MARCKS mediates vascular contractility through regulating interactions between voltage-gated Ca2+ channels and PIP2

Research output: Contribution to journalArticlepeer-review

  • External authors:
  • K.S. Jahan
  • J. Shi
  • H.Z.E. Greenberg
  • M.M.-A. Baudel
  • V. Barrese
  • I.A. Greenwood
  • A.P. Albert

Abstract

Phosphatidylinositol 4,5-bisphosphate (PIP 2) acts as substrate and unmodified ligand for Gq-protein-coupled receptor signalling in vascular smooth muscle cells (VSMCs) that is central for initiating contractility. The present work investigated how PIP 2 might perform these two potentially conflicting roles by studying the effect of myristoylated alanine-rich C kinase substrate (MARCKS), a PIP 2-binding protein, on vascular contractility in rat and mouse mesenteric arteries. Using wire myography, MANS peptide (MANS), a MARCKS inhibitor, produced robust contractions with a pharmacological profile suggesting a predominantly role for L-type (CaV1.2) voltage-gated Ca 2+ channels (VGCC). Knockdown of MARCKS using morpholino oligonucleotides reduced contractions induced by MANS and stimulation of α 1-adrenoceptors and thromboxane receptors with methoxamine (MO) and U46619 respectively. Immunocytochemistry and proximity ligation assays demonstrated that MARCKS and CaV1.2 proteins co-localise at the plasma membrane in unstimulated tissue, and that MANS and MO reduced these interactions and induced translocation of MARCKS from the plasma membrane to the cytosol. Dot-blots revealed greater PIP 2 binding to MARCKS than CaV1.2 in unstimulated tissue, with this binding profile reversed following stimulation by MANS and MO. MANS evoked an increase in peak amplitude and shifted the activation curve to more negative membrane potentials of whole-cell voltage-gated Ca 2+ currents, which were prevented by depleting PIP 2 levels with wortmannin. This present study indicates for the first time that MARCKS is important regulating vascular contractility and suggests that disinhibition of MARCKS by MANS or vasoconstrictors may induce contraction through releasing PIP 2 into the local environment where it increases voltage-gated Ca 2+ channel activity.

Bibliographical metadata

Original languageEnglish
JournalVascular Pharmacology
DOIs
Publication statusPublished - 21 Jul 2020