Iegorova O. High affinity positive modulation of P-type calcium channel by m-opioid agonists.

P-type calcium channels play a key role in the synaptic transmission between mammalian central neurons: major part of calcium entering pre-synaptic terminals delivered via these channels. Using standard whole-cell patch clamp techniques we have studied the effect of m-opioids on P-type calcium channels in acutely isolated Purkinje neurons from rat cerebellum. Extracellular application of opioid receptors agonists in the concentrations between 1 nM and 1000 nM induced excitatory or inhibitory effects on high-threshold VGCCs. The alkaloid agonist morphine is fairly selective for m-opioid receptor, although at micromolar concentrations it can interact also with k- and d-opioid receptors. In our experiments, facilitation of P-current by morphine was observed in 59% of the neurons, whereas in 12% of the neurons, this current was inhibited and in 29% of the neurons, no effect was observed. Inhibitory effect of morphine, which we observed in some cells, could be due to the previously described negative effect of k- or d-opioid receptors on VGCCs. It is also possible, that oppositely directed effects of morphine were masked in some cases. Selective -opioid agonist DAMGO in 10 nM produced a small, but consistent facilitation of current through P-type calcium channels (10±1%, n=27, p<0.001). The effect of DAMGO was rapid (less than 10 sec) and fully reversible. This effect was both concentration and voltage-dependent. The EC50 for the effect of DAMGO was 1.3±0.4 nM and the saturating concentration was 100 nM. Endogenous selective agonist of -opioid receptors, endomorphin-1 demonstrated similar action. The mechanisms that are involved in this modulation are different from the well-known mechanism involving G-proteins or processes dependent of phosphorylation. In summary, opioids exert bi-directional effects on P-type calcium channels: inhibition - through G-protein dependent pathway activated by k- or d-opioid receptors or via direct action on the P-channels, and facilitation - via G-protein-independent pathway involving the interaction with high affinity binding site resembling -opioid receptor. Our data do not exclude the possibility that opioids may interact with both m-, and with the other opioid receptors in the same cell, causing the competing effects on P-type calcium channels. It indicates the existence of a fundamentally new mechanism of regulation of calcium channels.