imaging-plasma-membrane-deformations-with-ptirfm

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Wayne State University

Studies of endogenous G-protein-mediated pathways in neurons by whole-cell electrophysiology.

Biochemical approaches to study interaction of calcium channels with RGS12 in primary neuronal cultures.

The physiological properties of most ion channels are defined experimentally by functional expression of their pore-forming alpha subunits in Xenopus laevis oocytes. Here, we cloned a family of Xenopus KCNE genes that encode MinK-related peptide K(+) channel beta subunits (xMiRPs) and demonstrated their constitutive expression in oocytes. Electrophysiological analysis of xMiRP2 revealed that when overexpressed this gene modulates human cardiac K(+) channel alpha subunits HERG (human ether-a-go-go-related gene) and KCNQ1 by suppressing HERG currents and removing the voltage dependence of KCNQ1 activation. The ability of endogenous levels of xMiRP2 to contribute to the biophysical attributes of overexpressed mammalian K(+) channels in oocyte studies was assessed next. Injection of an xMiRP2 sequence-specific short interfering RNA (siRNA) oligo reduced endogenous xMiRP2 expression 5-fold, whereas a control siRNA oligo had no effect, indicating the effectiveness of the RNA interference technique in Xenopus oocytes. The functional effects of endogenous xMiRP2 silencing were tested using electrophysiological analysis of heterologously expressed HERG channels. The RNA interference-mediated reduction of endogenous xMiRP2 expression increased macroscopic HERG current as much as 10-fold depending on HERG cRNA concentration. The functional effects of human MiRP1 (hMiRP1)/HERG interaction were also affected by endogenous xMiRP2. At high HERG channel density, at which the effects of endogenous xMiRP2 are minimal, hMiRP1 reduced HERG current. At low HERG current density, hMiRP1 paradoxically up-regulated HERG current, a result consistent with hMiRP1 rescuing HERG from suppression by endogenous xMiRP2. Thus, endogenous Xenopus MiRP subunits contribute to the base-line properties of K(+) channels like HERG in oocyte expression studies, which could explain expression level- and expression system-dependent variation in K(+) channel function.

RNA interference reveals that endogenous Xenopus MinK-related peptides govern mammalian K+ channel function in oocyte expression studies.

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.

Pharmacogenetic considerations in diseases of cardiac ion channels.

&#x4FE1;&#x53F7;&#x8F6C;&#x5BFC;&#x4E2D;&#x7684;&#x4E00;&#x4E2A;&#x65B0;&#x5174;&#x6982;&#x5FF5;&#x662F;&#x795E;&#x7ECF;&#x5143;&#x53D7;&#x4F53;&#x548C;&#x6E20;&#x9053;&#x7684;&#x5FAE;&#x533A;&#x7684;&#x4FE1;&#x53F7;&#x86CB;&#x767D;&#x88AB;&#x5E26;&#x5230;&#x672C;&#x7EC4;&#x7EC7;&#x5728;&#x4E00;&#x8D77;&#xFF0C;&#x89C4;&#x7BA1;&#x529F;&#x80FD;&#x53CD;&#x5E94;&#x3002;&#x591A;&#x91CD;&#x6027;&#x7684;&#x6F5C;&#x5728;&#x7684;&#x86CB;&#x767D;&#x8D28;-&#x86CB;&#x767D;&#x8D28;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x4EA7;&#x751F;&#x7684;&#x89C4;&#x7BA1;&#x53CA;&#x8FD9;&#x4E9B;&#x4EA4;&#x4E92;&#x7684;&#x65F6;&#x95F4;&#x9700;&#x8981;&#x3002;&#x6211;&#x4EEC;&#x5DF2;&#x786E;&#x5B9A;&#x5728;&#x4E0D;&#x540C;&#x65F6;&#x95F4;&#x540E;&#x6FC0;&#x6D3B;&#x7684; &gamma;-&#x6C28;&#x57FA;&#x4E01;&#x9178;&#xFF0C;B &#x578B;&#x53D7;&#x4F53;&#x901A;&#x9053;&#x4FE1;&#x53F7;&#x5206;&#x5B50;&#x914D;&#x5408;&#x7269;&#x5F62;&#x6210;&#x7684; N &#x578B;&#x7ECF;&#x3002;&#x7B2C;&#x4E00;&#x6B21;&#x4EA4;&#x4E92;&#x7684;&#x7C7B;&#x578B;&#x4EE5;&#x53CA;&#x6D89;&#x53CA;&#x9884;&#x4FE1;&#x53F7;&#x86CB;&#x767D;&#x7B49; Src &#x6FC0;&#x9176;&#x4E0E;&#x7ECF;&#x901A;&#x9053;&#xFF0C;&#x56E0;&#x4E3A;&#x5B83;&#x8FC5;&#x901F;&#x6FC0;&#x6D3B;&#x7684;&#x53D7;&#x4F53;&#x548C;&#x89C4;&#x7BA1;&#x7ECF;&#x901A;&#x9053;&#x7684;&#x6291;&#x5236;&#x4F5C;&#x7528;&#x7684;&#x7A0B;&#x5EA6;&#x3002;&#x7B2C;&#x4E8C;&#x79CD;&#x7C7B;&#x578B;&#x7684;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x6D89;&#x53CA;&#x4FE1;&#x53F7;&#x7684;&#x5206;&#x5B50;&#xFF0C;&#x62DB;&#x8058;&#x5230;&#x901A;&#x9053;&#x7684;&#x53D7;&#x4F53;&#x6FC0;&#x6D3B;&#x548C;&#x63A7;&#x5236;&#x901A;&#x9053;&#x54CD;&#x5E94;&#x7684;&#x901F;&#x7387;&#x3002;&#x62DB;&#x52DF;&#x6210;&#x5458;&#x7684; Ras &#x9014;&#x5F84;&#x4EA7;&#x751F;&#x4E24;&#x4E2A;&#x6548;&#x679C;&#xFF0C;&#x5982;&#x4E0B;&#x6240;&#x793A;&#xFF1A; 1&#xFF09; 2&#xFF09; &#x7684; MAP &#x6FC0;&#x9176;&#x6FC0;&#x6D3B;&#x4E0E; &gamma;-&#x6C28;&#x57FA;&#x4E01;&#x9178;&#x4ECB;&#x5BFC;&#x6291;&#x5236;&#x7684;&#x7ECF;&#x7535;&#x6D41;&#x7684;&#x53D1;&#x75C5;&#x6BD4;&#x7387;&#x7684;&#x8C03;&#x5236;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x8868;&#x660E;&#x7ECF;&#x901A;&#x9053; alpha(1) &#x4E9A;&#x5355;&#x4F4D;&#x804C;&#x80FD;&#x4F5C;&#x4E3A;&#x52A8;&#x6001;&#x7684;&#x811A;&#x624B;&#x67B6;&#x5141;&#x8BB8;&#x7A0B;&#x5E8F;&#x96C6;&#x7684;&#x7EC6;&#x80DE;&#x5185;&#x4FE1;&#x53F7;&#x8F6C;&#x5BFC;&#x548C;&#x7EC4;&#x4EF6;&#xFF0C;&#x6539;&#x53D8;&#x901A;&#x9053;&#x6D3B;&#x52A8;&#x8DEF;&#x7531;&#x5230;&#x5730;&#x56FE;&#x6FC0;&#x9176;&#x901A;&#x8DEF;&#x7684;&#x4FE1;&#x53F7;&#x3002;

N &#x578B; Ca2 + &#x901A;&#x9053;&#x4F5C;&#x4E3A;&#x652F;&#x67B6;&#x86CB;&#x767D;&#x4E2D;&#x7684;&#x4FE1;&#x53F7;&#x5206;&#x5B50; GABAB &#x53D7;&#x4F53;&#x5F71;&#x54CD;&#x7684;&#x5927;&#x4F1A;&#x3002;

An emerging concept in signal transduction is the organization of neuronal receptors and channels into microdomains in which signaling proteins are brought together to regulate functional responses. With the multiplicity of potential protein-protein interactions arises the need for the regulation and timing of these interactions. We have identified N-type Ca(2+) channel-signaling molecule complexes formed at different times upon activation of gamma-aminobutyric acid, type B, receptors. The first type of interaction involves pre-association of signaling proteins such as Src kinase with the Ca(2+) channel, because it is rapidly activated by the receptors and regulates the magnitude of the inhibition of the Ca(2+) channel. The second type of interaction involves signaling molecules that are recruited to the channel by receptor activation and control the rate of the channel response. Recruitment of members of the Ras pathway has two effects as follows: 1) modulation of the rate of onset of the gamma-aminobutyric acid-mediated inhibition of Ca(2+) current, and 2) activation of MAP kinase. Our results suggest that the Ca(2+) channel alpha(1) subunit functions as a dynamic scaffold allowing assembly of intracellular signaling components that alter channel activity and route signals to the MAP kinase pathway.

N-type Ca2+ channels as scaffold proteins in the assembly of signaling molecules for GABAB receptor effects.

Un nouveau concept dans la transduction du signal est l&#x26;#39;organisation des r&#xE9;cepteurs neuronaux et les canaux en microdomaines dans laquelle les prot&#xE9;ines de signalisation sont r&#xE9;unis pour r&#xE9;guler les r&#xE9;ponses fonctionnelles. Avec la multiplicit&#xE9; des possibles interactions prot&#xE9;ine-prot&#xE9;ine se pose la n&#xE9;cessit&#xE9; de la r&#xE9;glementation et le calendrier de ces interactions. Nous avons identifi&#xE9; de type N Ca (2 +) du canal de signalisation complexes mol&#xE9;cule form&#xE9;e &#xE0; diff&#xE9;rents moments lors de l&#x26;#39;activation de l&#x26;#39;acide gamma-aminobutyrique, de type B, les r&#xE9;cepteurs. Le premier type d&#x26;#39;interaction consiste &#xE0; pr&#xE9;-association de prot&#xE9;ines de signalisation telles que la kinase Src avec le Ca (2 +) de canal, car il est rapidement activ&#xE9;e par les r&#xE9;cepteurs et r&#xE9;glemente l&#x26;#39;ampleur de l&#x26;#39;inhibition de la Ca (2 +) du canal. Le second type d&#x26;#39;interaction comprend mol&#xE9;cules de signalisation qui sont recrut&#xE9;es au canal par l&#x26;#39;activation du r&#xE9;cepteur et de contr&#xF4;ler le taux de la r&#xE9;ponse de canal. Le recrutement des membres de la voie Ras a deux effets comme suit: 1) de modulation de la vitesse d&#x26;#39;apparition de l&#x26;#39;acide gamma-aminobutyrique inhibition de la Ca (2 +) en cours, et 2) l&#x26;#39;activation de la MAP kinase. Nos r&#xE9;sultats sugg&#xE8;rent que le Ca (2 +) du canal alpha (1) les fonctions de sous-unit&#xE9;s comme un ensemble dynamique d&#x26;#39;&#xE9;chafaudage permettant de intracellulaires de signalisation composants qui modifient l&#x26;#39;activit&#xE9; du canal et acheminer des signaux &#xE0; la voie des MAP kinases.

De type N canaux Ca 2 + que les prot&#xE9;ines d&#x26;#39;&#xE9;chafaudage &#xE0; l&#x26;#39;Assembl&#xE9;e de mol&#xE9;cules de signalisation pour les effets des r&#xE9;cepteurs GABAB

N-Typ-Ca 2 +-Kan&#xE4;le als Ger&#xFC;stproteine &#x200B;&#x200B;in der Anordnung von Signalmolek&#xFC;len f&#xFC;r GABAB-Rezeptor Effekte.

Un concetto emergente nella trasduzione del segnale &egrave; l&#39;organizzazione dei recettori neuronali e canali in microdomains in cui le proteine di segnalazione sono portate insieme a regolare le risposte funzionali. Con la molteplicit&agrave; delle potenziali interazioni proteina-proteina si pone la necessit&agrave; per la regolazione e la tempistica di queste interazioni. Ca(2+) N-tipo canale di segnalazione complessi molecola formate in tempi diversi all&#39;attivazione dei recettori di tipo B, acido gamma - aminobutirrico, abbiamo identificato. Il primo tipo di interazione coinvolge preassociazione di segnalazione proteine come la chinasi Src con il canale di Ca(2+), perch&eacute; esso viene rapidamente attivato da recettori e regola la grandezza dell&#39;inibizione del canale Ca(2+). Il secondo tipo di interazione coinvolge molecole che sono reclutati per il canale di attivazione del recettore e controllano il tasso di risposta il canale di segnalazione. Reclutamento di membri del percorso Ras ha due effetti come segue: 1) modulazione del tasso di insorgenza di inibizione mediata da acido gamma - aminobutirrico della corrente Ca(2+) e 2) attivazione della chinasi della mappa. I nostri risultati suggeriscono che la subunit&agrave; Ca(2+) canale alpha(1) funziona come un&#39;impalcatura dinamica che permette il montaggio di componenti di segnalazione intracellulare che alterare l&#39;attivit&agrave; del canale e instradano i segnali per il pathway della chinasi mappa.

Canali Ca2 + N-tipo come proteine impalcatura nell&#39;assemblaggio di molecole per gli effetti del recettore GABAB di segnalazione.

&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x306B;&#x304A;&#x3051;&#x308B;&#x65B0;&#x305F;&#x306A;&#x6982;&#x5FF5;&#x306F;&#x3001;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x304C;&#x6A5F;&#x80FD;&#x7684;&#x5FDC;&#x7B54;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x7D50;&#x96C6;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x3078;&#x306E;&#x795E;&#x7D4C;&#x53D7;&#x5BB9;&#x4F53;&#x3084;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x7D44;&#x7E54;&#x3067;&#x3059;&#x3002;&#x6F5C;&#x5728;&#x7684;&#x306A;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA; - &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306E;&#x591A;&#x69D8;&#x3067;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306E;&#x5236;&#x5FA1;&#x3068;&#x30BF;&#x30A4;&#x30DF;&#x30F3;&#x30B0;&#x306E;&#x5FC5;&#x8981;&#x6027;&#x304C;&#x751F;&#x3058;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005;&#x306F;&#x3001;&#x3B3;-&#x30A2;&#x30DF;&#x30CE;&#x916A;&#x9178;&#x3001;B&#x578B;&#x3001;&#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x6D3B;&#x6027;&#x5316;&#x306B;&#x5225;&#x3005;&#x306E;&#x30BF;&#x30A4;&#x30DF;&#x30F3;&#x30B0;&#x3067;&#x5F62;&#x6210;&#x3055;&#x308C;&#x305F;N&#x578B;Ca&#xFF08;2 +&#xFF09;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x30B7;&#x30B0;&#x30CA;&#x30EA;&#x30F3;&#x30B0;&#x5206;&#x5B50;&#x8907;&#x5408;&#x4F53;&#x3092;&#x540C;&#x5B9A;&#x3057;&#x305F;&#x3002;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306E;&#x6700;&#x521D;&#x306E;&#x30BF;&#x30A4;&#x30D7;&#x306F;&#x3001;&#x305D;&#x308C;&#x304C;&#x6025;&#x901F;&#x306B;&#x53D7;&#x5BB9;&#x4F53;&#x306B;&#x3088;&#x3063;&#x3066;&#x6D3B;&#x6027;&#x5316;&#x3068;Ca&#xFF08;2 +&#xFF09;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x963B;&#x5BB3;&#x306E;&#x5927;&#x304D;&#x3055;&#x3092;&#x8ABF;&#x7BC0;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x305F;&#x3081;&#x3001;&#x4F8B;&#x3048;&#x3070;Ca&#xFF08;2 +&#xFF09;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x3068;&#x306E;Src&#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x306A;&#x3069;&#x306E;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x30B7;&#x30B0;&#x30CA;&#x30EA;&#x30F3;&#x30B0;&#x306E;&#x4E8B;&#x524D;&#x4F1A;&#x5408;&#x304C;&#x542B;&#x307E;&#x308C;&#x307E;&#x3059;&#x3002;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306E;&#x4E8C;&#x3064;&#x76EE;&#x306E;&#x30BF;&#x30A4;&#x30D7;&#x306F;&#x3001;&#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x6D3B;&#x6027;&#x5316;&#x3068;&#x5236;&#x5FA1;&#x306B;&#x3088;&#x3063;&#x3066;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306B;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x5FDC;&#x7B54;&#x306E;&#x30EC;&#x30FC;&#x30C8;&#x3092;&#x63A1;&#x7528;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x5206;&#x5B50;&#x304C;&#x542B;&#x307E;&#x308C;&#x307E;&#x3059;&#x3002; Ca&#x306E;&#x3B3;-&#x30A2;&#x30DF;&#x30CE;&#x916A;&#x9178;&#x5A92;&#x4ECB;&#x963B;&#x5BB3;&#xFF08;2 +&#xFF09;&#x96FB;&#x6D41;&#x3001;&#x304A;&#x3088;&#x3073;MAP&#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x306E;2&#xFF09;&#x6D3B;&#x6027;&#x5316;&#x306E;&#x767A;&#x75C7;&#x7387;&#x306E;1&#xFF09;&#x5909;&#x8ABF;&#xFF1A;Ras&#x7D4C;&#x8DEF;&#x306E;&#x30E1;&#x30F3;&#x30D0;&#x30FC;&#x306E;&#x52DF;&#x96C6;&#x306F;&#x3001;&#x6B21;&#x306E;&#x3088;&#x3046;&#x306B;2&#x3064;&#x306E;&#x52B9;&#x679C;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005;&#x306E;&#x7D50;&#x679C;&#x306F;&#x793A;&#x5506;&#x3057;&#x3066;&#x3044;&#x308B;&#x306E;Ca&#xFF08;2 +&#xFF09;&#x30C1;&#x30E3;&#x30F3;&#x30CD;&#x30EB;&#x3B1;&#xFF08;1&#xFF09;MAP&#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x7D4C;&#x8DEF;&#x306B;&#x30C1;&#x30E3;&#x30F3;&#x30CD;&#x30EB;&#x6D3B;&#x6027;&#x3068;&#x4FE1;&#x53F7;&#x7D4C;&#x8DEF;&#x3092;&#x5909;&#x66F4;&#x3059;&#x308B;&#x7D30;&#x80DE;&#x5185;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x306E;&#x52D5;&#x7684;&#x306A;&#x8DB3;&#x5834;&#x3092;&#x53EF;&#x80FD;&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x3068;&#x3057;&#x3066;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x306E;&#x6A5F;&#x80FD;&#x3002;

GABAB&#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x52B9;&#x679C;&#x306E;&#x305F;&#x3081;&#x306E;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x5206;&#x5B50;&#x306E;&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x306B;&#x304A;&#x3051;&#x308B;&#x8DB3;&#x5834;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3068;&#x3057;&#x3066;&#x306E;N&#x578B;Ca2 +&#x30C1;&#x30E3;&#x30CD;&#x30EB;

&#xC2E0;&#xD638; &#xBCC0;&#xD658;&#xC5D0; &#xB098;&#xC624;&#xB294; &#xAC1C;&#xB150;&#xC740; microdomains &#xC2E0;&#xD638; &#xB2E8;&#xBC31;&#xC9C8; &#xAC00;&#xC838;&#xC5D0; &#xC2E0;&#xACBD; &#xC218;&#xC6A9; &#xCCB4;&#xC640; &#xCC44;&#xB110;&#xC758; &#xC870;&#xC9C1; &#xAE30;&#xB2A5; &#xC751;&#xB2F5; &#xADDC;&#xC81C;&#xB97C; &#xD568;&#xAED8;. &#xC7A0;&#xC7AC;&#xC801;&#xC778; &#xB2E8;&#xBC31;&#xC9C8; &#xB2E8;&#xBC31;&#xC9C8; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC758; &#xBCF5;&#xD569;&#xC131;&#xC73C;&#xB85C; &#xADDC;&#xC815; &#xBC0F; &#xC774;&#xB7EC;&#xD55C; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC758; &#xD0C0;&#xC774;&#xBC0D;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC694;&#xAC00; &#xBC1C;&#xC0DD;&#xD569;&#xB2C8;&#xB2E4;. N &#xD615; Ca(2+) &#xCC44;&#xB110; &#xC2E0;&#xD638; &#xBD84;&#xC790; &#xB2E8;&#xC9C0; &#xD615;&#xC131; &#xAC10;&#xB9C8;-aminobutyric &#xC0B0; B &#xD615; &#xC218;&#xC6A9; &#xCCB4;&#xC758; &#xD65C;&#xC131;&#xD654;&#xC5D0; &#xB530;&#xB77C; &#xC11C;&#xB85C; &#xB2E4;&#xB978; &#xC2DC;&#xAC04;&#xC5D0; &#xD30C;&#xC545; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC758; &#xCCAB; &#xBC88;&#xC9F8; &#xC720;&#xD615;&#xC740; &#xBE60;&#xB974;&#xAC8C; &#xC218;&#xC6A9; &#xCCB4;&#xC5D0; &#xC758;&#xD574; &#xD65C;&#xC131;&#xD654; Ca(2+) &#xCC44;&#xB110; &#xC5B5;&#xC81C;&#xC758; &#xD06C;&#xAE30;&#xB97C; &#xC870;&#xC808; &#xD558;&#xAE30; &#xB54C;&#xBB38;&#xC5D0; Ca(2+) &#xCC44;&#xB110; Src &#xB2C8; &#xAC19;&#xC740; &#xB2E8;&#xBC31;&#xC9C8; &#xC2E0;&#xD638; pre-association&#xB97C; &#xD3EC;&#xD568; &#xD55C;&#xB2E4;. &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC758; &#xB450; &#xBC88;&#xC9F8; &#xC720;&#xD615;&#xC740; &#xC2E0;&#xD638; &#xBD84;&#xC790; &#xC218;&#xC6A9; &#xCCB4; &#xD65C;&#xC131;&#xD654; &#xCC44;&#xB110;&#xC5D0; &#xCC44;&#xC6A9; &#xB418; &#xACE0; &#xCC44;&#xB110; &#xC751;&#xB2F5;&#xC758; &#xC18D;&#xB3C4; &#xC81C;&#xC5B4; &#xD558;&#xB294; &#xD3EC;&#xD568; &#xB429;&#xB2C8;&#xB2E4;. Ras &#xD1B5;&#xB85C;&#xC758; &#xAD6C;&#xC131;&#xC6D0;&#xC758; &#xCC44;&#xC6A9;&#xC740; &#xB2E4;&#xC74C;&#xACFC; &#xAC19;&#xC740; &#xB450; &#xAC00;&#xC9C0; &#xD6A8;&#xACFC;: Ca(2+) &#xC804;&#xB958;&#xC758; &#xAC10;&#xB9C8;-aminobutyric &#xC0B0; &#xC911;&#xC7AC; &#xC800;&#xD574; &#xBC0F; 2) &#xC9C0;&#xB3C4; &#xD0A4; &#xB2C8; &#xC544; &#xC81C; &#xD65C;&#xC131;&#xD654;&#xC758; &#xBC1C;&#xBCD1; &#xB960;&#xC758; 1) &#xBCC0;&#xC870;. &#xC6B0;&#xB9AC;&#xC758; &#xACB0;&#xACFC; &#xAC83; Ca(2+) &#xCC44;&#xB110; alpha(1) &#xC18C; &#xB2E8;&#xC704; &#xB3D9;&#xC801; &#xBE44; &#xACC4; &#xD5C8;&#xC6A9; &#xD558; &#xCC44;&#xB110; &#xD65C;&#xB3D9;&#xC744; &#xBCC0;&#xACBD; &#xD558; &#xACE0; &#xC9C0;&#xB3C4; &#xB2C8; &#xD1B5;&#xB85C;&#xB97C; &#xC2E0;&#xD638;&#xB97C; &#xB77C;&#xC6B0;&#xD305;&#xD558;&#xB294; &#xC138;&#xD3EC;&#xB0B4; &#xC2E0;&#xD638; &#xBD80;&#xD488;&#xC758; &#xC870;&#xB9BD;&#xC73C;&#xB85C; &#xC791;&#xB3D9; &#xD558;&#xB294; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;.

N &#xD615; Ca2 + &#xCC44;&#xB110; &#xC2E0;&#xD638; GABAB &#xC218;&#xC6A9; &#xCCB4; &#xD6A8;&#xACFC; &#xB300; &#xD55C; &#xBD84;&#xC790;&#xC758; &#xC870;&#xB9BD; &#xBE44; &#xACC4; &#xB2E8;&#xBC31;&#xC9C8;&#xC73C;&#xB85C;.

Un nuevo concepto en la transducci&#xF3;n de la se&#xF1;al es la organizaci&#xF3;n de los receptores neuronales y canales en microdominios en la que las prote&#xED;nas de se&#xF1;alizaci&#xF3;n se re&#xFA;nen para regular las respuestas funcionales. Con la multiplicidad de posibles interacciones prote&#xED;na-prote&#xED;na se plantea la necesidad de la regulaci&#xF3;n y el calendario de estas interacciones. Hemos identificado la N-tipo Ca (2 +) del canal de se&#xF1;alizaci&#xF3;n complejos de mol&#xE9;culas formadas en diferentes momentos tras la activaci&#xF3;n del &#xE1;cido gamma-aminobut&#xED;rico, tipo B, los receptores. El primer tipo de interacci&#xF3;n implica pre-asociaci&#xF3;n de prote&#xED;nas de se&#xF1;alizaci&#xF3;n tales como quinasa Src con el Ca (2 +) del canal, debido a que es r&#xE1;pidamente activada por los receptores y regula la magnitud de la inhibici&#xF3;n de la Ca (2 +) del canal. El segundo tipo de interacci&#xF3;n implica mol&#xE9;culas de se&#xF1;alizaci&#xF3;n que son reclutados al canal por la activaci&#xF3;n del receptor y controlar la velocidad de la respuesta del canal. El reclutamiento de miembros de la v&#xED;a Ras tiene dos efectos de la siguiente manera: 1) la modulaci&#xF3;n de la velocidad de aparici&#xF3;n del &#xE1;cido gamma-aminobut&#xED;rico mediada por la inhibici&#xF3;n de la Ca (2 +) actual, y 2) la activaci&#xF3;n de MAP quinasa. Nuestros resultados sugieren que el Ca (2 +) del canal alfa (1) las funciones de las subunidades de una din&#xE1;mica de conjunto del andamio, permitiendo de componentes de se&#xF1;alizaci&#xF3;n intracelulares que alteran la actividad del canal y las se&#xF1;ales de ruta de la v&#xED;a MAP cinasa.

N-tipo canales de Ca2 + como las prote&#xED;nas andamio en la Asamblea de mol&#xE9;culas de se&#xF1;alizaci&#xF3;n para efectos del receptor de GABAB

The voltage-gated potassium channel formed by hERG pore-forming alpha subunits generates the IKr cardiac potassium current, and is considered essential for human ventricular repolarization. What is not certain is whether human IKr channels contain ancillary subunits in vivo. Two chief contenders for this role are MinK (encoded by KCNE1) and MiRP1 (KCNE2). MinK and MiRP1 are single transmembrane domain peptides that can co-assemble with hERG in heterologous systems. MinK increases hERG currents by an unknown mechanism. MiRP1 alters hERG current density and gating, although no consensus has been reached as to the precise extent of these effects. Here we discuss key aspects of the debate surrounding the potential roles of MinK and MiRP1 in IKr: inconsistencies between reports of the effects of MiRP1 on hERG in vitro; association with long QT syndrome of inherited mutations in MinK and MiRP1; and a role for MiRP1 polymorphisms in acquired arrhythmia despite the apparent inability of MiRP1 to impinge upon the unique inner vestibule drug-binding site that dominates hERG pharmacology.

Does hERG coassemble with a beta subunit? Evidence for roles of MinK and MiRP1.

The regulation of epithelial Na(+) channel (ENaC) activity by Na(+) was studied in Xenopus oocytes using two-electrode voltage clamp and patch-clamp recording techniques. Here we show that amiloride-sensitive Na(+) current (I(Na)) is downregulated when ENaC-expressing cells are exposed to high extracellular [Na(+)]. The reduction in macroscopic Na(+) current is accompanied by an increase in the concentration of intracellular Na(+) ([Na(+)](i)) and is only slowly reversible. At the single-channel level, incubating oocytes in high-Na(+) solution reduces open probability (P(o)) approximately twofold compared to when [Na(+)] is kept low, by increasing mean channel closed times. However, increasing P(o) by introducing a mutation in the beta-subunit (S518C) which, in the presence of [2-(trimethylammonium) ethyl] methane thiosulfonate (MTSET), locks the channel in an open state, could not alone abolish the downregulation of macroscopic current measured with exposure to high external [Na(+)]. Inhibition of the insertion of new channels into the plasma membrane using Brefeldin A revealed that surface channel lifetime is also markedly reduced under these conditions. In channels harbouring a beta-subunit mutation, R564X, associated with Liddle's syndrome, open probability in both high- and low-Na(+) conditions is significantly higher than in wild-type channels. Increasing the P(o) of these channels with an activating mutation abrogated the difference in macroscopic current observed between groups of oocytes incubated in high- and low-Na(+) conditions. These findings demonstrate that reduction of ENaC P(o) is a physiological mechanism limiting Na(+) entry when [Na(+)](i) is high.

Open probability of the epithelial sodium channel is regulated by intracellular sodium.

G&egrave;nes de la famille des KCNE codent seul domaine transmembranaire auxiliaires sous-unit&eacute;s qui co-assembl&eacute;es avec les sous-unit&eacute;s alpha canal de potassium voltage-d&eacute;pendants (Kv) de modifier leur fonction. KCNE2 (&eacute;galement connu comme MiRP1) est exprim&eacute; dans le c&oelig;ur, est associ&eacute;e &agrave; une arythmie cardiaque humaine et module cardiaque Kv sous-unit&eacute;s alpha hERG et KCNQ1 in vitro. KCNE2 et KCNQ1 sont &eacute;galement exprim&eacute;s dans les cellules pari&eacute;tales, menant &agrave; la sp&eacute;culation, qu&#39;ils forment une cha&icirc;ne native complexe l&agrave;. Ici, nous avons perturb&eacute; le g&egrave;ne murin kcne2 et trouv&eacute; que kcne2 (- / -) souris pr&eacute;sentent un ph&eacute;notype gastrique s&eacute;v&egrave;re avec s&eacute;cr&eacute;tion de proton profond&eacute;ment r&eacute;duite des cellules pari&eacute;tales, morphologie anormale des cellules pari&eacute;tales, achlorhydrie, hypergastrin&eacute;mie et frappant hyperplasie glandulaire gastrique r&eacute;sultant d&#39;une augmentation du nombre de cellules s&eacute;cr&eacute;trices non acide. KCNQ1 expos&eacute;es une distribution anormale des glandes gastriques de kcne2 (- / -) souris, avec l&#39;augmentation de l&#39;expression dans les cellules s&eacute;cr&eacute;trices non acide. Les cellules pari&eacute;tales de kcne2 (+/-) souris expos&eacute;es architecture normale mais r&eacute;duit la s&eacute;cr&eacute;tion de proton, et kcne2 (+/-) de souris ont &eacute;t&eacute; hypochlorhydric, ce qui indique un effet dose-g&egrave;ne et un d&eacute;faut primaire dans la s&eacute;cr&eacute;tion d&#39;acide gastrique. Ces donn&eacute;es d&eacute;montrent que KCNE2 est essentielle pour la s&eacute;cr&eacute;tion d&#39;acide gastrique, la premi&egrave;re preuve g&eacute;n&eacute;tique qu&#39;un membre de la famille de g&egrave;ne KCNE est n&eacute;cessaire pour la fonction gastro-intestinale normale.

La sous-unit&eacute; auxiliaire de canal du potassium KCNE2 est essentielle pour la s&eacute;cr&eacute;tion d&#39;acide gastrique.

KCNE &#x5BB6;&#x65CF;&#x4E2D;&#x7684;&#x57FA;&#x56E0;&#x7F16;&#x7801;&#x4E9A;&#x5355;&#x8DE8;&#x819C;&#x57DF;&#x9644;&#x5C5E;&#x7ED3;&#x6784;&#xFF0C;co-assemble (Kv) &#x7535;&#x538B;&#x95E8;&#x63A7;&#x6027;&#x94BE;&#x901A;&#x9053; alpha &#x4E9A;&#x57FA;&#x6539;&#x53D8;&#x5176;&#x529F;&#x80FD;&#x3002;KCNE2 &#xFF08;&#x4E5F;&#x79F0;&#x4E3A; MiRP1&#xFF09; &#x8868;&#x793A;&#xFF0C;&#x5728;&#x5FC3;&#x810F;&#x3001; &#x4E0E;&#x4EBA;&#x7C7B;&#x7684;&#x5FC3;&#x5F8B;&#x5931;&#x5E38;&#xFF0C;&#x5173;&#x8054;&#xFF0C;&#x8C03;&#x8282;&#x5FC3;&#x810F; Kv alpha &#x4E9A;&#x57FA; hERG &#x548C; KCNQ1 &#x4F53;&#x5916;&#x3002;KCNE2 &#x548C; KCNQ1 &#x90FD;&#x4E5F;&#x8868;&#x793A;&#x5728;&#x80C3;&#x58C1;&#x7EC6;&#x80DE;&#xFF0C;&#x5BFC;&#x81F4;&#x5B83;&#x4EEC;&#x5F62;&#x6210;&#x4E00;&#x4E2A;&#x590D;&#x6742;&#x7684;&#x672C;&#x673A;&#x901A;&#x9053;&#x90A3;&#x91CC;&#x6295;&#x673A;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#xFF0C;kcne2 &#x548C;&#x4E2D;&#x65AD;&#x5C0F;&#x9F20; kcne2 &#x57FA;&#x56E0; (-/-) &#x5C0F;&#x9F20;&#x6709;&#x4E00;&#x4E2A;&#x4E25;&#x91CD;&#x7684;&#x80C3;&#x8868;&#x578B;&#x4E0E;&#x8D28;&#x5B50;&#x6DF1;&#x523B;&#x5730;&#x51CF;&#x5C11;&#x7684;&#x80C3;&#x58C1;&#x7EC6;&#x80DE;&#x5206;&#x6CCC;&#x3001; &#x5F02;&#x5E38;&#x80C3;&#x58C1;&#x7EC6;&#x80DE;&#x5F62;&#x6001;&#x3001; achlorhydria&#x3001; hypergastrinemia&#x3001; &#x548C;&#x7A81;&#x51FA;&#x80C3;&#x817A;&#x4F53;&#x589E;&#x751F;&#x5F15;&#x8D77;&#x7684;&#x975E;&#x9178;&#x5206;&#x6CCC;&#x7EC6;&#x80DE;&#x7684;&#x6570;&#x91CF;&#x589E;&#x52A0;&#x3002;KCNQ1 &#x5C55;&#x51FA;&#x4ECE; kcne2 &#x7684;&#x80C3;&#x817A;&#x4F53;&#x5F02;&#x5E38;&#x5206;&#x5E03; (-/-) &#x5C0F;&#x9F20;&#xFF0C;&#x589E;&#x52A0;&#x975E;&#x9178;&#x5206;&#x6CCC;&#x7EC6;&#x80DE;&#x4E2D;&#x7684;&#x8868;&#x8FBE;&#x3002;&#x4ECE; kcne2 (+ /-&#xFF09; &#x5C0F;&#x9F20;&#x7684;&#x80C3;&#x58C1;&#x7EC6;&#x80DE;&#x8868;&#x73B0;&#x51FA;&#x6B63;&#x5E38;&#x7684;&#x4F53;&#x7CFB;&#x7ED3;&#x6784;&#xFF0C;&#x4F46;&#x51CF;&#x5C11;&#x5206;&#x6CCC;&#x8D28;&#x5B50;&#xFF0C;&#x548C; kcne2 (+ /-&#xFF09; &#x5C0F;&#x9F20;&#x662F; hypochlorhydric&#xFF0C;&#x8868;&#x660E;&#x57FA;&#x56E0;&#x5242;&#x91CF;&#x6548;&#x5E94;&#x548C;&#x80C3;&#x9178;&#x5206;&#x6CCC;&#x4E2D;&#x7684;&#x4E3B;&#x8981;&#x7F3A;&#x9677;&#x3002;&#x8FD9;&#x4E9B;&#x6570;&#x636E;&#x8868;&#x660E; KCNE2 &#x662F;&#x80C3;&#x9178;&#x7684;&#x5206;&#x6CCC;&#xFF0C;KCNE &#x57FA;&#x56E0;&#x5BB6;&#x65CF;&#x7684;&#x6210;&#x5458;&#x662F;&#x5BF9;&#x80C3;&#x80A0;&#x9053;&#x7684;&#x6B63;&#x5E38;&#x529F;&#x80FD;&#x6240;&#x9700;&#x7684;&#x7B2C;&#x4E00;&#x4E2A;&#x9057;&#x4F20;&#x8BC1;&#x636E;&#x7684;&#x5FC5;&#x8981;&#x6761;&#x4EF6;&#x3002;

KCNE2 &#x94BE;&#x901A;&#x9053;&#x8F85;&#x52A9;&#x4E9A;&#x57FA;&#x662F;&#x80C3;&#x9178;&#x5206;&#x6CCC;&#x7684;&#x5FC5;&#x8981;&#x6761;&#x4EF6;&#x3002;

Genes in the KCNE family encode single transmembrane domain ancillary subunits that co-assemble with voltage-gated potassium (Kv) channel alpha subunits to alter their function. KCNE2 (also known as MiRP1) is expressed in the heart, is associated with human cardiac arrhythmia, and modulates cardiac Kv alpha subunits hERG and KCNQ1 in vitro. KCNE2 and KCNQ1 are also expressed in parietal cells, leading to speculation they form a native channel complex there. Here, we disrupted the murine kcne2 gene and found that kcne2 (-/-) mice have a severe gastric phenotype with profoundly reduced parietal cell proton secretion, abnormal parietal cell morphology, achlorhydria, hypergastrinemia, and striking gastric glandular hyperplasia arising from an increase in the number of non-acid secretory cells. KCNQ1 exhibited abnormal distribution in gastric glands from kcne2 (-/-) mice, with increased expression in non-acid secretory cells. Parietal cells from kcne2 (+/-) mice exhibited normal architecture but reduced proton secretion, and kcne2 (+/-) mice were hypochlorhydric, indicating a gene-dose effect and a primary defect in gastric acid secretion. These data demonstrate that KCNE2 is essential for gastric acid secretion, the first genetic evidence that a member of the KCNE gene family is required for normal gastrointestinal function.

The KCNE2 potassium channel ancillary subunit is essential for gastric acid secretion.

La famiglia KCNE i geni codificano per singolo dominio transmembrana ausiliari subunit&agrave; che co-assemble con potassio voltaggio-dipendenti (Kv) canale alfa subunit&agrave; a modificare la loro funzione. KCNE2 (noto anche come MiRP1) &egrave; espresso nel cuore, &egrave; associata a disturbi del ritmo cardiaco umano e modula cardiaco Kv subunit&agrave; alfa hERG e KCNQ1 in vitro. KCNE2 e KCNQ1 sono anche espressi in cellule parietali, conducendo alla speculazione che formano un complesso nativo del canale ci. Qui, abbiamo interrotto il gene kcne2 murino e trovato quello kcne2 (- / -) topi hanno un fenotipo grave gastrico con cellule parietali profondamente ridotta secrezione protonica, morfologia anormale delle cellule parietali, acloridria, hypergastrinemia e sorprendente iperplasia ghiandolare gastrico derivanti da un aumento del numero di cellule secretorie non acido. KCNQ1 esposto distribuzione anomala delle ghiandole gastriche da kcne2 (- / -) topi, con maggiore espressione in cellule secretorie non acido. Cellule parietali da kcne2 (+ /-) topi esposti normale architettura ma ridotta secrezione protonica e kcne2 (+ /-) topi sono stati hypochlorhydric, che indica un effetto gene-dose e un difetto primario nella secrezione di acido gastrico. Questi dati dimostrano che KCNE2 &egrave; essenziale per la secrezione di acido gastrico, le prime prove genetiche che un membro della famiglia del gene KCNE &egrave; richiesto per la normale funzione gastrointestinale.

La secondaria ausiliario della scanalatura del potassio KCNE2 &egrave; essenziale per la secrezione di acido gastrico.

KCNE &#x30D5;&#x30A1;&#x30DF;&#x30EA;&#x30FC;&#x907A;&#x4F1D;&#x5B50; co-assemble 1 &#x3064;&#x306E;&#x819C;&#x8CAB;&#x901A;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x88DC;&#x52A9;&#x7684;&#x306A;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x306E;&#x6A5F;&#x80FD;&#x3092;&#x5909;&#x66F4;&#x3059;&#x308B;&#x30AB;&#x30EA;&#x30A6;&#x30E0; (Kv) &#x30C1;&#x30E3;&#x30CD;&#x30EB; &#x30A2;&#x30EB;&#x30D5;&#x30A1; &#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x3068;&#x30A8;&#x30F3;&#x30B3;&#x30FC;&#x30C9;&#x3057;&#x307E;&#x3059;&#x3002;&#xFF08;MiRP1 &#x3068;&#x3057;&#x3066;&#x3082;&#x77E5;&#x3089;&#x308C;&#x308B;&#xFF09; KCNE2 &#x306E;&#x5FC3;&#x3092;&#x8868;&#x73FE;&#x3001;&#x4EBA;&#x9593;&#x306E;&#x5FC3;&#x81D3;&#x306E;&#x4E0D;&#x6574;&#x8108;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x3001;&#x5FC3;&#x81D3;&#x306E; Kv &#x30A2;&#x30EB;&#x30D5;&#x30A1; &#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8; hERG &#x3068; in vitro KCNQ1 &#x3092;&#x8ABF;&#x7BC0;&#x3057;&#x307E;&#x3059;&#x3002;KCNE2 &#x3068; KCNQ1 &#x3082;&#x7D30;&#x80DE;&#x3067;, &#x6295;&#x6A5F;&#x5F7C;&#x3089;&#x304C;&#x30CD;&#x30A4;&#x30C6;&#x30A3;&#x30D6; &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x8907;&#x5408;&#x4F53;&#x3092;&#x5F62;&#x6210;&#x3059;&#x308B;&#x30EA;&#x30FC;&#x30C9;&#x3092;&#x8868;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x6211;&#x3005; &#x306F;&#x30DE;&#x30A6;&#x30B9; kcne2 &#x907A;&#x4F1D;&#x5B50;&#x3092;&#x7834;&#x58CA;&#x3057;&#x3001;&#x305D;&#x306E; kcne2 &#x304C;&#x898B;&#x3064;&#x304B;&#x308A;&#x307E;&#x3057;&#x305F; (-/-) &#x30DE;&#x30A6;&#x30B9;&#x306F;&#x6DF1;&#x523B;&#x306A;&#x80C3;&#x8868;&#x73FE;&#x578B;&#x6DF1;&#x304F;&#x524A;&#x6E1B;&#x58C1;&#x7D30;&#x80DE;&#x306E;&#x30D7;&#x30ED;&#x30C8;&#x30F3;&#x306E;&#x5206;&#x6CCC;&#x7570;&#x5E38;&#x306E;&#x982D;&#x9802;&#x306E;&#x7D30;&#x80DE;&#x306E;&#x5F62;&#x614B;&#x3001;&#x53D7;&#x3051;&#x3066;&#x3001;&#x9AD8;&#x30AC;&#x30B9;&#x30C8;&#x30EA;&#x30F3;&#x8840;&#x75C7;&#x3001;&#x975E;&#x9178;&#x5206;&#x6CCC;&#x7D30;&#x80DE;&#x306E;&#x5897;&#x52A0;&#x304B;&#x3089;&#x751F;&#x3058;&#x308B;&#x5370;&#x8C61;&#x7684;&#x306A;&#x80C3;&#x817A;&#x904E;&#x5F62;&#x6210;&#x3068;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;KCNQ1 &#x5C55;&#x793A; kcne2 &#x304B;&#x3089;&#x80C3;&#x817A;&#x306E;&#x7570;&#x5E38;&#x5206;&#x5E03; (-/-) &#x30DE;&#x30A6;&#x30B9;&#x975E;&#x9178;&#x5206;&#x6CCC;&#x7D30;&#x80DE;&#x306B;&#x304A;&#x3051;&#x308B;&#x767A;&#x73FE;&#x306E;&#x5897;&#x52A0;&#x3067;&#x3002;&#x58C1;&#x7D30;&#x80DE; (+/-) &#x306E;&#x30DE;&#x30A6;&#x30B9; kcne2 &#x304B;&#x3089;&#x901A;&#x5E38;&#x30A2;&#x30FC;&#x30AD;&#x30C6;&#x30AF;&#x30C1;&#x30E3;&#x3067;&#x3059;&#x304C;&#x30D7;&#x30ED;&#x30C8;&#x30F3;&#x306E;&#x5206;&#x6CCC;&#x304C;&#x4F4E;&#x4E0B;&#x3057;&#x3001;kcne2 (+/-) &#x306E;&#x30DE;&#x30A6;&#x30B9;&#x3060;&#x3063;&#x305F; hypochlorhydric&#x3001;&#x907A;&#x4F1D;&#x5B50;&#x7DDA;&#x91CF;&#x52B9;&#x679C;&#x3068;&#x80C3;&#x9178;&#x5206;&#x6CCC;&#x306E;&#x4E3B;&#x8981;&#x306A;&#x6B20;&#x9665;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x3001;KCNE2 &#x304C;&#x80C3;&#x9178;&#x5206;&#x6CCC;, KCNE &#x907A;&#x4F1D;&#x5B50;&#x30D5;&#x30A1;&#x30DF;&#x30EA;&#x30FC;&#x306E;&#x30E1;&#x30F3;&#x30D0;&#x30FC;&#x304C;&#x901A;&#x5E38;&#x306E;&#x6D88;&#x5316;&#x7BA1;&#x6A5F;&#x80FD;&#x306B;&#x5FC5;&#x8981;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x6700;&#x521D;&#x306E;&#x907A;&#x4F1D;&#x5B66;&#x7684;&#x8A3C;&#x62E0;&#x306F;&#x4E0D;&#x53EF;&#x6B20;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

KCNE2 &#x30AB;&#x30EA;&#x30A6;&#x30E0; &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x88DC;&#x52A9;&#x7684;&#x306A;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x306F;&#x80C3;&#x9178;&#x306E;&#x5206;&#x6CCC;&#x306E;&#x305F;&#x3081;&#x4E0D;&#x53EF;&#x6B20;&#x3067;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

KCNE &#xAC00;&#xC871;&#xC5D0; &#xC788;&#xB294; &#xC720;&#xC804;&#xC790; &#xD558;&#xB098;&#xC758; &#xB9C9; &#xD6A1;&#xB2E8; &#xB3C4;&#xBA54;&#xC778; &#xBCF4;&#xC870; subunits co-assemble&#xB294; &#xADF8;&#xB4E4;&#xC758; &#xAE30;&#xB2A5;&#xC744; &#xBCC0;&#xACBD;&#xD560; &#xD558; &#xC804;&#xC555; &#xAC1C;&#xD3D0; &#xCE7C;&#xB968; (Kv) &#xCC44;&#xB110; &#xC54C;&#xD30C; &#xC18C; &#xB2E8;&#xC704;&#xC640; &#xC778;&#xCF54;&#xB529;. KCNE2 (MiRP1 &#xB77C;&#xACE0;&#xB3C4; &#xD568;)&#xC5D0; &#xD45C;&#xD604; &#xD558; &#xACE0; &#xC778;&#xAC04;&#xC758; &#xC2EC;&#xC7A5; &#xBD80;&#xC815;&#xB9E5;&#xACFC; &#xAD00;&#xB828; &#xB41C; &#xC2EC;&#xC7A5; Kv &#xC54C;&#xD30C; subunits hERG &#xBC0F; &#xCCB4; &#xC678; KCNQ1 &#xBCC0;&#xC870;. KCNE2 &#xBC0F; kcnq1&#xB3C4; &#xD45C;&#xD604; &#xB429;&#xB2C8;&#xB2E4; &#xC815;&#xC218; &#xB9AC; &#xC138;&#xD3EC;&#xC5D0; &#xD22C;&#xAE30; &#xAC70;&#xAE30; &#xAE30;&#xBCF8; &#xCC44;&#xB110; &#xBCF5;&#xC7A1; &#xD55C; &#xD615;&#xC131;&#xC744; &#xC120;&#xB3C4;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xAC00; murine kcne2 &#xC720;&#xC804;&#xC790;&#xB97C; &#xC911;&#xB2E8; &#xD558; &#xACE0; &#xADF8; kcne2 &#xBC1C;&#xACAC; (-/-) &#xC0DD;&#xC950;&#xAC00; &#xC2EC;&#xD788; &#xAC10;&#xC18C; &#xB41C; &#xC815;&#xC218; &#xB9AC; &#xC140; &#xC591;&#xC131;&#xC790; &#xBD84; &#xBE44;, &#xBE44;&#xC815;&#xC0C1;&#xC801;&#xC778; &#xC815;&#xC218; &#xB9AC; &#xC138;&#xD3EC; &#xD615;&#xD0DC;&#xD559;, achlorhydria, hypergastrinemia, &#xBC0F; &#xB208;&#xC5D0; &#xB744;&#xB294; &#xC704; &#xC120;&#xC758; &#xC99D;&#xC2DD; &#xBE44; &#xC0B0; &#xBD84; &#xC138;&#xD3EC; &#xC218;&#xC758; &#xC99D;&#xAC00;&#xC5D0;&#xC11C; &#xBC1C;&#xC0DD; &#xD558;&#xB294; &#xC2EC;&#xAC01;&#xD55C; &#xC704; &#xD615;. KCNQ1 &#xC804;&#xC2DC; kcne2&#xC5D0;&#xC11C; &#xC704; &#xC0D8;&#xC758; &#xBE44;&#xC815;&#xC0C1;&#xC801;&#xC778; &#xBD84;&#xD3EC; (-/-) &#xB9C8;&#xC6B0;&#xC2A4;, &#xBE44; &#xC0B0; &#xBD84; &#xC140; &#xC99D;&#xAC00; &#xC2DD;&#xC73C;&#xB85C;. &#xC815;&#xC218; &#xB9AC; &#xC138;&#xD3EC; (+) &#xB9C8;&#xC6B0;&#xC2A4; kcne2 &#xC77C;&#xBC18; &#xAC74;&#xCD95; &#xC804;&#xC2DC; &#xD558;&#xC9C0;&#xB9CC; &#xC591;&#xC131;&#xC790; &#xBD84; &#xBE44; &#xAC10;&#xC18C;&#xC640; (+) &#xB9C8;&#xC6B0;&#xC2A4; kcne2 &#xD588;&#xB2E4; hypochlorhydric, &#xC720;&#xC804;&#xC790; &#xD22C;&#xC5EC; &#xD6A8;&#xACFC; &#xC704; &#xC0B0; &#xBD84; &#xBE44;&#xC758; &#xC8FC;&#xC694; &#xACB0;&#xD568;&#xC744; &#xB098;&#xD0C0;&#xB0B4;&#xB294;. &#xC774;&#xB7EC;&#xD55C; &#xB370;&#xC774;&#xD130; KCNE2 &#xC704; &#xC0B0; &#xBD84; &#xBE44;, KCNE &#xC720;&#xC804;&#xC790; &#xAC00;&#xC871;&#xC758; &#xC77C;&#xC6D0;&#xC740; &#xC815;&#xC0C1;&#xC801;&#xC778; &#xC704;&#xC7A5; &#xAE30;&#xB2A5;&#xC5D0; &#xD544;&#xC694;&#xD55C; &#xCCAB; &#xBC88;&#xC9F8; &#xC720;&#xC804;&#xC790; &#xC99D;&#xAC70;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC218;&#xC801;&#xC785;&#xB2C8;&#xB2E4; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;.

KCNE2 &#xCE7C;&#xB968; &#xCC44;&#xB110; &#xBCF4;&#xC870; &#xC18C; &#xB2E8;&#xC704; &#xC704; &#xC0B0; &#xBD84; &#xBE44;&#xC5D0; &#xD544;&#xC218;&#xC801;&#xC785;&#xB2C8;&#xB2E4;.

Genes de la familia KCNE codifican solo dominio transmembrana auxiliares subunidades que co-assemble con subunidades alfa de canales de potasio voltaje (Kv) para alterar su funci&oacute;n. KCNE2 (tambi&eacute;n conocido como MiRP1) se expresa en el coraz&oacute;n, se asocia con arritmia cardiaca humana y modula cardiaca Kv subunidades alfa hERG y KCNQ1 in vitro. KCNE2 y KCNQ1 se expresan tambi&eacute;n en las c&eacute;lulas parietales, conduce a la especulaci&oacute;n que forman un complejo de canales nativos all&iacute;. Aqu&iacute;, nos interrumpe el gen murino kcne2 y eso kcne2 (- / -) ratones tienen un fenotipo severo g&aacute;strico con secreci&oacute;n de protones profundamente reducida c&eacute;lula parietal, morfolog&iacute;a anormal de la c&eacute;lula parietal, aclorhidria, Hipergastrinemia y llamativa hiperplasia glandular g&aacute;strica derivadas de un aumento en el n&uacute;mero de c&eacute;lulas secretoras no &aacute;cido. KCNQ1 exhibi&oacute; distribuci&oacute;n anormal en gl&aacute;ndulas g&aacute;stricas de kcne2 (- / -) ratones, con aumento de la expresi&oacute;n en c&eacute;lulas secretoras no &aacute;cido. Las c&eacute;lulas parietales de kcne2 (+/-) ratones exhibieron arquitectura normal pero reduce la secreci&oacute;n de protones, y kcne2 (+/-) ratones fueron hypochlorhydric, lo que indica un efecto de la dosis g&eacute;nica y un defecto primario en la secreci&oacute;n &aacute;cida g&aacute;strica. Estos datos demuestran que KCNE2 es esencial para la secreci&oacute;n &aacute;cida g&aacute;strica, la primera evidencia gen&eacute;tica que un miembro de la familia de genes KCNE es necesario para la funci&oacute;n gastrointestinal normal.

La subunidad auxiliar de la canal de potasio KCNE2 es esencial para la secreci&oacute;n &aacute;cida g&aacute;strica.

The epithelial Na(+) channel (ENaC) is a multimeric membrane protein consisting of three subunits, alpha, beta, and gamma. The total number of subunits per functional channel complex has been described variously to follow either a tetrameric arrangement of 2alpha:1beta:1gamma or a higher-ordered stoichiometry of 3alpha:3beta:3gamma. 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 alpha, beta, or gamma subunits. Recordings from patches in oocytes expressing a single species, wild type or mutant, of alpha, beta, and gamma 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 alpha-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 alpha-subunit. However, intermediate or hybrid conductances were not observed with coexpression of wild-type and mutant betaG529A or gammaG534E subunits. Our results support a tetrameric arrangement of ENaC subunits where 2alpha, 1beta, and 1gamma come together around central pore.

Determination of epithelial Na+ channel subunit stoichiometry from single-channel conductances.

&#x5168;&#x5185;&#x53CD;&#x5C04;&#x8367;&#x5149;&#x663E;&#x5FAE;&#x955C; (TIRFM) &#x56FE;&#x50CF;&#x7684;&#x7EC6;&#x80DE;&#x819C;&#x7EC6;&#x80DE;&#x8D28;&#x6DB2;&#x754C;&#x9762;&#x548C;&#x4E4B;&#x524D;&#x548C;&#x671F;&#x95F4;&#x80DE;&#x5DF2;&#x5141;&#x8BB8;&#x4E2A;&#x522B;&#x5206;&#x6CCC;&#x9897;&#x7C92;&#x7684;&#x884C;&#x4E3A;&#x7684;&#x89C1;&#x89E3;&#x3002;&#x5BF9;&#x52A8;&#x6001;&#x7684;&#x80DE;&#xFF0C;&#x7EC6;&#x80DE;&#x8D28;&#x819C;&#x7684;&#x5176;&#x4ED6;&#x4F19;&#x4F34;&#x77E5;&#x5C11;&#x5F97;&#x591A;&#x3002;&#x5728;&#x8FD9;&#x9879;&#x7814;&#x7A76;&#xFF0C;&#x6211;&#x4EEC;&#x62A5;&#x544A;&#x7531;&#x4E8E;&#x80DE;&#x7EC6;&#x80DE;&#x819C;&#x62D3;&#x6251;&#x4E2D;&#x68C0;&#x6D4B;&#x5FEB;&#x901F;&#x7684;&#x4E9A;&#x5FAE;&#x7C73;&#x7EA7;&#x66F4;&#x6539; TIRFM &#x57FA;&#x4E8E;&#x6781;&#x5316;&#x6280;&#x672F;&#x7684;&#x6267;&#x884C;&#x60C5;&#x51B5;&#x3002;Postfusion &#x9897;&#x7C92;&#x819C;&#x7B49;&#x79BB;&#x5B50;&#x819C;&#x590D;&#x5408;&#x9884;&#x6D4B;&#x62D3;&#x6251;&#x7ED3;&#x6784;&#x7684;&#x56FE;&#x50CF;&#x6A21;&#x62DF;&#x540C;&#x65F6;&#x51FA;&#x793A;&#x65F6;&#x6280;&#x672F;&#x7684;&#x7406;&#x8BBA;&#x5206;&#x6790;&#x3002;DiI &#x67D3;&#x8272;&#x725B;&#x80BE;&#x4E0A;&#x817A;&#x55DC;&#x94EC;&#x7EC6;&#x80DE;&#x4F7F;&#x7528;&#x504F;&#x632F;&#x7684; TIRFM &#x5B9E;&#x9A8C;&#x8BC1;&#x660E;&#x5FEB;&#x901F;&#x548C;&#x591A;&#x6837;&#x7684;&#x4E9A;&#x5FAE;&#x7C73;&#x7EA7;&#x62D3;&#x6251;&#x66F4;&#x6539;&#x4E2D;&#x7EC6;&#x80DE;&#x819C;&#x7684;&#x80DE;&#x878D;&#x5408;&#x540E;&#x7ACB;&#x5373;&#x53D1;&#x751F;&#x7684;&#x5730;&#x70B9;&#x3002;&#x6211;&#x4EEC;&#x63D0;&#x4F9B;&#x8BE5;&#x5730;&#x533A;&#x9AD8;&#x5C14;&#x57FA; dynamin &#x9E1F;&#x82F7;&#x4E09;&#x78F7;&#x9178;&#x9176;&#x6D3B;&#x6027;&#x7684;&#x6291;&#x5236;&#x4F5C;&#x7528;&#x6539;&#x53D8;&#x548C;&#x65F6;&#x95F4;&#x4E0A;&#x6709;&#x522B;&#x4E8E;&#x8861;&#x91CF;&#x7684; VMAT2 pHluorin &#x7684;&#x5185;&#x541E;&#x4F5C;&#x7528;&#x7684;&#x6301;&#x4E45;&#x6027;&#x66F2;&#x7387;&#x7684;&#x76F4;&#x63A5;&#x8BC1;&#x636E;&#x3002;

&#x672C;&#x5730;&#x5316;&#x7684;&#x7EC6;&#x80DE;&#x819C;&#x540E;&#x80DE;&#x7684;&#x6781;&#x5316; TIRFM &#x53EF;&#x89C6;&#x5316;&#x62D3;&#x6251;&#x7684;&#x53D8;&#x5316;&#x3002;

Total internal reflection fluorescence microscopy (TIRFM) images the plasma membrane-cytosol interface and has allowed insights into the behavior of individual secretory granules before and during exocytosis. Much less is known about the dynamics of the other partner in exocytosis, the plasma membrane. In this study, we report the implementation of a TIRFM-based polarization technique to detect rapid submicrometer changes in plasma membrane topology as a result of exocytosis. A theoretical analysis of the technique is presented together with image simulations of predicted topologies of the postfusion granule membrane-plasma membrane complex. Experiments on diI-stained bovine adrenal chromaffin cells using polarized TIRFM demonstrate rapid and varied submicrometer changes in plasma membrane topology at sites of exocytosis that occur immediately upon fusion. We provide direct evidence for a persistent curvature in the exocytotic region that is altered by inhibition of dynamin guanosine triphosphatase activity and is temporally distinct from endocytosis measured by VMAT2-pHluorin.

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

Microscopie de fluorescence de r&eacute;flexion totale interne (TIRFM), images de l&#39;interface de la membrane plasmique-cytosol et a permis de mieux comprendre le comportement des granules s&eacute;cr&eacute;teurs individuels avant et pendant l&#39;exocytose. Beaucoup moins est connu au sujet de la dynamique de l&#39;autre partenaire dans l&#39;exocytose, la membrane plasmique. Dans cette &eacute;tude, nous pr&eacute;sentons la mise en &oelig;uvre d&#39;une technique de polarisation de base TIRFM pour d&eacute;tecter les changements de SUBMICROMETRIQUES rapide dans la topologie de la membrane plasmique &agrave; la suite de l&#39;exocytose. Une analyse th&eacute;orique de la technique est pr&eacute;sent&eacute;e ainsi que des simulations d&#39;image des topologies pr&eacute;dites de la membrane de plasma membrane des granules post&eacute;rieurement complexe. Exp&eacute;riences sur teint&eacute; diI cellules chromaffines surr&eacute;nales bovines en utilisant TIRFM polaris&eacute;e d&eacute;montrent les changements SUBMICROMETRIQUES rapide et vari&eacute; dans la topologie de la membrane plasmique dans les sites d&#39;exocytose survenant imm&eacute;diatement apr&egrave;s la fusion. Nous fournissons une preuve directe pour une courbure persistante dans la r&eacute;gion d&#39;exocytotique qui est alt&eacute;r&eacute;e par l&#39;inhibition de l&#39;activit&eacute; de triphosphatase dynamine guanosine et temporellement distinct d&#39;endocytose mesur&eacute;e par VMAT2-pHluorin.

Changements topologiques localis&eacute;es de la membrane plasmique apr&egrave;s exocytose visualis&eacute;e par TIRFM polaris&eacute;e.

Totalreflexion Fluoreszenzmikroskopie (TIRFM) Bildern die Plasmamembran-Zytosol-Schnittstelle und erm&ouml;glichte Einblicke in das Verhalten der einzelnen sekretorisches Granulat, vor und w&auml;hrend der Exozytose. &Uuml;ber die Dynamik des anderen Partners in Exozytose, der Plasmamembran ist weit weniger bekannt. In dieser Studie berichten wir die Durchf&uuml;hrung einer Polarisation TIRFM basierende Technik Plasmamembran Topologie durch Exozytose rapid seiner &Auml;nderungen erkennen. Eine theoretische Analyse der Technik wird zusammen mit Bild-Simulationen der vorhergesagten Topologien der postfusion Untermodul Membran-Plasmamembran Komplex pr&auml;sentiert. Experimente an DiI-befleckten bovine adrenal chromaffin Zellen mit polarisierten TIRFM zeigen schnelle und abwechslungsreiche seiner &Auml;nderungen in Plasmamembran Topologie an Standorten der Exozytose, die sofort nach Verschmelzung auftreten. Wir bieten direkte Beweise f&uuml;r eine dauerhafte Kr&uuml;mmung in der exocytotic Region, die durch Hemmung der Dynamin Guanosin Adenosintriphosphatase Aktivit&auml;t ver&auml;ndert und unterscheidet sich zeitlich von Endozytose von VMAT2-pHluorin gemessen.

Lokalisierte topologischen &Auml;nderungen der Plasmamembran nach Exozytose von polarisierten TIRFM visualisiert.

Microscopia di fluorescenza di riflessione interna totale (TIRFM) immagini dell&#39;interfaccia membrana plasmatica-citosol e ha permesso intuizioni il comportamento dei singoli granuli secretori prima e durante la esocitosi. Molto meno &egrave; conosciuto circa la dinamica di altro partner in esocitosi, la membrana plasmatica. In questo studio, segnaliamo l&#39;implementazione di una tecnica di polarizzazione basata su TIRFM per rilevare i cambiamenti rapidi submicrometriche nella topologia della membrana plasmatica a seguito di esocitosi. Analisi teorica della tecnica presentata insieme alle simulazioni di immagine del predetti topologie della membrana plasma-membrana granulo postfusion complessa. Esperimenti su tinto diI bovina surrenale cellule cromaffini utilizzando polarizzata TIRFM dimostrano cambiamenti submicrometriche rapida e varia nella topologia della membrana plasmatica presso siti di esocitosi che si verificano immediatamente dopo la fusione. Forniamo prove dirette per una curvatura persistente nella regione questo che viene alterata dall&#39;inibizione dell&#39;attivit&agrave; di dynamin guanosina trifosfatasi e temporalmente distinto da endocitosi misurata da VMAT2-pHluorin.

Cambiamenti topologici localizzate della membrana del plasma su esocitosi visualizzati da TIRFM polarizzata.

&#x5168;&#x5185;&#x90E8;&#x53CD;&#x5C04;&#x86CD;&#x5149;&#x9855;&#x5FAE;&#x93E1; (TIRFM) &#x306E;&#x7D30;&#x80DE;&#x819C;-&#x7D30;&#x80DE;&#x8CEA;&#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30D5;&#x30A7;&#x30A4;&#x30B9; &#x30A4;&#x30E1;&#x30FC;&#x30B8;&#x304A;&#x3088;&#x3073;&#x524D;&#x306B;&#x3001;&#x3068;&#x30A8;&#x30AD;&#x30BD;&#x30B5;&#x30A4;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x306E;&#x4E2D;&#x306B;&#x500B;&#x3005; &#x306E;&#x5206;&#x6CCC;&#x9846;&#x7C92;&#x306E;&#x884C;&#x52D5;&#x306B;&#x6D1E;&#x5BDF;&#x529B;&#x3092;&#x8A31;&#x53EF;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x306F;&#x308B;&#x304B;&#x306B;&#x5C11;&#x306A;&#x3044;&#x7D30;&#x80DE;&#x819C;&#x30A8;&#x30AD;&#x30BD;&#x30B5;&#x30A4;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x306E;&#x4ED6;&#x306E;&#x30D1;&#x30FC;&#x30C8;&#x30CA;&#x30FC;&#x306E;&#x30C0;&#x30A4;&#x30CA;&#x30DF;&#x30AF;&#x30B9;&#x306B;&#x3064;&#x3044;&#x3066;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;, &#x539F;&#x5F62;&#x8CEA;&#x819C;&#x30C8;&#x30DD;&#x30ED;&#x30B8;&#x30FC; &#x30A8;&#x30AD;&#x30BD;&#x30B5;&#x30A4;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x306E;&#x7D50;&#x679C;&#x3068;&#x3057;&#x3066;&#x6025;&#x901F;&#x306A;&#x30B5;&#x30D6;&#x30DE;&#x30A4;&#x30AF;&#x30ED; &#x30E1;&#x30FC;&#x30BF;&#x30FC;&#x306E;&#x5909;&#x66F4;&#x3092;&#x691C;&#x51FA;&#x3059;&#x308B;&#x504F;&#x5149;&#x89B3;&#x5BDF;&#x306B;&#x57FA;&#x3065;&#x304F;&#x624B;&#x6CD5;&#x306E;&#x5B9F;&#x88C5;&#x3092;&#x5831;&#x544A;&#x3057;&#x307E;&#x3059;&#x3002;&#x7406;&#x8AD6;&#x89E3;&#x6790;&#x624B;&#x6CD5;&#x306E; postfusion &#x9846;&#x7C92;&#x819C;&#x30D7;&#x30E9;&#x30BA;&#x30DE;&#x819C;&#x8907;&#x5408;&#x4F53;&#x306E;&#x4E88;&#x6E2C;&#x3055;&#x308C;&#x305F;&#x30C8;&#x30DD;&#x30ED;&#x30B8;&#x306E;&#x753B;&#x50CF;&#x30B7;&#x30DF;&#x30E5;&#x30EC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3068;&#x3068;&#x3082;&#x306B;&#x63D0;&#x793A;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x504F;&#x5149;&#x89B3;&#x5BDF;&#x3092;&#x7528;&#x3044;&#x305F; diI &#x30B9;&#x30C6;&#x30F3;&#x30C9; &#x30A6;&#x30B7;&#x526F;&#x814E;&#x9AC4;&#x8CEA;&#x7D30;&#x80DE;&#x306E;&#x5B9F;&#x9A13;&#x306F;&#x6025;&#x901F;&#x3067;&#x591A;&#x69D8;&#x306A;&#x30B5;&#x30D6;&#x30DE;&#x30A4;&#x30AF;&#x30ED; &#x30E1;&#x30FC;&#x30BF;&#x30FC; &#x30C8;&#x30DD;&#x30ED;&#x30B8;&#x306E;&#x5909;&#x66F4;&#x306B;&#x539F;&#x5F62;&#x8CEA;&#x819C;&#x878D;&#x5408;&#x6642;&#x306B;&#x3059;&#x3050;&#x306B;&#x767A;&#x751F;&#x3059;&#x308B;&#x30B5;&#x30A4;&#x30C8; &#x30A8;&#x30AD;&#x30BD;&#x30B5;&#x30A4;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x6C38;&#x7D9A;&#x7684;&#x306A;&#x66F2;&#x7387;&#x5206;&#x6CCC;&#x306B;&#x304A;&#x3051;&#x308B;&#x30C0;&#x30A4;&#x30CA;&#x30DF;&#x30F3; &#x30B0;&#x30A2;&#x30CE;&#x30B7;&#x30F3; &#x30B0;&#x30A2;&#x30CE;&#x30B7;&#x30F3;&#x6D3B;&#x6027;&#x306E;&#x963B;&#x5BB3;&#x306B;&#x3088;&#x3063;&#x3066;&#x5909;&#x66F4;&#x3055;&#x308C;&#x308B;&#x3068;&#x306F;&#x7570;&#x306A;&#x308B;&#x4E00;&#x6642;&#x7684; VMAT2-pHluorin &#x306B;&#x3088;&#x3063;&#x3066;&#x6E2C;&#x5B9A;&#x3057;&#x305F;&#x30A8;&#x30F3;&#x30C9;&#x30B5;&#x30A4;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x306E;&#x76F4;&#x63A5;&#x7684;&#x8A3C;&#x62E0;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;

&#x7D30;&#x80DE;&#x819C;&#x30A8;&#x30AD;&#x30BD;&#x30B5;&#x30A4;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x504F;&#x5149;&#x89B3;&#x5BDF;&#x306B;&#x3088;&#x308B;&#x53EF;&#x8996;&#x5316;&#x6642;&#x306E;&#x30ED;&#x30FC;&#x30AB;&#x30E9;&#x30A4;&#x30BA;&#x3055;&#x308C;&#x305F;&#x30C8;&#x30DD;&#x30ED;&#x30B8;&#x30FC;&#x306E;&#x5909;&#x5316;&#x3002;

&#xCD1D; &#xB0B4;&#xBD80; &#xBC18;&#xC0AC; &#xD615;&#xAD11; &#xD604;&#xBBF8;&#xACBD; (TIRFM) &#xD50C;&#xB77C;&#xC988;&#xB9C8; &#xBA64;&#xBE0C;&#xB808;&#xC778; cytosol &#xC778;&#xD130;&#xD398;&#xC774;&#xC2A4; &#xC774;&#xBBF8;&#xC9C0;&#xC640; &#xC804;&#xACFC; exocytosis &#xC911; &#xAC1C;&#xBCC4; &#xBD84;&#xACFC; &#xB9BD;&#xC758; &#xB3D9;&#xC791;&#xC5D0; &#xB300; &#xD55C; &#xD1B5;&#xCC30;&#xB825;&#xC744; &#xD5C8;&#xC6A9; &#xD558;&#xACE0;&#xC788;&#xB2E4;. &#xD6E8;&#xC52C; &#xB35C; exocytosis, &#xC6D0;&#xD615;&#xC9C8; &#xB9C9;&#xC5D0;&#xC11C;&#xC5D0;&#xC11C; &#xB2E4;&#xB978; &#xD30C;&#xD2B8;&#xB108;&#xC758; &#xC5ED;&#xD559;&#xC5D0; &#xB300; &#xD55C; &#xC54C;&#xB824;&#xC838; &#xC788;&#xB2E4;. &#xC774; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; &#xC6B0;&#xB9AC; exocytosis &#xACB0;&#xACFC;&#xB85C; &#xD50C;&#xB77C;&#xC988;&#xB9C8; &#xBA64;&#xBE0C;&#xB808;&#xC778; &#xD1A0;&#xD3F4;&#xB85C;&#xC9C0;&#xC758; &#xBE60;&#xB978; submicrometer &#xBCC0;&#xD654;&#xB97C; &#xAC10;&#xC9C0; &#xD558;&#xB294; TIRFM &#xAE30;&#xBC18; &#xC591;&#xADF9; &#xD654; &#xAE30;&#xC220; &#xAD6C;&#xD604;&#xC744; &#xBCF4;&#xACE0;. &#xAE30;&#xC220;&#xC5D0; &#xB300; &#xD55C; &#xC774;&#xB860;&#xC801; &#xBD84;&#xC11D; &#xBCF5;&#xC7A1; &#xD55C; postfusion&#xACFC; &#xB9BD; &#xB9C9; &#xD50C;&#xB77C;&#xC988;&#xB9C8; &#xBA64;&#xBE0C;&#xB808;&#xC778;&#xC758; &#xC608;&#xCE21; &#xB41C; &#xD1A0;&#xD3F4;&#xB85C;&#xC9C0;&#xC758; &#xC2DC;&#xBBAC;&#xB808;&#xC774;&#xC158;&#xC744; &#xC774;&#xBBF8;&#xC9C0;&#xC640; &#xD568;&#xAED8; &#xC81C;&#xACF5; &#xB429;&#xB2C8;&#xB2E4;. DiI &#xC5BC;&#xB8E9;&#xC9C4; &#xC18C; &#xBD80; &#xC2E0; &#xD06C;&#xB86C; &#xCE5C;&#xD654; &#xC138;&#xD3EC;&#xC5D0; &#xD3B8;&#xAD11;&#xB41C; TIRFM&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC2E4;&#xD5D8; exocytosis&#xC758; &#xC735;&#xD569;&#xC5D0; &#xB530;&#xB77C; &#xC989;&#xC2DC; &#xBC1C;&#xC0DD; &#xD558;&#xB294; &#xC0AC;&#xC774;&#xD2B8;&#xC5D0;&#xC11C; &#xD50C;&#xB77C;&#xC988;&#xB9C8; &#xBA64;&#xBE0C;&#xB808;&#xC778; &#xD1A0;&#xD3F4;&#xB85C;&#xC9C0; &#xBCC0;&#xACBD; &#xC2E0;&#xC18D; &#xD558; &#xACE0; &#xB2E4;&#xC591; &#xD55C; submicrometer&#xB97C; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. Dynamin guanosine triphosphatase &#xD65C;&#xB3D9;&#xC758; &#xC800;&#xD574;&#xC5D0; &#xC758;&#xD574; &#xBCC0;&#xACBD; &#xB418; &#xACE0; &#xC77C;&#xC2DC;&#xC801;&#xC73C;&#xB85C; &#xB2E4;&#xB978; endocytosis VMAT2 Phluorin&#xC5D0; &#xC758;&#xD574; &#xCE21;&#xC815; &#xD558;&#xB294; exocytotic &#xC601;&#xC5ED;&#xC5D0; &#xC601;&#xAD6C; &#xACE1;&#xB960;&#xC5D0; &#xB300; &#xD55C; &#xC9C1;&#xC811;&#xC801;&#xC778; &#xC99D;&#xAC70;&#xB97C; &#xC81C;&#xACF5; &#xD558; &#xACE0; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

Exocytosis &#xD3B8;&#xAD11;&#xB41C; TIRFM&#xC5D0; &#xC758;&#xD574; &#xC2DC;&#xAC01;&#xC5D0; &#xB530;&#xB77C; &#xD50C;&#xB77C;&#xC988;&#xB9C8; &#xBA64;&#xBE0C;&#xB808;&#xC778;&#xC758; &#xC9C0;&#xC5ED;&#xD654; &#xB41C; &#xD1A0;&#xD3F4;&#xB85C;&#xC9C0; &#xBCC0;&#xACBD;.

Microscop&iacute;a de fluorescencia de reflexi&oacute;n interna total (TIRFM) im&aacute;genes de la interfaz de la membrana plasm&aacute;tica-citosol y ha permitido penetraciones en el comportamiento de los gr&aacute;nulos secretores antes y durante la exocitosis. Mucho menos se sabe sobre la din&aacute;mica del otro socio en la exocitosis, la membrana plasm&aacute;tica. En este estudio, divulgamos la aplicaci&oacute;n de una t&eacute;cnica de polarizaci&oacute;n basadas en TIRFM para detectar Submicrom&eacute;trica r&aacute;pida cambios en la topolog&iacute;a de la membrana plasm&aacute;tica por exocitosis. Un an&aacute;lisis te&oacute;rico de la t&eacute;cnica se presenta junto con simulaciones de imagen de topolog&iacute;as previstas de la membrana de la membrana de plasma de gr&aacute;nulo postfusion compleja. Experimentos en diI te&ntilde;ido bovinos suprarrenales c&eacute;lulas cromafines usando polarizado TIRFM demuestran cambios Submicrom&eacute;trica r&aacute;pido y variado en la topolog&iacute;a de la membrana plasm&aacute;tica en sitios de exocitosis que ocurren inmediatamente sobre la fusi&oacute;n. Proporcionamos evidencia directa de una curvatura persistente en la regi&oacute;n exocit&oacute;tico que es alterada por la inhibici&oacute;n de la actividad de Dinamina guanosina trifosfatasa y temporal difiere de endocitosis midiendo VMAT2-pHluorin.

Cambios topol&oacute;gicos localizados de la membrana plasm&aacute;tica en exocitosis visualizado por TIRFM polarizado.

We have recently developed a combination of polarization and total internal reflection fluorescence microscopy (pTIRFM) to monitor changes in plasma membrane topology occurring after fusion of chromaffin granules. In this report, pTIRFM is further exploited to reveal two major findings in regards to the secretory pathway in bovine chromaffin cells. First, we show that changes in membrane topology are sometimes detected even prior to fusion. This occurs with high probability in a small subset of granules that appear in the evanescent field during the experiment. On these occasions, the plasma membrane invaginates with the movement just preceding the appearance of a granule in the evanescent field. Such events may represent a direct interaction of the granule with the plasma membrane. Second, we show that the topological fate of the post-fusion, granule/plasma membrane intermediate is regulated by divalent cation. When Sr2+ is used instead of Ca2+ to trigger exocytosis, membrane topology in the exocytotic region is stabilized with significant curvature and indentation.

Polarized TIRFM reveals changes in plasma membrane topology before and during granule fusion.

Dynamin is a master regulator of membrane fission in endocytosis. However, a function for dynamin immediately upon fusion has also been suspected from a variety of experiments that measured release of granule contents. The role of dynamin guanosine triphosphate hydrolase (GTPase) activity in controlling fusion pore expansion and postfusion granule membrane topology was investigated using polarization optics and total internal reflection fluorescence microscopy (pTIRFM) and amperometry. A dynamin-1 (Dyn1) mutant with increased GTPase activity resulted in transient deformations consistent with rapid fusion pore widening after exocytosis; a Dyn1 mutant with decreased activity slowed fusion pore widening by stabilizing postfusion granule membrane deformations. The experiments indicate that, in addition to its role in endocytosis, GTPase activity of dynamin regulates the rapidity of fusion pore expansion from tens of milliseconds to seconds after fusion. These findings expand the membrane-sculpting repertoire of dynamin to include the regulation of immediate postfusion events in exocytosis that control the rate of release of soluble granule contents.

A new role for the dynamin GTPase in the regulation of fusion pore expansion.

Voltage-gated potassium (Kv) channels are critical for neuronal excitability and are targeted to specific subcellular compartments to carry out their unique functions. While it is widely believed that Kv channels exist as heteromeric complexes in neurons, direct tests of the hypothesis that specific heteromeric channel populations display divergent spatial and temporal dynamics are limited. Using a bimolecular fluorescence complementation approach, we monitored the assembly and localization of cell surface channel complexes in living cells. While PSD95-mediated clustering was subunit independent, selective visualization of heteromeric Kv complexes in rat hippocampal neurons revealed subunit-dependent localization that was not predicted by analyzing individual subunits. Assembly of Kv1.1 with Kv1.4 prevented axonal localization but not surface expression, while inclusion of Kv1.2 imparted clustering at presynaptic sites and decreased channel mobility within the axon. This mechanism by which specific Kv channel subunits can act in a dominant manner to impose unique trafficking properties to heteromeric complexes extended to Shab-related family of Kv channels. When coexpressed, Kv2.1 and Kv2.2 heteromultimers did not aggregate in somatodendritic clusters observed with expression of Kv2.1 alone. These studies demonstrate selective axonal trafficking and surface localization of distinct Kv channels based on their subunit composition.

Subunit-dependent axonal trafficking of distinct alpha heteromeric potassium channel complexes.

Assays for real-time investigation of exocytosis typically measure what is released from the granule. From this, inferences are made about the dynamics of membrane remodeling as fusion progresses from start to finish. We have recently undertaken a different approach to investigate the fusion process, by focusing not primarily on the granule, but rather its partner in exocytosis - the plasma membrane. We have been guided by the idea that biochemical interactions between the granule and plasma membranes before and during fusion, cause changes in membrane conformation. To enable study of membrane conformation, a novel imaging technique was developed combining polarized excitation of an oriented membrane probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI) with total internal reflection fluorescence microscopy (pTIRFM). Because this technique measures changes in membrane conformation (or deformations) directly, its usefulness persists even after granule cargo reporter (catecholamine, or protein), is no longer present. In this mini-review, we first summarize the workings of pTIRFM. We then discuss the application of the technique to investigate deformations in the membrane preceding fusion, and later, during fusion pore expansion. Finally, we discuss how expansion of the fusion pore may be regulated by the GTPase activity of dynamin.

Real-time imaging of plasma membrane deformations reveals pre-fusion membrane curvature changes and a role for dynamin in the regulation of fusion pore expansion.

Carbon fiber microelectrodes provide the ideal platform for performing ultrafast, selective measurements of electroactive brain molecules. This article highlights the current status of the use of carbon fiber microelectrodes in neurochemical measurements, outlining the most cutting edge findings and technological advances in amperometry and fast-scan cyclic voltammetry.

Article	Year
Studies of endogenous G-protein-mediated pathways in neurons by whole-cell electrophysiology. Methods in enzymology\| PubMed ID: 11665634	2002
Biochemical approaches to study interaction of calcium channels with RGS12 in primary neuronal cultures. Methods in enzymology\| PubMed ID: 11665642	2002
RNA interference reveals that endogenous Xenopus MinK-related peptides govern mammalian K+ channel function in oocyte expression studies. The Journal of biological chemistry\| PubMed ID: 12529362	2003
Pharmacogenetic considerations in diseases of cardiac ion channels. The Journal of pharmacology and experimental therapeutics\| PubMed ID: 14561846	2003
N-type Ca2+ channels as scaffold proteins in the assembly of signaling molecules for GABAB receptor effects. The Journal of biological chemistry\| PubMed ID: 15047708	2004
Does hERG coassemble with a beta subunit? Evidence for roles of MinK and MiRP1. Novartis Foundation symposium\| PubMed ID: 16050264	2005
Open probability of the epithelial sodium channel is regulated by intracellular sodium. The Journal of physiology\| PubMed ID: 16690707	2006
The KCNE2 potassium channel ancillary subunit is essential for gastric acid secretion. The Journal of biological chemistry\| PubMed ID: 16754665	2006
Determination of epithelial Na+ channel subunit stoichiometry from single-channel conductances. The Journal of general physiology\| PubMed ID: 17562820	2007
Localized topological changes of the plasma membrane upon exocytosis visualized by polarized TIRFM. The Journal of cell biology\| PubMed ID: 20142424	2010
Polarized TIRFM reveals changes in plasma membrane topology before and during granule fusion. Cellular and molecular neurobiology\| PubMed ID: 21061164	2010
A new role for the dynamin GTPase in the regulation of fusion pore expansion. Molecular biology of the cell\| PubMed ID: 21460182	2011
Subunit-dependent axonal trafficking of distinct alpha heteromeric potassium channel complexes. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 21917805	2011
Real-time imaging of plasma membrane deformations reveals pre-fusion membrane curvature changes and a role for dynamin in the regulation of fusion pore expansion. Journal of neurochemistry\| PubMed ID: 22671293	2012
Ultrafast detection and quantification of brain signaling molecules with carbon fiber microelectrodes. Analytical chemistry\| PubMed ID: 22881278	2012

Arun Anantharam

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