CA2-INDUCED CA2+ RELEASE IN CHINESE-HAMSTER OVARY (CHO) CELLS CO-EXPRESSING DIHYDROPYRIDINE AND RYANODINE RECEPTORS()

Combined patch-clamp and Fura-2 measurements were performed on chinese hamster ovary (CHO) cells co-expressing two channel proteins involved in skeletal muscle excitation-contraction (E-C) coupling, the ryanodi ne receptor (RyR)-Ca2+ release channel (in the membrane of internal Ca 2+ stores) and the dihydropyridine receptor (DHPR)-Ca2+ channel (in th e plasma membrane). To ensure expression of functional L-type Ca2+ cha nnels, rye expressed alpha(2), beta, and gamma DHPR subunits and a chi meric DHPR alpha(1) subunit in which the putative cytoplasmic loop bet ween repeats II and III is of skeletal origin and the remainder is car diac. There was no clear indication of skeletal-type coupling between the DHPR and the RyR; depolarization failed to induce a Ca2+ transient (CaT) in the absence of extracellular Ca2+ ([Ca2+](0)). However, in t he presence of [Ca2+](0), depolarization evoked CaTs with a hell-shape d voltage dependence. About 30% of the cells tested exhibited two kine tic components: a fast transient increase in intracellular Ca2+ concen tration ([Ca2+],) (the first component; reaching 95% of its peak <0.6 s after depolarization) followed by a second increase in [Ca2+](i) whi ch lasted for 5-10 s (the second component). Our results suggest that the first component primarily reflected Ca2+ influx through Ca2+ chann els, whereas the second component resulted from Ca2+ release through t he RyR expressed in the membrane of internal Ca2+ stores. However, the onset and the rate of Ca2+ release appeared to be much slower than in native cardiac myocytes, despite a similar activation rate of Ca2+ cu rrent. These results suggest that the skeletal muscle RyR isoform supp orts Ca2+-induced Ca2+ release but that the distance between the DHPRs and the RyRs is, on average, much larger in the cotransfected CHO cel ls than in cardiac myocytes. We conclude that morphological properties of T-tubules and/or proteins other than the DHPR and the RyR are requ ired for functional ''close coupling'' like that observed in skeletal or cardiac muscle. Nevertheless, some of our results imply that these two channels are potentially able to directly interact with each other .