title: Modification of L-type calcium current by intracellularly applied trypsin in guinea-pig ventricular myocytes.
abstract: 1. The L-type Ca2+ current was recorded in guinea-pig ventricular myocytes by the patch clamp technique in the whole-cell configuration. The modification of the current by intracellular application of proteases was studied. 2. During the first phase of action, trypsin, an endopeptidase, increased the amplitude of Ca2+ current about 3-fold. 3. Thereafter, there was a drastic slowing of the inactivation time course of the enhanced Ca2+ current. The half-time of inactivation increased from a control value of about 25 ms to values larger than 200 ms. 4. Cell dialysis with carboxypeptidase A, an exopeptidase, also enlarged the amplitude of Ca2+ current, but did not affect the kinetics of Ca2+ current. Leuaminopeptidase did not modify the Ca2+ current. 5. The hypothesis that Ca2+ channels are affected by the protease is supported by the fact that alterations of the extracellular Na+ or K+ concentration did not influence the modification of the membrane current. Another argument for the involvement of Ca2+ channels is that the modified membrane current could be blocked by inorganic and organic Ca2+ channel blockers (e.g. 10 microM-Cd2+, 100 microM-La3+ or 1 microM-D600). 6. Although the actions of trypsin and maximal concentrations of isoprenaline on the amplitude of the Ca2+ current were not additive, the slowing of inactivation by trypsin occurred independently from beta-adrenergic stimulation. 7. The effect of trypsin on the Ca2+ current could not be blocked by intracellular 5'-adenylyl-imidodiphosphate (AMP-PNP) or Rp-adenosine 3'5'-monothionophosphate (Rp-cAMPS), both of which are known to suppress the cyclic AMP-dependent phosphorylation of the Ca2+ channel. 8. It was concluded that trypsin may directly modify the membrane protein which forms the Ca2+ channel. Since the increment in peak Ca2+ current resembled the action of cyclic AMP-dependent phosphorylation, it may be related to the removal of a 'chemical' inactivation gate which is normally controlled by phosphorylation. The slowing of the time course of Ca2+ current inactivation by trypsin could be due to a modification of the voltage-dependent inactivation gate. Alternatively, the endopeptidase might remove an internal Ca2+ binding site normally responsible for Ca2+-dependent inactivation.
authors.affiliation: II. Physiologisches Institut der Universität des Saarlandes, Homburg/Saar, F.R.G.
authors.forename: J
authors.initials: J
authors.lastname: Hescheler
authors.forename: W
authors.initials: W
authors.lastname: Trautwein
mesh_terms: D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002268:Carboxypeptidases / Q000494:pharmacology; D043422:Carboxypeptidases A; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D006168:Guinea Pigs; D006321:Heart / Q000502:physiology*; D007545:Isoproterenol / Q000494:pharmacology; D008564:Membrane Potentials; D010766:Phosphorylation; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology*
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D013873:Thionucleotides; C016957:adenosine-3',5'-cyclic phosphorothioate; D000266:Adenylyl Imidodiphosphate; D000242:Cyclic AMP; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D014357:Trypsin; D007545:Isoproterenol