Ca2+ current-gated focal and local ca2+ release in rat atrial myocytes: evidence from rapid 2-d confocal imaging

Contraction of mammalian cardiac myocytes is controlled by a sequence of events (E-C coupling) that include ICa-gated opening of Ca2+ release channels (RyRs) in the sarcoplasmic reticulum (SR) (Beukelmann & Wier, 1988; Nabauer et al. 1989; Niggli & Lederer, 1990; Cleemann & Morad, 1991; for review see Morad, 2001). In specialized junctions (dyads and peripheral couplings), the RyRs are found in close proximity (< 20 nm, Sun et al. 1995) to dihydropyridine (DHP)-sensitive Ca2+ channels, providing for privileged cross-signalling between the DHP receptors (DHPRs) and RyRs (Sham et al. 1995; Adachi-Akahane et al. 1996). In cell types lacking t-tubules (atrial cells, Purkinje cells, avian ventricular cells) RyRs are also found in non-junctional or corbular SR throughout the central regions of the cell with no associated DHP receptors. The mode of gating and the contribution of this set of RyRs to cytosolic rise of Ca2+ remains uncertain. It has been suggested that non-junctional RyRs might be activated by diffusion of Ca2+ from neighbouring Ca2+ release sites. Such a mechanism is likely to be regenerative (for review see Berridge, 1997) as it would lack the features that are associated with peripheral or dyadic junctions to provide the negative feedback control over the Ca2+-induced Ca2+ release process (Sham et al. 1995; Soldatov et al. 1998; Morad, 2001). Confocal Ca2+ imaging in field stimulated atrial cells suggests that Ca2+ release initiated at the periphery propagates into the interior of the myocyte (Berlin, 1995; Huser et al. 1996; Mackenzie et al. 2001; Kockskamper et al. 2001). What remains uncertain, however, is the extent to which junctional and non-junctional Ca2+ release sites are controlled by ICa. Earlier measurements, in this regard, demonstrate discrepancy between the voltage-dependence of global Ca2+ transients and ICa in human and rabbit atrial myocytes (Hatem et al. 1997; Mitcheson et al. 1997). In the absence of detailed confocal Ca2+ imaging in voltage-clamped myocytes, however, it is difficult to quantify the extent to which the peripheral or central Ca2+ release sites are controlled by ICa. In this respect, in cultured guinea-pig atrial myocytes, the delayed component of Ca2+ release was effectively de-coupled from ICa-gated release with a large concentration of Ca2+ buffer (≈65 mm citrate) (Lipp et al. 1990, 1996). Since these experiments were carried out using a weak and mobile Ca2+ buffer (citrate) and a slow (8 Hz) Ca2+ imaging approach (Lipp et al. 1996), neither the temporal resolution nor the effectiveness of cytosolic Ca2+ buffering were sufficient to determine whether the peripheral junctional sites in atrial cells are as immune to Ca2+ buffers as have been reported for the ventricular dyadic junctions (Sham et al. 1995; Adachi-Akahane et al. 1996; Cleemann et al. 1998). To address these issues we used rapid (240 Hz) 2-D confocal microscopy in voltage-clamped atrial myocytes dialysed with different concentrations of Ca2+ buffer (fluo-3, EGTA) to monitor simultaneously the Ca2+ release process near the periphery and at the interior during ICa. Our results suggest that the peripheral Ca2+ release was mediated by focal Ca2+ release which like ventricular dyadic Ca2+ release was directly gated by ICa (Shacklock et al. 1995; Cleemann et al. 1998). Unexpectedly, however, we found that both the slow and fast components of central Ca2+ release of atrial myocytes were also under continuous control of ICa.