The broad goal of this proposal is to define the in vivo roles of syntaxin 1A (STX1 A) with emphasis on its modulation of CFTR chloride channels and gut physiology. CFTR is essential to normal human physiology;defects in its synthesis or regulation cause multiple disorders including cystic fibrosis (too little activity in lung) or secretory diarrhea (too much CFTR activity in gut). STX1A has been proposed to serve two roles in mammals: (i) as a key mediator of neurotransmitter release by participating in SNARE-mediated vesicle fusion at the synapse and (ii) as an inhibitor of certain ion channels including CFTR. CFTR-STX1 interactions are potentially significant at two levels. First, they may provide novel targets for the development of drugs to treat CFTR-related disorders if they substantially limit CFTR function in vivo, as implied by our tissue culture data. Second, these interactions may integrate CFTR activity with other cell functions (e.g., membrane traffic). In spite of these potential implications the proposed functional roles of STX1A in regulating neuronal secretion or CFTR function have not yet been established in a mammalian animal model. We have generated homozygous STX1A knock-out (KO) mice as a first step toward defining the in vivo significance of CFTR interactions with STX1A. These mice are viable and fertile but exhibit a detectable gut phenotype that is characterized by increased fecal output and abnormally moist feces (possible indicators of hyperactive CFTR channels). Surprisingly, the STX1A KO mice exhibit no obvious behavioral phenotype, which seems inconsistent with the proposed role of this SNARE as a key mediator of neuronal secretion. The lack of a strong neuronal phenotype may be due to the existence of a second closely related isoform (STX1B) in brain and gut. We propose to specifically define the in vivo roles of syntaxins 1A/1B in controlling gut physiology, CFTR function and neuronal secretion by pursuing three specific aims: (i) to characterize the gut and neuronal phenotypes of our existing STX1A KO mice with emphasis on CFTR function in the intestine;(ii) to generate STX1A knock-in animals which harbor mutations that disrupt the CFTR interaction but not SNARE complex assembly and (iii) to define the role of the closely related STX1B in CFTR regulation and neuronal secretion. The results should establish the biologic roles of STX1A/1B in vivo and the physiologic significance of their interactions with the CFTR channel.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL058341-12
Application #
7644917
Study Section
Gastrointestinal Cell and Molecular Biology Study Section (GCMB)
Program Officer
Banks-Schlegel, Susan P
Project Start
1996-09-30
Project End
2012-06-30
Budget Start
2009-07-01
Budget End
2010-06-30
Support Year
12
Fiscal Year
2009
Total Cost
$326,250
Indirect Cost
Name
University of Alabama Birmingham
Department
Physiology
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Hwang, Tzyh-Chang; Kirk, Kevin L (2013) The CFTR ion channel: gating, regulation, and anion permeation. Cold Spring Harb Perspect Med 3:a009498
Bernard, Karen; Kirk, Kevin L (2010) Cross-linking of ?F508-CFTR promotes its trafficking to the plasma membrane. Channels (Austin) 4:251-4
Kirk, Kevin L (2010) CFTR channels and wound healing. Focus on ""Cystic fibrosis transmembrane conductance regulator is involved in airway epithelial wound repair"". Am J Physiol Cell Physiol 299:C888-90
Thibodeau, Patrick H; Richardson 3rd, John M; Wang, Wei et al. (2010) The cystic fibrosis-causing mutation deltaF508 affects multiple steps in cystic fibrosis transmembrane conductance regulator biogenesis. J Biol Chem 285:35825-35
Ganeshan, Radhika; Nowotarski, Krzysztof; Di, Anke et al. (2007) CFTR surface expression and chloride currents are decreased by inhibitors of N-WASP and actin polymerization. Biochim Biophys Acta 1773:192-200
Ganeshan, Radhika; Di, Anke; Nelson, Deborah J et al. (2003) The interaction between syntaxin 1A and cystic fibrosis transmembrane conductance regulator Cl- channels is mechanistically distinct from syntaxin 1A-SNARE interactions. J Biol Chem 278:2876-85
Cormet-Boyaka, Estelle; Di, Anke; Chang, Steven Y et al. (2002) CFTR chloride channels are regulated by a SNAP-23/syntaxin 1A complex. Proc Natl Acad Sci U S A 99:12477-82