Mg2+- and MgATP-inhibited and Ca2+/calmodulin-sensitive TRPM7-like current in hepatoma and hepatocytes.
Although understood to be ubiquitously expressed, the functional identification and significance of Mg(2+)-inhibited, nonspecific cation currents has been established in only a few cell types. Here we identified an outwardly rectifying nonspecific cation current in quiescent rat hepatocytes and the proliferating and polarized rat hepatoma, WIF-B. Under whole cell recording conditions in which cells were bathed and dialyzed with Na-gluconate solutions, the latter Ca(2+) and Mg(2+) free, current reversed close to 0 mV, was time independent, and was greater than 10 times higher at +120 mV compared with -120 mV. Outward current at -120 mV developed slowly, from 17.7 +/- 10.3 pA/pF at patch rupture to 106.6 +/- 15.6 pA/pF at 12 min in WIF-B cells, and 4.9 +/- 2.7 to 20.6 +/- 5.6 pA/pF in rat hepatocytes. The nonspecific TRP channel inhibitor, 2-aminoethoxyphenylborate (2-APB), inhibited current (IC(50) = 72 +/- 13 microM) and caused apoptotic cell death in WIF-B cells. Rat hepatocyte survival was more resistant to 2-APB. Dialysis of WIF-B cells with physiological concentrations of Mg(2+) and Mg-ATP, but not ATP alone, inhibited current development, suggesting that Trpm7 rather than Trpm6 underlies this current. RT-PCR demonstrated that both Trpm6 and Trpm7 are expressed at similar levels in both cell types, suggesting that the functional differences noted are not transcript dependent. Intracellular Ca(2+) (IC(50) = 125 +/- 35 nM) also inhibited current development, and this could be partially relieved by the calmodulin and Ca(2+)/calmodulin-dependent kinase inhibitors W-7, staurosporine, KN-93, or calmodulin kinase II (CaMKII) inhibitory peptide. To summarize, our results show that in addition to their established Mg(2+) sensitivity, Trpm7-like channels are inhibited by cytosolic Ca(2+) in a CaMKII-dependent manner and may support hepatocellular survival during proliferation.
Mishra
Ravi
R
Department of Biology and Medicine, Hotel Dieu Hospital and Queen's University, Kingston, Ontario, Canada.
Rao
Vidya
V
Ta
Robert
R
Shobeiri
Navid
N
Hill
Ceredwyn E
CE
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis; D001596:Benzylamines / Q000494:pharmacology; D001896:Boron Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D016764:Cell Polarity; D049109:Cell Proliferation; D002470:Cell Survival; D003956:Dialysis; D004305:Dose-Response Relationship, Drug; D005260:Female; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D008274:Magnesium / Q000378:metabolism*; D008564:Membrane Potentials; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D019311:Staurosporine / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D050053:TRPM Cation Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D000096926:Benzenesulfonamides
D000255:Adenosine Triphosphate; D001596:Benzylamines; D001896:Boron Compounds; D002118:Calcium; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002147:Calmodulin; D008274:Magnesium; D047428:Protein Kinase Inhibitors; D019311:Staurosporine; D013449:Sulfonamides; D050053:TRPM Cation Channels; C507531:TRPM6 protein, rat; C072105:KN 93; D000096926:Benzenesulfonamides; C017967:W 7; C109986:2-aminoethoxydiphenyl borate; C486584:Trpm7 protein, rat