The Interaction between Syntaxin 1A and Cystic Fibrosis Transmembrane Conductance Regulator Cl− Channels Is Mechanistically Distinct from Syntaxin 1A-SNARE Interactions

Radhika Ganeshan,Anke Di,Deborah J Nelson,Michael W Quick,Kevin L Kirk

doi:10.1074/jbc.m211790200

Abstract

Syntaxin 1A binds to and inhibits epithelial cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels and synaptic Ca(2+) channels in addition to participating in SNARE complex assembly and membrane fusion. We exploited the isoform-specific nature of the interaction between syntaxin 1A and CFTR to identify residues in the H3 domain of this SNARE (SNARE motif) that influence CFTR binding and regulation. Mutating isoform-specific residues that map to the surface of syntaxin 1A in the SNARE complex led to the identification of two sets of hydrophilic residues that are important for binding to and regulating CFTR channels or for binding to the syntaxin regulatory protein Munc-18a. None of these mutations affected syntaxin 1A binding to other SNAREs or the assembly and stability of SNARE complexes in vitro. Conversely, the syntaxin 1A-CFTR interaction was unaffected by mutating hydrophobic residues in the H3 domain that influence SNARE complex stability and Ca(2+) channel regulation. Thus, CFTR channel regulation by syntaxin 1A involves hydrophilic interactions that are mechanistically distinct from the hydrophobic interactions that mediate SNARE complex formation and Ca(2+) channel regulation by this t-SNARE.

Highlights

The cystic fibrosis transmembrane conductance regulator (CFTR)1 is a cyclic AMP-activated anion channel that mediates salt and fluid transport across epithelial cells [1]
We replaced Glu238 with leucine, since human syntaxins 4 and 5 have leucine at the corresponding position. The abilities of these syntaxin 1A mutants to bind to CFTR as well as to SNAP-23 and to Munc-18 were tested using the indicated GST-syn1A⌬C proteins to pull down native proteins from HT29-Cl19A epithelial cell lysates (Fig. 2A)
CFTR Binding Is Not Inhibited by Mutating Hydrophobic Residues Implicated in SNARE Complex Formation and Ca2ϩ Channel Regulation (A240V/V244A)—Since the preceding results indicate that two hydrophilic residues (Ser225 and Glu238) of syntaxin 1A are important for its interaction with CFTR, we examined the effect of mutating residues buried in the hydrophobic layers of the SNARE complex on CFTR binding

Summary

Introduction

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated anion channel that mediates salt and fluid transport across epithelial cells [1]. Syntaxin 1A inhibits CFTR-mediated chloride currents in a variety of cell types and expression systems This effect of syntaxin 1A on CFTR channel activity may be due to the fact that the amino-terminal cytoplasmic tail of CFTR, to which syntaxin 1A binds, regulates channel gating apparently by interacting with the regulatory domain and/or nucleotide binding domain 1 [4]. It is not clear if the structural basis of the interactions between syntaxin 1A and different ion channels is similar In this regard, Bezprozvanny et al [14] have reported that the regulation of voltage-gated Ca2ϩ channels by syntaxin 1A is disrupted by point mutations in specific hydrophobic residues in the H3 domain that are implicated in SNARE complex stability

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Conclusion