteristics of alpha10-containing receptors. In addition, alpha9- and alpha10-subunit mRNAs show limited similar tissue distribution patterns and are expressed in cochlea, pituitary gland, and keratinocytes. These data suggest that, in vivo, alpha9-containing receptors coassemble with alpha10-subunit.

Candoxin, a novel toxin from Bungarus candidus, is a reversible antagonist of muscle (alphabetagammadelta ) but a poorly reversible antagonist of neuronal alpha 7 nicotinic acetylcholine receptors.

In contrast to most short and long chain curaremimetic neurotoxins that produce virtually irreversible neuromuscular blockade in isolated nerve-muscle preparations, candoxin, a novel three-finger toxin from the Malayan krait Bungarus candidus, produced postjunctional neuromuscular blockade that was readily and completely reversible. Nanomolar concentrations of candoxin (IC(50) = approximately 10 nm) also blocked acetylcholine-evoked currents in oocyte-expressed rat muscle (alphabetagammadelta) nicotinic acetylcholine receptors in a reversible manner. In contrast, it produced a poorly reversible block (IC(50) = approximately 50 nm) of rat neuronal alpha7 receptors, clearly showing diverse functional profiles for the two nicotinic receptor subsets. Interestingly, candoxin lacks the helix-like segment cyclized by the fifth disulfide bridge at the tip of the middle loop of long chain neurotoxins, reported to be critical for binding to alpha7 receptors. However, its solution NMR structure showed the presence of some functionally invariant residues involved in the interaction of both short and long chain neurotoxins to muscle (alphabetagammadelta) and long chain neurotoxins to alpha7 receptors. Candoxin is therefore a novel toxin that shares a common scaffold with long chain alpha-neurotoxins but possibly utilizes additional functional determinants that assist in recognizing neuronal alpha7 receptors.