title: 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-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.
authors.affiliation: Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, United Kingdom.
authors.forename: J L
authors.initials: JL
authors.lastname: Weiss
authors.forename: R D
authors.initials: RD
authors.lastname: Burgoyne
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism* / Q000502:physiology; D002417:Cattle; D019439:Chromaffin Cells / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004594:Electrophysiology; D004789:Enzyme Activation; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism*; D009538:Nicotine / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D037342:Pertussis Toxin; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D004268:DNA-Binding Proteins; C083693:PP1 protein, Physcomitrella patens; D010940:Plant Proteins; D010566:Virulence Factors, Bordetella; D019000:Phosphotyrosine; D009538:Nicotine; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D019061:src-Family Kinases; D011493:Protein Kinase C; D002118:Calcium