Referenced in: none

Automatically associated channels: Cav2.1

Title: Protein kinase Cα and P-type Ca channel CaV2.1 in red blood cell calcium signalling.

Authors: Lisa Wagner-Britz, Jue Wang, Lars Kaestner, Ingolf Bernhardt

Journal, date & volume: Cell. Physiol. Biochem., 2013 , 31, 883-91

PubMed link: http://www.ncbi.nlm.nih.gov/pubmed/23817128

Abstract
Protein kinase Cα (PKCα) is activated by an increase in cytosolic Ca(2+) in red blood cells (RBCs). Previous work has suggested that PKCα directly stimulates the CaV2.1 channel, whereas other studies revealed that CaV2.1 is insensitive to activation by PKC. The aim of this study was to resolve this discrepancy.We performed experiments based on a single cell read-out of the intracellular Ca(2+) concentration in terms of Fluo-4 fluorescence intensity and phosphatidylserine exposure to the external membrane leaflet. Measurement modalities included flow cytometry and live cell imaging.Treatment of RBCs with phorbol 12-myristate 13-acetate (PMA) led to two distinct populations of cells with an increase in intracellular Ca(2+): a weak-responding and a strong-responding population. The EC50 of PMA for the number of cells with Ca(2+) elevation was 2.7±1.2 µM; for phosphatidylserine exposure to the external membrane surface, it was 2.8±0.5 µM; and for RBC haemolysis, it was 2.9±0.5 µM. Using pharmacological manipulation with the CaV2.1 inhibitor ω-agatoxin TK and the broad protein kinase C inhibitor Gö6983, we are able to show that there are two independent PMA-activated Ca(2+) entry processes: the first is independent of CaV2.1 and directly PKCα-activated, while the second is associated with a likely indirect activation of CaV2.1. Further studies using lysophosphatidic acid (LPA) as a stimulation agent have provided additional evidence that PKCα and CaV2.1 are not directly interconnected in a signalling chain.Although we provide evidence for a lack of interaction between PKCα and CaV2.1 in RBCs, further studies are required to decipher the signalling relationship between LPA, PKCα and CaV2.1.