Second messenger pathways involved in up-regulation of an L-type calcium channel

Catfish cone horizontal cells contain a voltage-gated L-type calcium channe l that is modulated by activation of metabotropic glutamate receptors (mGlu Rs). Activation of group I mGluRs with the mGluR I agonist. (S)-3,5-dihydro xyphenylglycine [(S) 3,5-DHPG], potentiated peak calcium current amplitude, shifted the membrane potential corresponding to peak current activity. and widened the calcium current's activation range. In this study, we have exa mined the mechanisms linking: activation of the mGluRs with "up-regulation" of calcium current activity. Under whole-cell voltage-clamp conditions fav oring expression of the L-type calcium current, we provide evidence that ac tivation of mGluRs initiate the diacylglyceral (DG) second messenger pathwa y to activate protein kinase C (PKC) and up-regulate calcium channel activi ty. This evidence was based on results using a number of PKC activators and inhibitors. PKC activators mimicked the effect of (S) 3,5-DHPG on calcium current activity. Up-regulation of the calcium channel by PKC activators or (S) 3,5-DHPG was eliminated if PKC inhibitors were present. These results also demonstrated that activation of group I mGluRs were linked to a pertus sis toxin sensitive G-protein. When the GTP analog, guanosine 5-0-(3-thiotr iphosphate (GTP gamma S), was allowed to diffuse into voltage-clamp cells, up-regulation of the calcium channel occurred and mimicked the effect of (S ) 3,5-DHPG. However, when pertussis toxin (PTX) was allowed to diffuse into the cell along with GTP gamma S, GTP gamma S failed to modulate calcium cu rrent activity. IP3 (inositol 1,4,5 triphosphate) is a second product produ ced by activation of group I mGluRs. Once formed, IP3 can trigger calcium r elease from IP3-sensitive intracellular stores. To determine if the IP3 sec ond messenger system was involved in up-regulation of calcium channel, (S) 3,5-DHPG was applied to voltage-clamped cone horizontal cells containing di fferent concentrations of the calcium buffer, EGTA. Low concentrations of E GTA failed to buffer calcium released from intracellular stores. In the pre sence of low EGTA concentrations, (S) 3,5-DHPG's enhancement of the calcium current amplitude was reduced. Inhibition of the calcium current amplitude in low concentrations of EGTA was eliminated in the presence of the intrac ellular calcium store blocker, heparin. These results suggest that both the DG and IP3 second messenger pathways are involved in modulation of the vol tage-gated calcium channel in catfish cone horizontal cells. The DG pathway up-regulates the voltage-gated calcium channel activity whereas calcium re leased from IP3 intracellular stores inhibits peak current amplitude.