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Immunogold-labeled L-type Calcium Channels are Clustered in the Surface Plasma Membrane Overlying Junctional Sarcoplasmic Reticulum in Guinea-pig Myocytes—Implications for Excitation–contraction Coupling in Cardiac Muscle

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Abstract

Ca2+release through ryanodine receptors, located in the membrane of the junctional sarcoplasmic reticulum (SR), initiates contraction of cardiac muscle. Ca2+influx through plasma membrane L-type Ca2+channels is thought to be an important trigger for opening ryanodine receptors (««Ca2+-induced Ca2+-release»»). Optimal transmission of the transmembrane Ca2+influx signal to SR release is predicted to involve spatial juxtaposition of L-type Ca2+channels to the ryanodine receptors of the junctional SR. Although such spatial coupling has often been implicitly assumed, and data from immunofluorescence microscopy are consistent with its existence, the definitive demonstration of such a structural organization in mammalian tissue is lacking at the electron-microscopic level. To determine the spatial distribution of plasma membrane L-type Ca2+channels and their location in relation to underlying junctional SR, we applied two high-resolution immunogold-labeling techniques, label-fracture and cryothin-sectioning, combined with quantitative analysis, to guinea-pig ventricular myocytes. Label-fracture enabled visualization of colloidal gold-labeled L-type Ca2+channels in planar freeze-fracture electron-microscopic views of the plasma membrane. Mathematical analysis of the gold label distribution (by nearest-neighbor distance distribution and the radial distribution function) demonstrated genuine clustering of the labeled channels. Gold-labeled cryosections showed that labeled L-type Ca2+channels quantitatively predominated in domains of the plasma membrane overlying junctional SR. These findings provide an ultrastructural basis for functional coupling between L-type Ca2+channels and junctional SR and for excitation–contraction coupling in guinea-pig cardiac muscle.

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      Citation Excerpt :

      The first immunohistochemical study of Ca2+ channel distribution, in rabbit myocytes, showed that in ventricular cells immunostaining occurred primarily at the t-tubules (Carl et al., 1995) whereas in rabbit atrial myocytes, L-type Ca2+ channel staining was observed in discrete spots along the sarcolemma but was absent from the interior of the fibers (Carl et al., 1995). Therefore, it appears that the L type Ca2+ channel is concentrated at the t-tubules; this has been confirmed in guinea pig (Gathercole et al., 2000) and rat (Scriven et al., 2000). Comparative studies suggest that the t-tubular concentration of the L-type Ca2+ channel is greater in rat ventricular myocytes than in those from the rabbit (Takagishi et al., 2000).

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    Please address all correspondence to: Professor N. J. Severs, Cardiac Medicine, National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, Royal Brompton Hospital, London SW3 6NP, UK. E-mail: [email protected]

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