High K⁺ Rapidly Stimulates Ca²⁺-Dependent Phosphorylation of Three Proteins Concomitant With Insulin Secretion From HIT Cells

We have previously shown in a glucose-responsive insulin-secreting β-cell line (HIT cells) that a cell membrane–depolarizing concentration of K⁺ causes an influx of extracellular Ca²⁺ that elevates the free cytosolic Ca²⁺ concentration ([Ca²⁺],) and activates insulin secretion. To identify the cellular mechanisms that couple Ca²⁺ influx to exocytosis, studies of protein phosphorylation were conducted. HIT cells were loaded with ³²P and stimulated with either 40 mM K⁺ or 19.4 mM glucose, both of which trigger the immediate release of insulin. Phosphoproteins were examined with two-dimensional polyacrylamide gel electrophoresis and autoradiography. With addition of 40 mM K⁺, after the rise in [Ca²⁺]₁ proteins of M_r 17,500, 20,000, and 24,000 were rapidly and specifically phosphorylated. The 17,000-M_r protein showed maximum phosphorylation by 1 min, whereas phosphorylation of the 24,000- and 20,000-M_r proteins continued to increase at 5 min. Despite stimulating the immediate release of insulin, high glucose did not result in a change in [Ca²⁺], or the phosphorylation of the three Ca²⁺-dependent phosphoproteins. Insulin secretion, the increase in [Ca²⁺]₁, and phosphorylation of the proteins by 40 mM K⁺ was inhibited by 100 μM verapamil, an organic Ca²⁺-channel–blocking drug. The rapid phosphorylation of three proteins, after a rise in [Ca²⁺]₁ and the coordinate inhibition of insulin secretion and phosphorylation, are consistent with the hypothesis that Ca²⁺ enters the β-cell through voltage-dependent Ca²⁺ channels, regulating insulin release by effects on protein phosphorylation. However, glucose activates the immediate release of insulin by another, as yet undefined, mechanism.