Requirement of the TRPC1 cation channel in the generation of transient Ca²⁺ oscillations by the calcium-sensing receptor

The calcium-sensing receptor (CaR) is an allosteric protein that responds to extracellular Ca²⁺ ([Ca²⁺]_o) and aromatic amino acids with the production of different patterns of oscillations in intracellular Ca²⁺ concentration ([Ca²⁺]_i). An increase in [Ca²⁺]_o stimulates phospholipase C-mediated production of inositol 1,4,5-trisphosphate and causes sinusoidal oscillations in [Ca²⁺]_i. Conversely, aromatic amino acid-induced CaR activation does not stimulate phospholipase C but engages an unidentified signaling mechanism that promotes transient oscillations in [Ca ²⁺]_i. We show here that the [Ca²⁺]_i oscillations stimulated by aromatic amino acids were selectively abolished by TRPC1 down-regulation using either a pool of small inhibitory RNAs (siRNAs) or two different individual siRNAs that targeted different coding regions of TRPC1. Furthermore, [Ca²⁺]_i oscillations stimulated by aromatic amino acids were also abolished by inhibition of TRPC1 function with an antibody that binds the pore region of the channel. We also show that aromatic amino acid-stimulated [Ca²⁺]_i oscillations can be prevented by protein kinase C (PKC) inhibitors or siRNA-mediated PKCα down-regulation and impaired by either calmodulin antagonists or by the expression of a dominant-negative calmodulin mutant. We propose a model for the generation of CaR-mediated transient [Ca²⁺]_i oscillations that integrates its stimulation by aromatic amino acids with TRPC1 regulation by PKC and calmodulin.