Protein Kinase Cα and P-Type Ca2+ Channel CaV2.1 in Red Blood Cell Calcium Signalling

Abstract

Background/Aims: Protein kinase Cα (PKCα) is activated by an increase in cytosolic Ca²⁺ in red blood cells (RBCs). Previous work has suggested that PKCα directly stimulates the Ca_V2.1 channel, whereas other studies revealed that Ca_V2.1 is insensitive to activation by PKC. The aim of this study was to resolve this discrepancy. Methods: We performed experiments based on a single cell read-out of the intracellular Ca²⁺ 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. Results: Treatment of RBCs with phorbol 12-myristate 13-acetate (PMA) led to two distinct populations of cells with an increase in intracellular Ca²⁺: a weak-responding and a strong-responding population. The EC₅₀ of PMA for the number of cells with Ca²⁺ 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 Ca_V2.1 inhibitor ω-agatoxin TK and the broad protein kinase C inhibitor Gö6983, we are able to show that there are two independent PMA-activated Ca²⁺ entry processes: the first is independent of Ca_V2.1 and directly PKCα-activated, while the second is associated with a likely indirect activation of Ca_V2.1. Further studies using lysophosphatidic acid (LPA) as a stimulation agent have provided additional evidence that PKCα and Ca_V2.1 are not directly interconnected in a signalling chain. Conclusion: Although we provide evidence for a lack of interaction between PKCα and Ca_V2.1 in RBCs, further studies are required to decipher the signalling relationship between LPA, PKCα and Ca_V2.1.