The effects of trypsin on ATP-sensitive potassium channel properties and sulfonylurea receptors in the CRI-G1 insulin-secreting cell line.

The effects of the proteolytic enzyme trypsin upon ATP-sensitive potassium (KATP) channel activity were examined in the CRI-G1 insulin-secreting cell line. Trypsin activated channels only when applied to the intracellular surface of the cell membrane. The activation could be prevented by the concomitant application of trypsin inhibitor or by heat inactivation of the enzyme. The trypsin-induced change in channel activity was accompanied by a reduction in the rate of channel rundown. However, trypsin did not affect the mean single channel conductance (55.2 pS), the ionic selectivity, or rectification of the KATP channel. Concentration response curves for various KATP channel inhibitors were constructed in the presence and absence of intracellular trypsin. The EC50 for tolbutamide was shifted from 30.0 +/- 4.5 microM, with 100 micrograms/ml heat-inactivated trypsin present to 9.7 +/- 1.0 mM with active trypsin in the intracellular solution. Treatment of the cells' external surface with 1 mg/ml trypsin did not alter the potency of tolbutamide. Intracellular trypsin also produced a significant fall in the potency of glibenclamide, meglitinide, and phentolamine but did not alter the effectiveness of thiopentone. Radioligand binding studies demonstrated a total loss of 3H-labeled glibenclamide binding when the intracellular surface of the cells was exposed to trypsin. In contrast, 3H-labeled glibenclamide binding was not affected when the enzyme was applied to the external surface. Trypsin treatment, therefore, alters a number of characteristics of KATP channel pharmacology, and we suggest that this is due to action at possibly more than one site but includes the functional cleavage of the sulfonylurea receptor from the KATP channel.