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 potas sium (K-ATP) channel activity were examined in the CRI-G1 insulin-secr eting cell line. Trypsin activated channels only when applied to the i ntracellular surface of the cell membrane. The activation could be pre vented by the concomitant application of trypsin inhibitor or by heat inactivation of the enzyme. The trypsin-induced change in channel acti vity was accompanied by a reduction in the rate of channel rundown. Ho wever, trypsin did not affect the mean single channel conductance (55. 2 pS), the ionic selectivity, or rectification of the K-ATP channel. C oncentration response curves for various K-ATP channel inhibitors were constructed in the presence and absence of intracellular trypsin. The EC(50) for tolbutamide was shifted from 30.0 +/- 4.5 mu M, with 100 m u g/ml heat-inactivated trypsin present to 9.7 +/- 1.0 mM with active trypsin in the intracellular solution. Treatment of the cells' externa l surface with 1 mg/ml trypsin did not alter the potency of tolbutamid e. Intracellular trypsin also produced a significant fall in the poten cy of glibenclamide, meglitinide, and phentolamine but did not alter t he effectiveness of thiopentone. Radioligand binding studies demonstra ted a total loss of H-3-labeled glibenclamide binding when the intrace llular surface of the cells was exposed to trypsin. In contrast, H-3-l abeled glibenclamide binding was not affected when the enzyme was appl ied to the external surface. Trypsin treatment, therefore, alters a nu mber of characteristics of K-ATP 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 K-ATP c hannel.