Rab25 influences functional Cav1.2 channel surface expression in arterial smooth muscle cells

Plasma membrane-localized Ca(V)1.2 channels are the primary calcium (Ca(V)1.2 ) influx pathway in arterial smooth muscle cells (myocytes). Ca(V)1.2 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 Ca(V)1.2 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 Forster resonance energy transfer (immunoFRET) microscopy demonstrated that Rab25 locates in close spatial proximity to Ca(V)1.2 channels in myocytes. Rab25 knockdown using siRNA reduced Ca(V)1.2 surface and intracellular abundance in arteries, as determined using arterial biotinylation. In contrast, Ca(V)1.2 was not located nearby Rab11A or Rab4 and Ca(V)1.2 protein was unaltered by Rab11A or Rab4A knockdown. Rab25 knockdown resulted in Ca(V)1.2 degradation by a mechanism involving both lysosomal and proteasomal pathways and reduced whole cell Ca(V)1.2 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 Ca(V)1.2 channels to control pressure- and depolarization-induced vasoconstriction.