Determination of Epithelial Na+ Channel Subunit Stoichiometry from Single-Channel Conductances

The epithelial Na+ channel (ENaC) is a multimeric membrane protein consisting of three subunits, α, β, and γ. The total number of subunits per functional channel complex has been described variously to follow either a tetrameric arrangement of 2α:1β:1γ or a higher-ordered stoichiometry of 3α:3β:3γ. Therefore, while it is clear that all three ENaC subunits are required for full channel activity, the number of the subunits required remains controversial. We used a new approach, based on single-channel measurements in Xenopus oocytes to address this issue. Individual mutations that alter single-channel conductance were made in pore-lining residues of ENaC α, β, or γ subunits. Recordings from patches in oocytes expressing a single species, wild type or mutant, of α, β, and γ showed a well-defined current transition amplitude with a single Gaussian distribution. When cRNAs for all three wild-type subunits were mixed with an equimolar amount of a mutant α-subunit (either S589D or S592T), amplitudes corresponding to pure wild-type or mutant conductances could be observed in the same patch, along with a third intermediate amplitude most likely arising from channels with at least one wild-type and at least 1 mutant α-subunit. However, intermediate or hybrid conductances were not observed with coexpression of wild-type and mutant βG529A or γG534E subunits. Our results support a tetrameric arrangement of ENaC subunits where 2α, 1β, and 1γ come together around central pore

Synaptotagmin‐7 enhances calcium‐sensing of chromaffin cell granules and slows discharge of granule cargos

Synaptotagmin‐7 (Syt‐7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin‐1 (Syt‐1). Despite a broad appreciation for the importance of Syt‐7, questions remain as to its localization, function in mediating discharge of dense core granule cargos, and role in triggering release in response to physiological stimulation. These questions were addressed using two distinct experimental preparations—mouse chromaffin cells lacking endogenous Syt‐7 (KO cells) and a reconstituted system employing cell‐derived granules expressing either Syt‐7 or Syt‐1. First, using immunofluorescence imaging and subcellular fractionation, it is shown that Syt‐7 is widely distributed in organelles, including dense core granules. Total internal reflection fluorescence (TIRF) imaging demonstrates that the kinetics and probability of granule fusion in Syt‐7 KO cells stimulated by a native secretagogue, acetylcholine, are markedly lower than in WT cells. When fusion is observed, fluorescent cargo proteins are discharged more rapidly when only Syt‐1 is available to facilitate release. To determine the extent to which the aforementioned results are attributable purely to Syt‐7, granules expressing only Syt‐7 or Syt‐1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt‐7 confers substantially greater calcium sensitivity to granule fusion than Syt‐1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium and effects on fusion pore expansion, Syt‐7 plays a central role in regulating secretory output from adrenal chromaffin cells.Syt‐7 is a high‐affinity calcium sensor expressed on chromaffin cell dense core granules. The purpose of this study was to assess the role of Syt‐7 in regulating the secretory response to cholinergic stimulation. Acetylcholine elicits secretion by elevating cytosolic calcium. The calcium sensitivity of exocytosis in cells lacking Syt‐7 is impaired. Cells that lack Syt‐7 also release peptide hormones at faster rates, implicating a role for Syt‐7 in regulating the exocytotic fusion pore. These data demonstrate that Syt‐7 has an important role in triggering exocytosis in cells and is likely to play a role in controlling hormone output, in situ.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162737/3/jnc14986.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162737/2/jnc14986-sup-0001-Supinfo.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162737/1/jnc14986_am.pd

Localized topological changes of the plasma membrane upon exocytosis visualized by polarized TIRFM

Imaging of individual secretory granules reveals how exocytosis curves the membrane

Pharmacogenetic considerations in diseases of cardiac ion channels

ABSTRACT Phenotypic variation within a species arises from differences in genetic makeup between individuals. This inherent diversity empowers the species as a whole to explore and expand into new environmental niches and also to survive new stressors within an ever-changing environment. Paradoxically, one class of stressors currently challenging the human population is therapeutic drugs: medications designed to combat disease are often associated with a host of nonspecific side effects. Following earlier studies of the involvement of some cardiac ion currents in unwanted drug interactions, recent reports have identified not only the ion channel subunits involved but also a range of mutations and single nucleotide polymorphisms in ion channel genes that predispose to both drug-induced and familial cardiac arrhythmia. The tendency for individuals harboring specific, often common, gene variants to succumb to life-threatening cardiac arrhythmia, and the contribution of other factors such as drug interaction to disease etiology in these cases, are discussed here together with potential pharmacogenetic strategies for arrhythmia circumvention and therapy. The tendency for genes to mutate is the key to evolution and the existence of distinct species of living organisms. Although some gene mutations arise and are selected against because of their adverse effects on the ability of an individual to survive and compete, other variants are not harmful, are not selected against, and thus either predominate or coexist as common sequence variants in the general population Cardiac Ion Currents and Arrhythmia The electrical activity required for cardiac contraction requires the precise, concerted action of cardiac ion channels. The depolarizing upstroke of the action potential is carried by 831 the movement of positively charged sodium ions through voltage-gated sodium-selective channels into the cardiac myocyte cytoplasm. This depolarization initiates calcium influx and calcium release from intracellular stores, facilitating muscular contraction. Subsequently, delayed rectifier outward potassium currents govern the timing, rate, and completion of cellular repolarization Long QT syndrome can be divided into two broad categories: inherited and acquired. Inherited long QT syndrome, classified LQT1 to 7 according to the underlying gene defect Six ion channel genes have been associated with human long QT syndrome: the voltage-gated sodium (Na v ) channel ␣ subunit SCN5A KCNQ1 and MinK Mutations Cause Inherited Long QT Syndrome and Deafness K v channels are formed by noncovalent association of four K v ␣ subunits, each containing six transmembrane or membraneassociated domains (S1-S6) and a membrane-embedded pore region K v LQT1, now more commonly referred to as KCNQ1, is a K v channel ␣ subunit expressed in human heart and other tissues Familial long QT syndrome is most often observed as one of two forms: Romano-Ward syndrome, the most common, is an autosomal dominant trait with no obvious noncardiac abnormalities that can be caused by mutations in a variety of genes Long QT syndrome-associated mutations in KCNQ1 are dispersed throughout the coding region, with particular hot spots in key functional domains such as the S4 -5 linker (channel gating and interaction with MinK), the pore (the active site of ion conduction and selectivity), and the S6 helix (part of the ion conduction pathway). Mutations in ␤ subunits such as MinK can also greatly impair channel function. The most studied of these is the D76N-MinK mutation linked to Summary of genes harboring mutations associated with inherited arrhythmia and common SNPs associated with increased risk of arrhythmia. A gene variant is classified as a common SNP rather than a mutation here if its estimated incidence is greater than 1% in the population studied. SNPs are listed as the amino acid number and its two known variants, with the risk-associated variant on the right. LQT(1-7) is the common nomenclature used to describe long QT syndromes based on genetic classification