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Removal of Ca current inactivation in dialysed guinea-pig atrial cardioballs by Ca chelators
Abstract:
Ca currents flowing during voltage clamp depolarizations were studied in cultured guinea-pig atrial cardioballs by means of single low resistance patch clamp pipettes. The pipettes were filled with solutions containing Cs+ as major cation in order to block K+ currents and high concentrations of various Ca chelating agents (EGTA, nitrilotriacetic acid, citrate, dipicolinic acid) to prevent rises of the intracellular Ca-activity by Ca-entry. Ca currents of myocytes loaded with 20 mM of either EGTA [(ethylenedioxy)-diethylenedinitrilo)tetra-acetic acid] or NTA (nitrilotriacetic acid) display a biphasic time course of inactivation at membrane potentials between -25 and +45 mV. The fast phase is reduced with increasingly positive membrane potentials. In cells loaded with either citrate or DPA (dipicolinic acid, pyridine-2,6-dicarboxylic acid) inactivation is negligible or absent for small depolarizations. In the range of membrane potentials where maximum current flows (0-+10 mV) a monophasic slow time course of inactivation is observed. At more positive membrane potentials inactivation is slowed. The amount of inactivation under this condition is related to the current density of the cell. Conditions, which for a given membrane potential reduce the amplitude of ICa such as extracellular application of blocking ions (Co2+, Cd2+), a conditioning depolarization, or 'rundown' of Ca-channels lead to a slowing or a complete removal of inactivation in cells dialysed with citrate or DPA respectively. Cells loaded with these Ca chelators did not show any symptom of voltage dependent inactivation of ICa. Under the conditions described action potentials were recorded in the current clamp mode. Upon dialysis with EGTA the typical 'triangular shaped' atrial action potential develops a plateau of 500 to 800 ms in duration. With citrate-containing pipette solutions the action potential duration usually is several seconds. The results for the first time demonstrate that inactivation of cardiac ICa can be considerably slowed or even removed. They provide further strong support for the hypothesis that inactivation of this current depends on Ca entry rather than membrane potential. The fast phase of inactivation observed with EGTA (NTA) possibly reflects the slow kinetics of the binding reaction of this type of Ca chelators.
Insights
Cardiac calcium (Ca) currents show varied inactivation. Using specific chelators like citrate or dipicolinic acid (DPA) can slow or remove inactivation, supporting Ca entry as the key factor, not membrane potential.
Area of Science:
- Cardiovascular Physiology
- Cellular Electrophysiology
- Ion Channel Function
Background:
- Cardiac calcium (Ca) currents (ICa) are crucial for atrial action potentials.
- Understanding ICa inactivation mechanisms is vital for cardiac function.
- Previous hypotheses suggested voltage dependence, but Ca entry's role was unclear.
Purpose of the Study:
- To investigate the role of intracellular Ca concentration in ICa inactivation.
- To determine if ICa inactivation is dependent on Ca entry or membrane potential.
- To explore methods for slowing or removing ICa inactivation.
Main Methods:
- Patch-clamp electrophysiology in cultured guinea-pig atrial myocytes.
- Utilized Cs+-filled pipettes to block K+ currents.
- Employed various Ca chelators (EGTA, NTA, citrate, DPA) to control intracellular Ca levels.
Main Results:
- EGTA/NTA loading resulted in biphasic ICa inactivation, dependent on membrane potential.
- Citrate/DPA loading showed negligible or slow inactivation, especially at potentials near peak current.
- Conditions reducing Ca entry (Co2+, Cd2+, depolarization, rundown) slowed or abolished inactivation with citrate/DPA.
- Action potential duration significantly prolonged with citrate/DPA compared to EGTA.
Conclusions:
- ICa inactivation is significantly influenced by intracellular Ca buffering.
- Results strongly support Ca entry, not solely membrane potential, as the primary driver of cardiac ICa inactivation.
- Specific Ca chelators can modulate ICa inactivation kinetics, impacting action potential duration.