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Mechanism of cardiac T-type Ca channel blockade by amiloride
J Tytgat1, J Vereecke, E Carmeliet
1Laboratory of Physiology, KU Leuven, Campus Gasthuisberg, Belgium.
Abstract:
Whole cell clamp experiments were used to study the mechanism of action of amiloride on cardiac T-type Ca channels in guinea pig ventricular myocytes. Two hundred fifty microM amiloride blocked the T-type Ca channel for approximately 50%, whereas this concentration did not suppress the L-type Ca channel. The Kd value for the block of the T-type Ca channel was 233 microM and the Hill coefficient was 1.1. The drug showed a rapid onset of action and a quick wash out with complete reversibility. Amiloride blocked the T-type Ca channel without frequency-dependency, i.e., block was established at the holding potential (-90 mV) without being dependent on activation and inactivation of the channel. Amiloride reduced the amplitude of the T-type Ca current at all potentials without changing steady-state activation and inactivation. Amiloride did not affect the T-type Ca current when the drug was applied intracellularly via the pipette solution. In summary, amiloride blocked the T-type Ca channel in a channel state- and voltage-independent way.
Insights
Amiloride effectively blocks cardiac T-type calcium channels, not L-type channels, in guinea pig heart cells. This action is rapid, reversible, and independent of channel state or voltage.
Area of Science:
- Cardiovascular Physiology
- Molecular Pharmacology
- Ion Channel Biophysics
Background:
- Cardiac T-type calcium channels (TTCCs) play a role in cardiac rhythm and contractility.
- Understanding the specific mechanisms of drug interactions with ion channels is crucial for developing targeted therapies.
Purpose of the Study:
- To investigate the mechanism of amiloride's action on cardiac T-type calcium channels (TTCCs).
- To determine the selectivity of amiloride for TTCCs over L-type calcium channels (LTCCs).
Main Methods:
- Whole-cell patch-clamp electrophysiology was performed on guinea pig ventricular myocytes.
- Amiloride was applied extracellularly at a concentration of 250 µM to assess its effect on TTCCs and LTCCs.
- Kinetic parameters, including dissociation constant (Kd) and Hill coefficient, were determined for amiloride block.
Main Results:
- Amiloride (250 µM) produced approximately 50% block of TTCCs, with a Kd of 233 µM and a Hill coefficient of 1.1.
- The same concentration of amiloride did not significantly affect LTCCs.
- Amiloride's block of TTCCs was rapid, reversible, frequency-independent, and occurred without altering steady-state activation or inactivation, suggesting a state- and voltage-independent mechanism. Intracellular application did not inhibit the current.
Conclusions:
- Amiloride selectively inhibits cardiac T-type calcium channels.
- The blockade is voltage- and state-independent, occurring via an extracellular mechanism.
- These findings highlight amiloride as a potential modulator of TTCC activity in cardiac tissue.