title: Role of SNAREs and H+-ATPase in the targeting of proton pump-coated vesicles to collecting duct cell apical membrane.
abstract: Recycling of H(+)-ATPase to the apical plasma membrane, mediated by vesicular exocytosis and endocytosis, is an important mechanism for controlling H(+) secretion by the collecting duct. We hypothesized that SNAREs (soluble N-ethylmaleimide-sensitive factor attachment proteins) may be involved in the targeting of H(+)-ATPase-coated vesicles. Using a tissue culture model of collecting duct H(+) secretory cells (inner medullary collecting duct (IMCD) cells), we demonstrated that they express the proteins required for SNARE-mediated exocytosis and form SNARE-fusion complexes upon stimulation of H(+)-ATPase exocytosis. Furthermore, exocytic amplification of apical H(+)-ATPase is sensitive to clostridial toxins that cleave SNAREs and thereby inhibit secretion. Thus, SNAREs are critical for H(+)-ATPase cycling to the plasma membrane. The process in IMCD cells has a feature distinct from that of neuronal cells: the SNARE complex includes and requires the vesicular cargo (H(+)-ATPase) for targeting. Using chimeras and truncations of syntaxin 1, we demonstrated that there is a specific cassette within the syntaxin 1 H3 domain that mediates binding of the SNAREs and a second distinct H3 region that binds H(+)-ATPase. Utilizing point mutations of the B1 subunit of the H(+)-ATPase, we document that this subunit contains specific targeting information for the H(+)-ATPase itself. In addition, we found that Munc-18-2, a regulator of exocytosis, plays a multifunctional role in this system: it regulates SNARE complex formation and the affinity of syntaxin 1 for H(+)-ATPase.
authors.affiliation: Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA. jhsch@bu.edu
authors.forename: J H
authors.initials: JH
authors.lastname: Schwartz
authors.forename: G
authors.initials: G
authors.lastname: Li
authors.forename: Q
authors.initials: Q
authors.lastname: Yang
authors.forename: V
authors.initials: V
authors.lastname: Suri
authors.forename: J J
authors.initials: JJ
authors.lastname: Ross
authors.forename: E A
authors.initials: EA
authors.lastname: Alexander
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D018699:Coated Vesicles / Q000502:physiology; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000502:physiology*; D023421:Models, Animal; D017494:Proton Pumps / Q000502:physiology; D006180:Proton-Translocating ATPases / Q000502:physiology*; D051381:Rats; D050600:SNARE Proteins / Q000502:physiology*