title: Activation of calcium-permeable cation channel by insulin in Chinese hamster ovary cells expressing human insulin receptors.
abstract: The present study was conducted to examine the ability of insulin receptor to activate the calcium signaling system in Chinese hamster ovary (CHO) cells expressing human insulin receptor (CHO-IR cells). In these cells, insulin evoked the elevation of cytoplasmic free calcium concentration, [Ca2+]c, measured by using fura-2. Insulin-induced increase in [Ca2+]c was blocked by reducing the extracellular calcium concentration to 1 microM or by adding nickel chloride, an inorganic inhibitor of calcium entry. Insulin did not elevate [Ca2+]c in parental CHO cells or in CHO cells expressing mutant insulin receptor lacking an ATP-binding site. When the transmembrane calcium current was measured by perforated whole-cell patch clamp, adding insulin to the bath solution markedly augmented the inward calcium current. In a cell-attached patch, a single channel activity appeared when insulin was included in the pipette. In contrast, insulin added outside the patch was ineffective. The current/voltage relationship demonstrated that insulin activated a voltage-independent calcium-permeable cation channel with a single-channel conductance of 10 pS. Exposing CHO-IR cells to pertussis toxin abolished the subsequent insulin effect on [Ca2+]c and activation of the calcium-permeable channel. Mastoparan activated the 10-pS calcium-permeable cation channel. In an inside-out patch, insulin activated the calcium-permeable channel when the bath solution contained both GTP and ATP. Nonhydrolyzable ATP could substitute for ATP. These results indicate that in CHO-IR cells, insulin elevates [Ca2+]c by activating the 10-pS calcium-permeable cation channel. Activation by the insulin receptor involves pertussis toxin-sensitive G protein.
authors.affiliation: Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan.
authors.forename: L
authors.initials: L
authors.lastname: Nie
authors.forename: M
authors.initials: M
authors.lastname: Kanzaki
authors.forename: H
authors.initials: H
authors.lastname: Shibata
authors.forename: I
authors.initials: I
authors.lastname: Kojima
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects*; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D037342:Pertussis Toxin; D011972:Receptor, Insulin / Q000032:analysis*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D015220:Calcium Channels; D007328:Insulin; D010566:Virulence Factors, Bordetella; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D037342:Pertussis Toxin; D011972:Receptor, Insulin; D002118:Calcium