Immunocytochemical and functional studies suggest that L-type Ca²⁺ channels are located predominantly in the t-tubules of cardiac ventricular myocytes. The purpose of the present study was to compare modulation of L-type Ca²⁺ current (I_CaL) by Ca²⁺ in the t-tubules (i.e. in control cells) and surface sarcolemma (i.e. in detubulated (DT) cells.

Wistar rats were killed humanely using a Schedule 1 method. Ventricular myocytes were enzymatically isolated, and detubulated using formamide as described by Kawai et al. (1999). The whole cell configuration of the patch clamp technique was used to record I_CaL. TEA/Cs-containing solutions were used, to avoid contamination by other currents; membrane potential was held at -80 mV and test pulses to 0 mV applied. Experiments were performed at 22-24 °C.

The rapid component of inactivation of I_CaL was significantly faster in control cells (t{special} = 9.5 ± 0.6 ms, mean ± S.E.M., n = 10) than in detubulated cells (16.8 ± 2.8 ms, n = 7, P < 0.05, Student’s unpaired t test), and frequency-dependent facilitation was present in control cells (10/12) but not in detubulated cells (12/12, see Fig. 1). Inactivation and facilitation of I_CaL are known to depend on local Ca²⁺ entry and release. Ryanodine (30 µM) slowed the fast inactivation phase of I_CaL in control cells so that it was no longer statistically different from in DT cells (control t{special}: 22.7 ± 2.1 ms, n = 10; detubulated: 23.8 ± 2.0 ms, n = 7; NS, unpaired t test) and abolished frequency-dependent facilitation in control cells. Formamide treatment of atrial myocytes, which lack t-tubules, did not change the fast inactivation phase of I_CaL (control t{special}: 9.8 ± 1.4 ms, n = 7; formamide-treated: 10.0 ± 1.6 ms, n = 7; NS, unpaired t test).

It appears likely that Ca²⁺-dependent modulation of I_CaL is due to local Ca²⁺ entry and release rather than bulk cytoplasmic Ca²⁺ (see for review Dirksen, 2002). Thus the observation that modulation of I_CaL by Ca²⁺ released from the SR is most marked at the t-tubules (i.e. in control cells) suggests that this difference may be due to differences in the structure of the dyad at the t-tubule and surface membrane.

This work was supported by project grants from the Wellcome Trust and British Heart Foundation, and a short-term travel grant to L.S. from the Wellcome Trust.