Regulation of CFTR chloride channels by syntaxin and Munc18 isoforms

Abstract

The cystic fibrosis gene encodes a cyclic AMP-gated chloride channel (CFTR) that mediates electrolyte transport across the luminal surfaces of a variety of epithelial cells. The molecular mechanisms that modulate CFTR activity in epithelial tissues are poorly understood. Here we show that CFTR is regulated by an epithelially expressed syntaxin (syntaxin 1A), a membrane protein that also modulates neurosecretion and calcium-channel gating in brain. Syntaxin 1A physically interacts with CFTR chloride channels and regulates CFTR-mediated currents both in Xenopus oocytes and in epithelial cells that normally express these proteins. The physical and functional interactions between syntaxin 1A and CFTR are blocked by a syntaxin-binding protein of the Munc18 protein family (also called n-Secl). Our results indicate that CFTR function in epithelial cells is regulated by an interplay between syntaxin and Munc18 isoforms.

New paradigms of CFTR chloride channel regulation

“…This integral membrane protein is a member of a large family of proteins that regulate membrane fusion in diverse tissues referred to as T-SNARES or Q-SNARES (reviewed in [53,54]). Syntaxin 1A protein is also expressed in gut and airway epithelial cells, although at much lower levels than in brain [55]. The coexpression of syntaxin 1A with CFTR in heterologous expression systems such as Xenopus oocytes leads to an inhibition of CFTR-mediated currents [55].…”

“…Syntaxin 1A protein is also expressed in gut and airway epithelial cells, although at much lower levels than in brain [55]. The coexpression of syntaxin 1A with CFTR in heterologous expression systems such as Xenopus oocytes leads to an inhibition of CFTR-mediated currents [55]. Moreover, CFTR can be rescued from this inhibition by multiple reagents, including (i) Munc-18a, a high-affinity syntaxin-binding protein [56,57] that is also expressed in gut epithelial cells [55]; (ii) soluble syntaxin 1A peptides; and (iii) botulinum neurotoxin C1, an endoprotease that cleaves membrane-anchored syntaxin 1A [51].…”

“…The coexpression of syntaxin 1A with CFTR in heterologous expression systems such as Xenopus oocytes leads to an inhibition of CFTR-mediated currents [55]. Moreover, CFTR can be rescued from this inhibition by multiple reagents, including (i) Munc-18a, a high-affinity syntaxin-binding protein [56,57] that is also expressed in gut epithelial cells [55]; (ii) soluble syntaxin 1A peptides; and (iii) botulinum neurotoxin C1, an endoprotease that cleaves membrane-anchored syntaxin 1A [51]. Importantly, each of these reagents also markedly stimulates CFTRmediated chloride currents in various colonic epithelial cell lines that normally express CFTR and syntaxin 1A [55,58].…”

Defects in Processing and Trafficking of Cystic Fibrosis Transmembrane Conductance Regulator

“…Further evidence came from studies demonstrating physical interaction of CFTR with syntaxin 1A, a protein known to be involved in neurosecretion and anchoring of CFTR to the cytoskeleton [142, 143]. The effects of CFTR on membrane turnover seem to be limited to clathrin-dependent endocytosis, i.e.…”

<em>In Vitro</em> Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

“…Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo [8][9][10][11][12][13][14][15][16][17][18][19] . In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains.…”