Study on the modulation mechanism of neuronal calcuim channels

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On the role of voltage-dependent calcium channels in the neurons, two lines of studies have been conducted : one was the experiment using Xenopus oocyte translation system in which modulation mechanism at the molecular level is to be studied ; the other was designed to investigate the modulation of calcium channel function at primary afferent nerve terminals in spinal cord slices.The outline of results are as follows :(1) Oocyte system : I have clarified that tonic inhibition of N-type channels by G-proteins is mediated by Gbetagamma subunit. The inhibition by Gbetagamma is competitive to that by protein kinase C, and washable by continuous intracellular perfusion with GTP-containing solution. Stimulation of coexpressed opioid receptors with agonist elicited a voltage-dependent inhibition of N-type channels, as shown in the tonic inhibition, however, the inhibition by opioids were voltage-resistant in the case of P/Q-type channels. The voltage-resistant inhibition could be isolated by applying GTPgammaS to N-type channels, and by intracellular perfusion with GTP-containing solution. I have also found two alternatively splicing variants of human N-type channels by RT-PCR.(2) Spinal slice : EPSPs were recorded from spinal cord slices with dorsal horn attached by blind patch clamp technique. The evoked responses were categorized into two groups : one is mediated by Adelta fibers ; the other is mediated by C fibers. Repetitive high-frequency stimulation of C fiber inputs induced a slow depolarization of spinal neurons. This slow depolarization was presynaptically inhibited either by agonists for m opioid, GABA_A, GABA_B, or 5-HT_<1A> receptors. I have also clarified that N-type channels are involved in the normal excitatory neurotransmission which is mediated by glutamic acid, and that P-type channels are involved in the slow depolarization of spinal neurons.