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

In common with many neurons, adrenal chromaffin cells possess distinct volt age-dependent and voltage-independent 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 v oltage-dependent pathway was pertussis toxin-sensitive, whereas the voltage -independent pathway was largely pertussis toxin-insensitive. Characterizat ion of the voltage-independent inhibition of Ca2+ currents revealed that it did not involve protein kinase C-dependent signaling pathways but did requ ire 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+ current s, and no further increase in Ca2+ currents was elicited by addition of nal oxone 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 volta ge-dependent facilitation elicited by a strong pre-pulse depolarization ind icating that this pathway was independent of Src kinase activity. PPI no lo nger increased Ca2+ currents after addition of the P/Q-type channel blocker omega -agatoxin TK. The alpha (1A) subunit of P/Q-type Ca2+ channels was i mmunoprecipitated from chromaffin cell extracts and found to be phosphoryla ted in a PP1-sensitive manner by endogenous kinases in the immunoprecipitat e. A high molecular mass (around 220 kDa) form of the alpha (1A) subunit wa s detected by anti-phosphotyrosine, suggesting a possible target for Src fa mily kinase action. These data demonstrate a voltage-independent mechanism for autocrine inhibition of P/Q-type Ca2+ channel currents in chromaffin ce lls that requires Src family kinase activity and suggests that this may be a widely distributed pathway for Ca2+ channel regulation.