Insensitivity of guinea pig ventricular delayed rectifier IK to intracellular trypsin: implications for channel structure and function.
OBJECTIVE
Intracellular application of proteolytic agents modifies the function of many voltage gated ion channels. The presence of a trypsin sensitive inhibitory domain in a channel protein may be important for G protein dependent activation. Guinea pig ventricular IK is modulated by a direct G protein pathway. The aim was to determine if guinea pig ventricular IK is also modified by intracellularly applied trypsin.


METHODS
Whole cell and excised inside out configurations of patch clamp were used to record IK from guinea pig ventricular myocytes before and after cytosolic application of trypsin (1 mg.ml-1). We used previously reported effects of trypsin on the L type calcium current (ICa) to monitor dialysis time and enzyme activity in whole cell experiments where IK and ICa were measured concomitantly.


RESULTS
Addition of trypsin to the solution bathing the cytosolic face of excised membrane patches had no effect on the amplitude or kinetics of IK. When added to the pipette solution and introduced by cell dialysis, trypsin had no effect on whole cell IK, even when significant effects on the amplitude and kinetics of ICa were evident.


CONCLUSIONS
Guinea pig ventricular IK is not enhanced or otherwise altered by intracellularly applied trypsin. Therefore direct phosphorylation independent enhancement of IK by guanine nucleotides cannot depend on interactions between G protein subunits and trypsin sensitive inhibitory channel domains. The lack of trypsin modification of cardiac ventricular IK suggests that the structure of the endogenous delayed rectifier K+ channel may be different than that of other voltage gated channels.
Freeman
L C
LC
Department of Physiology, University of Rochester, School of Medicine and Dentistry, NY 14642.
Kass
R S
RS
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D009132:Muscles / Q000166:cytology; D011188:Potassium / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology*
D015220:Calcium Channels; D015221:Potassium Channels; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014357:Trypsin; D011188:Potassium; D002118:Calcium