Quantification of calcium entry at the t-tubules and surface membrane in rat ventricular myocytes

¹ University of Bristol
² University of Caen

^* To whom correspondence should be addressed. E-mail: f.brette{at}bristol.ac.uk.

Submitted on June 17, 2005
Revised on July 18, 2005
Accepted on 27 September 2005

	Abstract

The action potential of cardiac ventricular myocytes is characterized by its long duration, mainly due to Ca flux through L-type Ca channels. Ca entry also serves to trigger the release of Ca from the sarcoplasmic reticulum. The aim of this study was to investigate the role of cell membrane invaginations called transverse (t)-tubules in determining Ca influx and action potential duration in cardiac ventricular myocytes. We used the whole cell patch clamp technique to record electrophysiological activity in intact rat ventricular myocytes (i.e. from the t-tubules and surface sarcolemma) and in detubulated myocytes (i.e. from the surface sarcolemma only). Action potentials were significantly shorter in detubulated cells than in control cells. In contrast, resting membrane potential and action potential amplitude was similar in control and detubulated myocytes. Experiments under voltage clamp using action potential waveforms were used to quantify Ca entry via the Ca current. Ca entry following detubulation was reduced by ~60%, a value similar to the decrease in action potential duration. We calculated that Ca influx at the t-tubules is 1.3 times that at the cell surface (4.9 versus 3.8 ìmol/L cytosol respectively) during a square voltage clamp pulse. In contrast, during an AP, Ca entry at the t-tubules is 2.2 times that at the cell surface (3.0 versus 1.4 ìmol/L cytosol respectively). However, more Ca entry occurs per µm2 of junctional membrane at the cell surface than in the t-tubules (in nM/µm2: 1.43 versus 1.06 during an AP). This difference is unlikely to be due to a difference in the number of Ca channels/junction at each site because we estimate that the same number of Ca channels is present at cell surface and t-tubule junctions (~35). This study provides the first evidence that the t-tubules are a key site for the regulation of action potential duration in ventricular cardiac myocytes. Our data also provide the first direct measurements of t-tubular Ca influx, which are consistent with the idea that cardiac excitation-contraction coupling largely occurs at the t-tubule dyadic clefts.

Key Words: action potential, calcium channel, calcium flux, cardiac, myocyte, transverse tubules

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