Abstract
In common with many neurons, adrenal chromaffin cells possess distinct voltage-dependent and voltage-independent pathways for Ca(2+) channel regulation. In this study, the voltage-independent pathway was revealed by addition of naloxone and suramin to remove tonic blockade of Ca(2+) currents via opioid and purinergic receptors due to autocrine feedback inhibition. This pathway requires the Ca(2+)-binding protein neuronal calcium sensor-1 (NCS-1). The voltage-dependent pathway was pertussis toxin-sensitive, whereas the voltage-independent pathway was largely pertussis toxin-insensitive. Characterization of the voltage-independent inhibition of Ca(2+) 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 Ca(2+) currents, and no further increase in Ca(2+) 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 omega-agatoxin TK. The alpha(1A) 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 alpha(1A) 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 Ca(2+) channel regulation.
Highlights
We have been examining the physiological roles of neuronal calcium sensor-1 (NCS-1) protein, a member of a family of neuronally expressed EF hand Ca2ϩ-binding proteins [16]
Our results indicate a requirement for Src family tyrosine kinase activity for this pathway acting on P/Q-type Ca2ϩ channels and that the ␣1A subunit of these channels is a substrate for Src family kinases
Voltage-dependent Inhibition of Ca2ϩ Channels in Chromaffin Cells Is Pertussis Toxin-insensitive—In this study, we generally recorded from adrenal chromaffin cells using a bath solution containing 10 mM Ba2ϩ and without perfusion of the cells
Summary
We have been examining the physiological roles of neuronal calcium sensor-1 (NCS-1) protein, a member of a family of neuronally expressed EF hand Ca2ϩ-binding proteins [16]. Autocrine inhibition of non-L-type Ca2ϩ currents, due to activation of ATP and/or opioid receptors, occurred mostly through a voltage-independent pathway, and NCS-1 appeared to be required for this pathway, but expression of NCS-1(E120Q) did not interfere with prepulse facilitation. Our results indicate a requirement for Src family tyrosine kinase activity for this pathway acting on P/Q-type Ca2ϩ channels and that the ␣1A subunit of these channels is a substrate for Src family kinases. This is the first demonstration of a regulatory pathway for P/Q-type Ca2ϩ channel inhibition involving Src family kinase activity
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