Modulation Of Hepatocellular Trpm7-like Currents By A Ca2+/calmodulin-sensitive Pathway

Abstract

Non-selective cation currents play integral roles in countering cell swelling, inducing apoptotic cell shrinkage, mediating Ca2+ influx and controlling Mg2+ homeostasis. The ‘chanzymes’ TRPM6 and TRPM7 are regulated by cytosolic Mg2+, and, under specific recording conditions, Mg-ATP or cell swelling. We recently identified a Mg2+-inhibited and outwardly rectifying cation current in both rat hepatocytes and the polarized rat hepatoma x human skin fibroblast cross, WIF-B. Here we investigated the regulation of these currents by cytosolic Ca2+. Under standard whole-cell recording conditions in which cells were bathed and dialyzed with Na-gluconate solutions, the latter Ca2+- and Mg2+-free, currents reversed close to 0 mV, showed no time dependence, and were 23 times higher at +120 mV as compared with -120 mV. Current at +120 mV developed slowly over dialysis, from 17.7±10.3 pA/pF at patch rupture to 106.5±15.6 pA/pF at 12 min. Inward current at -120 mV did not change significantly. Pipette solution containing Ca2+ inhibited current development with an IC50 of 125±35 nM. 50 μM W-7 or 200 nM staurosporine relieved the inhibition by 1 μM Ca2+, implicating channel regulation by a Ca2+/calmodulin-dependent kinase (CaMK). To address the nature of the kinase involved, we dialyzed the cells with more specific inhibitors of myosin light-chain kinase (MLCK) and CaMKII. 2 μM AIP, the CaMKII inhibitor, allowed full development of current, whereas 5 μM ML-7 did not affect the Ca2+-dependent channel inhibition ¼. Inspection of the rat Trpm7 primary structure identified a specific CaMKII substrate sequence in the proximal C-terminus. Conversely neither a CaM-binding domain nor a MLCK substrate consensus sequence was identified. The combined results support the conclusion that these channels are inhibited by cytosolic concentrations of Ca2+ in a CaMKII-dependent manner. Supported by NSERC & the Jeanne Mance Foundation, Hotel Dieu Hospital.