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 rece ptor to activate the calcium signaling system in Chinese hamster ovary (CHO) cells expressing human insulin receptor (CHO-IR cells). In thes e cells, insulin evoked the elevation of cytoplasmic free calcium conc entration, [Ca2+](c), measured by using fura-2. Insulin-induced increa se in [Ca2+](c) was blocked by reducing the extracellular calcium conc entration to 1 mu M or by adding nickel chloride, an inorganic inhibit or of calcium entry. Insulin did not elevate [Ca2+](c) in parental CHO cells or in CHO cells expressing mutant insulin receptor lacking an A TP-binding site. When the transmembrane calcium current was measured b y perforated whole-cell patch clamp, adding insulin to the bath soluti on markedly augmented the inward calcium current. In a cell-attached p atch, a single channel activity appeared when insulin was included in the pipette. In contrast, insulin added outside the patch was ineffect ive. The current/voltage relationship demonstrated that insulin activa ted a voltage-independent calcium-permeable cation channel with a sing le-channel conductance of 10 pS. Exposing CHO-IR cells to pertussis to xin abolished the subsequent insulin effect on [Ca2+](c) and activatio n of the calcium-permeable channel. Mastoparan activated the 10-pS cal cium-permeable cation channel. In an inside-out patch, insulin activat ed the calcium permeable channel when the bath solution contained both GTP and ATP. Nonhydrolyzable ATP could substitute for ATP. These resu lts indicate that in CHO-IR cells, insulin elevates [Ca2+](c) by activ ating the 10-pS calcium-permeable cation channel. Activation by the in sulin receptor involves pertussis toxin-sensitive G protein.