Potassium channels are key regulators of smooth muscle contractile state. Accumulating evidence suggests that they may act as signal transducers, translating extracellular stimuli by hormones, neurotransmitters and peptides, to intracellular signaling processes. To undertake this task, K+ channels must be in close proximity and forming macromolecular complexes with membrane receptors and intracellular signaling molecules. Moreover, this association should be dynamic and susceptible to change under different physiological conditions, for example, under the influence of sex hormones during pregnancy. Thus, our main hypothesis states that K+ channels localize to specific microdomains in smooth muscle (SM) and are intimately associated with receptors and signaling cascades either directly or indirectly via scaffolding proteins, and that, this association and subcellular distribution is influenced by sex hormones. To test this hypothesis, we will use as model system uterine SM that undergoes dramatic remodeling in structure and function during pregnancy and postpartum, and a multidisciplinary approach analyzing functional association, native subcellular colocalization, molecularity of protein-protein interactions, and the hormonal mechanisms leading to remodeling. Studies will focus on voltage and Ca2+-activated K+ (MaxiK, BKca) and Kv4.3 channels, 5-HT receptors, c-Src tyrosine kinase and caveolins, all critical regulators of SM function. Preliminary data indicate that, in myometrium: i) a new c-Src-related protein with tyrosine kinase activity is further induced in late pregnancy, ii) c-Src, Kv4.3 and caveolin comigrate in detergent resistant fractions and can be coimmunoprecipitated, iii) recombinant MaxiK carboxyl tail interacts with c-Src, iv) MaxiK/Kv4.3 in single myocytes are clustered mimicking c-Src, v) c-Stc and caveolin-la mRNAs are quadrupled during pregnancy, vi) spontaneous contractility is under the control of Src kinase <-> tyrosine phosphatase, and vii) its mechanical output is regulated by the new agonist ->c-Src tyrosine kinase ->MaxiK pathway. Thus, the Specific Aims are to investigate: 1) the identity of a new pregnancy-induced Src-like protein; 2) whether in SM MaxiK/Kv4.3 form macromolecular complexes with c-Src, caveolin and 5-HT receptors, their subcellular colocalization, and potential remodeling across gestation; 3) the molecular interactions of MaxiK-c-Src-caveolin-receptor comp/exes; 4) the mechanism(s) triggered by sex hormones leading to pregnancy-related changes of c-Src-caveolins- MaxiK/Kv4.3 associations, and their recovery in postpartum; and 5) the functional impact and mechanism of Src modulation of MaxiK/Kv4.3 channel activity, and their role in spontaneous or 5-HT induced contractility. These studies will increase our understanding of SM biology and may lead to better treatments of SM-related pathologies.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
1R01HD046510-01
Application #
6756694
Study Section
Biophysics of Synapses, Channels, and Transporters Study Section (BSCT)
Program Officer
Higgins, Rosemary
Project Start
2004-06-01
Project End
2009-03-31
Budget Start
2004-06-01
Budget End
2005-03-31
Support Year
1
Fiscal Year
2004
Total Cost
$345,188
Indirect Cost
Name
University of California Los Angeles
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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