Characterization and regulation of rat microglial Ca(2+) release-activated Ca(2+) (CRAC) channel by protein kinases.
We measured the activity of the Ca(2+) release-activated Ca(2+) (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 Ca(2+) 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 Na(+) 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 IP(3) 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.
Hahn
J
J
Department of Physiology, Chung-Ang University School of Medicine, Seoul, South Korea.
Jung
W
W
Kim
N
N
Uhm
D Y
DY
Chung
S
S
D000818:Animals; D000831:Animals, Newborn; D015220:Calcium Channels / Q000378:metabolism*; D002478:Cells, Cultured; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley
D015220:Calcium Channels; D011494:Protein Kinases