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

John P. Bannister, Simon Bulley, Marie Dennis Leo, Michael W. Kidd, Jonathan Jaggar

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Plasma membrane-localized CaV1.2 channels are the primary calcium (Ca2+) 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 depolarizationinduced vasoconstriction.

Original languageEnglish (US)
Pages (from-to)C885-C893
JournalAmerican Journal of Physiology - Cell Physiology
Volume310
Issue number11
DOIs
StatePublished - Jun 1 2016

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Muscle Cells
Smooth Muscle Myocytes
Cerebral Arteries
Vasoconstriction
rab4 GTP-Binding Proteins
rab GTP-Binding Proteins
Biotinylation
Pressure
Monomeric GTP-Binding Proteins
Endosomes
GTP Phosphohydrolases
Energy Transfer
Small Interfering RNA
Fluorescent Antibody Technique
Microscopy
Membrane Proteins
Proteins
Arteries
Western Blotting
Cell Membrane

All Science Journal Classification (ASJC) codes

  • Physiology
  • Cell Biology

Cite this

Rab25 influences functional Cav1.2 channel surface expression in arterial smooth muscle cells. / Bannister, John P.; Bulley, Simon; Leo, Marie Dennis; Kidd, Michael W.; Jaggar, Jonathan.

In: American Journal of Physiology - Cell Physiology, Vol. 310, No. 11, 01.06.2016, p. C885-C893.

Research output: Contribution to journalArticle

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AB - Plasma membrane-localized CaV1.2 channels are the primary calcium (Ca2+) 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 depolarizationinduced vasoconstriction.

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