| Popis: |
N-type voltage-gated calcium channels play key roles in many cellular functions including the regulation of neurotransmitter and hormone release. Ca2+ influx through these channels is tightly regulated by second messenger pathways and intrinsic channel properties including voltage-dependent and calcium-dependent inactivation. Less is known about the recovery of calcium channels following inactivation, or if the kinetics of recovery can be modulated by signaling pathways. Recombinant N-type channels (CaV2.2 (α1B), α2δ, and β1b) were expressed in HEK293 cells and barium used as the charge carrier to isolate voltage-dependent inactivation. An individual step depolarization lasting 1-10s, or trains of brief step depolarizations were used to produce inactivation, and recovery was tracked using brief steps at given intervals following the stimulus. Following “short” ( 10s). Acute application of the phorbol ester PMA (50-200nM) had little effect on the resting amplitude of IBa or inactivation during 5-50Hz stimulus trains. However, the slower time constant of IBa recovery was significantly prolonged by PMA. This action of PMA was not blocked by inhibitors of PKC that target the catalytic domain of the enzyme (bisindolylmaleimide I, Go6983, PKC inhibitory peptide 19-36), but was blocked by calphostin-C which targets the diacylglycerol binding C1 domain of PKC and other proteins. Intracellular dialysis with GDP-β-S also blocked the actions of PMA on recovery from inactivation. PMA had a similar effect on endogenous ICa recorded from bovine adrenal chromaffin cells. Our data identify a novel PKC-independent mechanism by which PMA (and perhaps diacylglycerol signaling) modulates the availability of N-type calcium channels during sustained or repetitive activity.Supported by NIH/NINDS (NS052446) and VUMC Discovery grant. |
