Trypsin increases availability and open probability of cardiac L-type Ca2+ channels without affecting inactivation induced by Ca2+.
The patch-clamp technique was employed to investigate the response of single L-type Ca2+ channels to the protease trypsin applied to the intracellular face of excised membrane patches from guinea pig ventricular myocytes. Calpastatin and ATP were used to prevent run-down of Ca2+ channel activity monitored with 96 mM Ba2+ as charge carrier in the presence of 2.5 microM (-)-BAYK 8644. Upon application of trypsin (100 micrograms/ml) channel activity was enhanced fourfold and remained elevated upon removal of trypsin, as expected of a proteolytic, irreversible modification. The trypsin effect was not mediated by a proteolytic activation of protein kinases, as evidenced by the insensitivity of this effect to protein kinase inhibitors. Trypsin-modified Ca2+ channels exhibited the usual run-down phanomenon upon removal of calpastatin and ATP. In ensemble average currents trypsin-induced changes of channel function are apparent as a threefold increase in peak current and a reduction in current inactivation. At the single channel level these effects were based on about a twofold increase in both Ca2+ channels' availability and open probability. Neither the actual number of channels in the patch nor their unitary conductance as well as reversal potential was changed by trypsin. The Ca(2+)-induced inactivation was not impaired, as judged by a comparable sensitivity of trypsin-modified Ca2+ channels to intracellular Ca2+. Similarly, trypsin treatment did not affect the sensitivity of Ca2+ channels to phenylalkylmine inhibition. The observed alterations in channel function are discussed in terms of possible structural correlates.
Schmid
R
R
Institute for Biophysics, University of Linz, Austria.
Seydl
K
K
Baumgartner
W
W
Groschner
K
K
Romanin
C
C
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000145:classification / Q000187:drug effects* / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D005711:Gallopamil / Q000031:analogs & derivatives / Q000494:pharmacology; D006168:Guinea Pigs; D006352:Heart Ventricles / Q000378:metabolism; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology*
D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D005711:Gallopamil; C033668:calpastatin; C041386:D 890; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D014357:Trypsin; D002118:Calcium