Syntaxin 1A has a specific binding site in the H3 domain that is critical for targeting of H+-ATPase to apical membrane of renal epithelial cells

1 Renal Section, Boston University Medical Center, and Departments of 2 Medicine, 3 Physiology, and 4 Pathology, Boston University School of Medicine, Boston, Massachusetts; and 5 Department of Nephrology, First Affiliated Hospital, Zhongshan University, Guangzhou, China Submitted 2 February 2005 ; accepted in final form 27 April 2005 H + transport in the collecting duct is regulated by exocytic insertion of H + -ATPase-laden vesicles into the apical membrane. The soluble N -ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptor (SNARE) proteins are critical for exocytosis. Syntaxin 1A contains three main domains, SNARE N, H3, and carboxy-terminal transmembrane domain. Several syntaxin isoforms form SNARE fusion complexes through the H3 domain; only syntaxin 1A, through its H3 domain, also binds H + -ATPase. This raised the possibility that there are separate binding sites within the H3 domain of syntaxin 1A for H + -ATPase and for SNARE proteins. A series of truncations in the H3 domain of syntaxin 1A were made and expressed as glutathione S -transferase (GST) fusion proteins. We determined the amount of H + -ATPase and SNARE proteins in rat kidney homogenate that complexed with GST-syntaxin molecules. Full-length syntaxin isoforms and syntaxin-1A C [amino acids (aa) 1–264] formed complexes with H + -ATPase and SNAP23 and vesicle-associated membrane polypeptide (VAMP). A cassette within the H3 portion was found that bound H + -ATPase (aa 235–264) and another that bound SNAP23 and VAMP (aa 190–234) to an equivalent degree as full-length syntaxin. However, the aa 235–264 cassette alone without the SNARE N (aa 1–160) does not bind but requires ligation to the SNARE N to bind H + -ATPase. When this chimerical construct was transected into inner medullary collecting duct cells it inhibited intracellular pH recovery, an index of H + -ATPase mediated secretion. We conclude that within the H3 domain of syntaxin 1A is a unique cassette that participates in the binding of the H + -ATPase to the apical membrane and confers specificity of syntaxin 1A in the process of H + -ATPase exocytosis. soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor proteins; exocytosis; H ++ transport Address for reprint requests and other correspondence: J. H. Schwartz, Evans Biomedical Research Center, 650 Albany St., Boston, MA 02118-2908 (e-mail: jhsch{at}bu.edu )