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.

Ca(2+) release through ryanodine receptors, located in the membrane of the junctional sarcoplasmic reticulum (SR), initiates contraction of cardiac muscle. Ca(2+)influx through plasma membrane L-type Ca(2+)channels is thought to be an important trigger for opening ryanodine receptors ("Ca(2+)-induced Ca(2+)-release"). Optimal transmission of the transmembrane Ca(2+)influx signal to SR release is predicted to involve spatial juxtaposition of L-type Ca(2+)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 Ca(2+)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 Ca(2+)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 Ca(2+)channels quantitatively predominated in domains of the plasma membrane overlying junctional SR. These findings provide an ultrastructural basis for functional coupling between L-type Ca(2+)channels and junctional SR and for excitation-contraction coupling in guinea-pig cardiac muscle.

Protein gene product 9.5 in the developing cochlea of the rat: cellular distribution and relation to the cochlear cytoskeleton.

Protein gene product 9.5 was immunolocalized in the adult and early postnatal (P2-P15) rat cochlea, and its distribution compared with a 200 kDa highly phosphorylated neurofilament subunit (neurofilament 200) and alpha-tubulin. In the adult, Protein gene product 9.5 was expressed exclusively in cochlear nerve fibres and ganglion cells, a small percentage of these (Type II ganglion cells and olivocochlear bundle fibres) being intensely positive for both protein gene product and neurofilament 200. In postnatal development, pillar and Deiters' cells were at first (P2-P15) strongly positive for protein gene product 9.5, and hair cells moderately so. At P2, all nerve fibres and ganglion cells showed co-expression of protein gene product 9.5 and neurofilament 200, but at later stages, the subset of intensely co-labelled neurons appeared, nerve fibres at P7 onwards and ganglion cells from P12. There was no overt correlation between the onset of protein gene product 9.5 and alpha-tubulin expression in any cochlear component. Protein gene product 9.5 expression in ganglion cells was at first (P2 and P7) mainly nuclear, and later also cytoplasmic. It is concluded that there is a clear correlation of high levels of protein gene product 9.5 and neurofilament protein expression, and that protein gene product 9.5 is expressed in some non-neuronal cells of the cochlea during its early development, persisting until after hearing has commenced.