title: Rab25 influences functional Cav1.2 channel surface expression in arterial smooth muscle cells.
abstract: Plasma membrane-localized CaV1.2 channels are the primary calcium (Ca(2+)) influx pathway in arterial smooth muscle cells (myocytes). CaV1.2 channels regulate several cellular functions, including contractility and gene expression, but the trafficking pathways that control the surface expression of these proteins are unclear. Similarly, expression and physiological functions of small Rab GTPases, proteins that control vesicular trafficking in arterial myocytes, are poorly understood. Here, we investigated Rab proteins that control functional surface abundance of CaV1.2 channels in cerebral artery myocytes. Western blotting indicated that Rab25, a GTPase previously associated with apical recycling endosomes, is expressed in cerebral artery myocytes. Immunofluorescence Förster resonance energy transfer (immunoFRET) microscopy demonstrated that Rab25 locates in close spatial proximity to CaV1.2 channels in myocytes. Rab25 knockdown using siRNA reduced CaV1.2 surface and intracellular abundance in arteries, as determined using arterial biotinylation. In contrast, CaV1.2 was not located nearby Rab11A or Rab4 and CaV1.2 protein was unaltered by Rab11A or Rab4A knockdown. Rab25 knockdown resulted in CaV1.2 degradation by a mechanism involving both lysosomal and proteasomal pathways and reduced whole cell CaV1.2 current density but did not alter voltage dependence of current activation or inactivation in isolated myocytes. Rab25 knockdown also inhibited depolarization (20-60 mM K(+)) and pressure-induced vasoconstriction (myogenic tone) in cerebral arteries. These data indicate that Rab25 is expressed in arterial myocytes where it promotes surface expression of CaV1.2 channels to control pressure- and depolarization-induced vasoconstriction.
authors.affiliation: Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee.
authors.forename: John P
authors.initials: JP
authors.lastname: Bannister
authors.affiliation: Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee.
authors.forename: Simon
authors.initials: S
authors.lastname: Bulley
authors.affiliation: Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee.
authors.forename: M Dennis
authors.initials: MD
authors.lastname: Leo
authors.affiliation: Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Kidd
authors.affiliation: Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee jjaggar@uthsc.edu.
authors.forename: Jonathan H
authors.initials: JH
authors.lastname: Jaggar
mesh_terms: D000818:Animals; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D002478:Cells, Cultured; D002536:Cerebral Arteries / Q000201:enzymology; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000201:enzymology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D021381:Protein Transport; D059748:Proteolysis; D034622:RNA Interference; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D014162:Transfection; D014661:Vasoconstriction; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C504728:Cacna1c protein, rat; D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); C509763:Rab25 protein, human; D046988:Proteasome Endopeptidase Complex; D020691:rab GTP-Binding Proteins
keywords: Rab GTPase; arterial smooth muscle cells; smooth muscle; vasoconstriction; voltage-dependent calcium channel