Modification of L-type calcium current by intracellularly applied trypsin in guinea-pig ventricular myocytes

• Published in: The Journal of physiology Vol. 404; no. 1; pp. 259 - 274
• Main Authors: Hescheler, J, Trautwein, W
• Format: Journal Article
• Language: English
• Published: Oxford The Physiological Society 01.10.1988; Blackwell
• Subjects: Adenylyl Imidodiphosphate - pharmacology; Animals; Biological and medical sciences; calcium; Calcium Channel Blockers - pharmacology; Calcium Channels - drug effects; Calcium Channels - physiology; Carboxypeptidases - pharmacology; Carboxypeptidases A; Cyclic AMP - analogs & derivatives; Cyclic AMP - pharmacology; Fundamental and applied biological sciences. Psychology; Guinea Pigs; Heart; Heart - physiology; Isoproterenol - pharmacology; Membrane Potentials; Phosphorylation; Thionucleotides - pharmacology; Time Factors; trypsin; Trypsin - pharmacology; ventricle; Vertebrates: cardiovascular system; Transmembrane current; Vertebrata; Trypsin; Mammalia; Guinea pig; Calcium; Enzyme; Rodentia; Electrophysiology; Heart ventricle; Inhibition; Heart cell
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1113/jphysiol.1988.sp017289

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.