We investigated, by using the patch clamp technique, Ca2+-mediated regulation of heterologously expressed TRPC6 and TRPC7 proteins in HEK293 cells, two closely related homologues of the transient receptor potential (TRP) family and molecular candidates for native receptor-operated Ca2+ entry channels. With nystatin-perforated recording, the magnitude and time courses of activation and inactivation of carbachol (CCh; 100 microM)-activated TRPC6 currents (I(TRPC6)) were enhanced and accelerated, respectively, by extracellular Ca2+ (Ca2+(o)) whether it was continuously present or applied after receptor stimulation. In contrast, Ca2+(o) solely inhibited TRPC7 currents (I(TRPC7)). Vigorous buffering of intracellular Ca2+ (Ca2+(i)) under conventional whole-cell clamp abolished the slow potentiating (i.e. accelerated activation) and inactivating effects of Ca2+(o), disclosing fast potentiation (EC50: approximately 0.4 mM) and inhibition (IC50: approximately 4 mM) of I(TRPC6) and fast inhibition (IC50: approximately 0.4 mM) of I(TRPC7). This inhibition of I(TRPC6) and I(TRPC7) seems to be associated with voltage-dependent reductions of unitary conductance and open probability at the single channel level, whereas the potentiation of I(TRPC6) showed little voltage dependence and was mimicked by Sr2+ but not Ba2+. The activation process of I(TRPC6) or its acceleration by Ca2+(o) probably involves phosphorylation by calmodulin (CaM)-dependent kinase II (CaMKII), as pretreatment with calmidazolium (3 microM), coexpression of Ca2+-insensitive mutant CaM, and intracellular perfusion of the non-hydrolysable ATP analogue AMP-PNP and a CaMKII-specific inhibitory peptide all effectively prevented channel activation. However, this was not observed for TRPC7. Instead, single CCh-activated TRPC7 channel activity was concentration-dependently suppressed by nanomolar Ca2+(i) via CaM and conversely enhanced by IP3. In addition, the inactivation time course of I(TRPC6) was significantly retarded by pharmacological inhibition of protein kinase C (PKC). These results collectively suggest that TRPC6 and 7 channels are multiply regulated by Ca2+ from both sides of the membrane through differential Ca2+-CaM-dependent and -independent mechanisms.

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In acutely isolated rat sacral dorsal commisural nucleus (SDCN) neurones, application of kainate (KA) reversibly potentiated glycine‐evoked Cl− currents (IGly) in a concentration‐dependent manner. The cellular events underlying the interaction between non‐NMDA receptors and glycine receptors were studied by using nystatin‐perforated patch and cell‐attached single‐channel recording modes. The action of KA was not accompanied by a shift in the reversal potential for IGly. In dose‐response curves, KA potentiated IGly without significantly changing glycine binding affinity. GYKI 52466 blocked while NS‐102 had no effect on the KA‐induced potentiation of IGly. The potentiation was reduced when KA was applied in a Ca2+‐free extracellular solution or in the presence of BAPTA AM, and was independent of the activation of voltage‐dependent Ca2+ channels. Pretreatment with KN‐62, a selective Ca2+‐calmodulin‐dependent protein kinase II (CaMKII) inhibitor, abolished the action of KA. Inhibition of calcineurin converted the KA‐induced potentiation to a sustained one. Single‐channel recordings revealed that KA decreased the mean closing time of glycine‐gated single‐channel activity, resulting in an increase in the probability of channel opening. It is proposed that Ca2+ entry through AMPA receptors modulates the glycine receptor function via coactivation of CaMKII and calcineurin in SDCN neurones. This interaction may provide a new postsynaptic mechanism for control of inhibitory synaptic signalling and represent one of the important regulatory mechanisms of spinal nociception.

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