Activation of calcium-permeable cation channel by insulin in Chinese hamster ovary cells expressing human insulin receptors.
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.
Nie
L
L
Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan.
Kanzaki
M
M
Shibata
H
H
Kojima
I
I
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
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