Characterization and regulation of rat microglial Ca2+ release-activated Ca2+ (CRAC) channel by protein kinases

초록

We measured the activity of the Ca2+ release-activated Ca2+ (CRAC) channel present in cultured rat microglia, using the whole-cell mode of patch clamp technique. When the concentration of divalent cations in external solution was reduced to the micromolar range, and Ca2+ chelating agent BAPTA was included in the pipette solution, we were able to record Na+ current through CRAC channels in single-channel levels. The unitary Na+ conductance through CRAC channel was 42.5 pS, which was similar to that of Jurkat cell. The Naf current activated slowly, reaching the maximal current level in about 10 min after whole-cell patches were made. The time required for the half-maximal activation of the current was 205 s (+/-31), while it was reduced to 84.3 s (+/-17.7) by including IP3 in the pipette solution as well. The peak currents ranged from 320 to 985 pA, which corresponded to 64-197 channels per cell. We studied the regulation of the current by protein kinase A (PKA) and protein kinase C (PKC). The current was enhanced by the addition of membrane-permeant analogue of cAMP, dibutyryl cAMP. Pretreating cells with PKA inhibitor, H-89, prevented the effect of dibutyryl cAMP. By contrast, the addition of PKC activator, PDBu, reduced the current. Staurosporine, a PKC inhibitor, prevented the effect of PDBu. These results suggest that CRAC channel in rat microglia is under the regulation of PKA and PKC in opposite directions. (C) 2000 Wiley-Liss, Inc.

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