engleski

Ca2+-dependent facilitation of Ca2+ current in sensory neurons: Loss of excitability regulation after nerve injury.

In dissociated sensory neurons, we have previously identified Ca2+-dependent facilitation (CDF) of ICa during a series of 5ms test pulses (0.4Hz, from Vh of -95mV to Vtest of -10mV) that followed a 2s depolarization (-10mV). After initial inhibition, ICa increased to 23±4% above pre-conditioning baseline at 22±1s after conditioning. Preventing Ca2+ influx eliminated CDF. We now report additional features of this CDF. Conditioning depolarizations as brief as 250ms also produced CDF (15±1%), and depolarization from a more natural Vh of -65mV similarly showed CDF (24±1%). To replicate typical neuronal activity, conditioning was applied as a train of action potential waveforms (100Hz for 1s), which also produced CDF (17%). These findings show that CDF is a natural phenomenon and not an artifact of a particular protocol. Reducing the series of test pulses to only a single pulse 20s after conditioning retained CDF (26±1%), indicating that CDF persists in a quiescent neuron. Since 5ms test pulses did not allow ICa to consistently reach peak, we employed a longer test depolarization (50ms). This demonstrated a 13±3% decreased time to peak ICa after conditioning. Altered kinetics alone does not account for CDF, since conditioning also increased peak ICa (23±4%). Release of Ca2+ from intracellular stores does not contribute to CDF since blockade with dantrolene (10µM) had no effect. CDF suppresses excitability through Ca2+ activation of K+ channels. Intracellular recording from intact DRGs have previously shown that AP generation (induced by 2nA current injection for 50ms) is decreased to 46±6% of baseline 10s after conditioning (2s, 0mV). We now report loss of this suppression (89±9% with conditioning, 90±6% without) 20d after peripheral axonal injury, consistent with CDF loss we have observed in dissociated neurons after SNL. Since CDF was sensitive to blockers of N-type Ca2+-channel and CaMKII, we tested the ability of CaMKII to phosphorylate this channel. We found that recombinant CaMKII phosphorylates endogenous Cav2.2 immunoprecipitated from DRG in an in vitro kinase reaction, but no phosphorylation was detected when CaMKII was omitted. This supports the proposed phosphorylation of endogenous Cav2.2 by CaMKII as an underlying mechanism for CDF in sensory neurons.

sensory neuron; nerve injury; CaMKII

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