Voltage-independent Inhibition of P/Q-type Ca2+ Channels in Adrenal Chromaffin Cells via a Neuronal Ca2+ Sensor-1-dependent Pathway Involves Src Family Tyrosine Kinase

Abstract

In common with many neurons, adrenal chromaffin cells possess distinct voltage-dependent and voltage-in-dependent pathways for Ca2+ channel regulation. In this study, the voltage-independent pathway was revealed by addition of naloxone and suramin to remove tonic blockade of Ca2+ currents via opioid and purinergic receptors due to autocrine feedback inhibition. This pathway requires the Ca2+-binding protein neuronal calcium sensor-1 (NCS-1). The voltage-dependent path. way was pertussis toxin-sensitive, whereas the voltage-independent pathway was largely pertussis toxin-insensitive. Characterization of the voltage-independent inhibition of Ca2+ currents revealed that it did not involve protein kinase C-dependent signaling pathways but did require the activity of a Src family tyrosine kinase. Two structurally distinct Src kinase inhibitors, 4-amino-5-(4-methylphenyl)7-(t-butyl)pyrazolo[3,4-d] pyrimidine (PP1) and a Src inhibitory peptide, increased the Ca2+ currents, and no further increase in Ca2+ currents was elicited by addition of naloxone and suramin. In addition, the Src-like kinase appeared to act in the same pathway as NCS-1. In contrast, addition of PP1 did not prevent a voltage-dependent facilitation elicited by a strong pre-pulse depolarization indicating that this pathway was independent of Src kinase activity. PPI no longer increased Ca 2+ currents after addition of the P/Q-type channel blocker ω-agatoxin TK. The α1A subunit of P/Q-type Ca 2+ channels was immunoprecipitated from chromaffin cell extracts and found to be phosphorylated in a PP1-sensitive manner by endogenous kinases in the immunoprecipitate. A high molecular mass (around 220 kDa) form of the α1A subunit was detected by anti-phosphotyrosine, suggesting a possible target for Src family kinase action. These data demonstrate a voltage-independent mechanism for autocrine inhibition of P/Q-type Ca 2+ channel currents in chromaffin cells that requires Src family kinase activity and suggests that this may be a widely distributed pathway for Ca2+ channel regulation.