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Trypsin and forskolin decrease the sensitivity of L-type calcium current to inhibition by cytoplasmic free calcium in
1Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada.
Abstract:
A key feature of trypsin action on ionic membrane currents including L-type Ca2+ current (ICa) is the removal of inactivation upon intracellular application. Here we report that trypsin also occludes the resting cytoplasmic free Ca2+ ([Ca2+]i)-induced inhibition of peak ICa in isolated guinea pig ventricular cardiomyocytes, using the whole-cell patch clamp in combination with the Fura-2 ratio-fluorescence technique. The effectiveness of trypsin to guard ICa against [Ca2+]i-induced inhibition was compared with that of forskolin, as cAMP-dependent phosphorylation had been suggested to confer protection against [Ca2+]i-induced inactivation. Intracellular dialysis of trypsin (1 mg/ml) augmented ICa by 7.2-fold, significantly larger than the threefold increase induced by forskolin (3 microM). Forskolin application after trypsin dialysis did not further enhance ICa. An increase in [Ca2+]i from resting levels (varied by 0.2, 10, and 40 mM EGTA dialysis) to submicromolar concentrations after replacement of external Na+ (Na(o)+) with tetraethylammonium (TEA+) resulted in monotonic inhibition of control ICa, elicited from a holding potential of -40 mV at 22 degrees C. AFter trypsin dialysis, however, ICa became less sensitive to submicromolar [Ca2+]i; the [Ca2+]i of half-maximal inhibition (K0.5, normally around 60 nM) increased by approximately 20-fold. Forskolin also increased the K0.5 by approximately threefold. These and accompanying kinetic data on ICa decay are compatible with a model in which it is assumed that Ca2+ channels can exist in two modes (a high open probability "willing" and a low open probability "reluctant" mode) that are in equilibrium with one another. An increase in [Ca2+]i places a larger fraction of channels in the reluctant mode. This interconversion is hindered by cAMP-dependent phosphorylation and becomes nearly impossible after tryptic digestion.
Insights
Trypsin prevents calcium channel inhibition by intracellular calcium, unlike forskolin. This suggests trypsin blocks a calcium-dependent inactivation mechanism in cardiac L-type calcium currents.
Area of Science:
- Cardiology
- Molecular Physiology
- Ion Channel Biophysics
Background:
- L-type Ca2+ current (ICa) inactivation is modulated by intracellular calcium ([Ca2+]i).
- Trypsin is known to remove inactivation of ionic membrane currents.
- cAMP-dependent phosphorylation is hypothesized to protect against [Ca2+]i-induced inactivation.
Purpose of the Study:
- To investigate trypsin's effect on [Ca2+]i-induced inhibition of peak ICa.
- To compare the protective efficacy of trypsin against [Ca2+]i-induced inhibition with forskolin.
- To explore the mechanism underlying trypsin's modulation of ICa.
Main Methods:
- Whole-cell patch clamp electrophysiology in isolated guinea pig ventricular cardiomyocytes.
- Fura-2 ratio-fluorescence technique to measure intracellular calcium concentrations.
- Intracellular dialysis of trypsin and forskolin, with varying external and internal calcium levels.
Main Results:
- Trypsin significantly augmented ICa (7.2-fold) compared to forskolin (3-fold).
- Trypsin rendered ICa less sensitive to [Ca2+]i-induced inhibition, increasing the K0.5 by ~20-fold.
- Forskolin also increased ICa and K0.5, but to a lesser extent than trypsin.
Conclusions:
- Trypsin effectively occludes the resting [Ca2+]i-induced inhibition of peak ICa.
- The findings support a model where trypsin prevents the shift of Ca2+ channels to a low open probability state induced by [Ca2+]i.
- Tryptic digestion appears to nearly abolish the interconversion between channel modes, unlike cAMP-dependent phosphorylation.