title: Ca2+-induced Ca2+ release in Chinese hamster ovary (CHO) cells co-expressing dihydropyridine and ryanodine receptors.
abstract: 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 ryanodine receptor (RyR)-Ca2+ release channel (in the membrane of internal Ca2+ stores) and the dihydropyridine receptor (DHPR)-Ca2+ channel (in the plasma membrane). To ensure expression of functional L-type Ca+ channels, we expressed alpha2, beta, and gamma DHPR subunits and a chimeric DHPR alpha(i) subunit in which the putative cytoplasmic loop between repeats II and III is of skeletal origin and the remainder is cardiac. 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+]o). However, in the presence of [Ca2+]o, depolarization evoked CaTs with a bell-shaped voltage dependence. About 30% of the cells tested exhibited two kinetic components: a fast transient increase in intracellular Ca2+ concentration ([Ca2+]i) (the first component; reaching 95% of its peak <0.6 s after depolarization) followed by a second increase in [Ca2+]i which lasted for 5-10 s (the second component). Our results suggest that the first component primarily reflected Ca2+ influx through Ca2+ channels, whereas the second component resulted from Ca2+ release through the 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+ current. These results suggest that the skeletal muscle RyR isoform supports Ca2+-induced Ca2+ release but that the distance between the DHPRs and the RyRs is, on average, much larger in the cotransfected CHO cells than in cardiac myocytes. We conclude that morphological properties of T-tubules and/or proteins other than the DHPR and the RyR are required 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.
authors.affiliation: Department of Membrane Biophysics, Max-Planck-Institut für biophysikalische Chemie, Göttingen, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Suda
authors.forename: D
authors.initials: D
authors.lastname: Franzius
authors.forename: A
authors.initials: A
authors.lastname: Fleig
authors.forename: S
authors.initials: S
authors.lastname: Nishimura
authors.forename: M
authors.initials: M
authors.lastname: Bödding
authors.forename: M
authors.initials: M
authors.lastname: Hoth
authors.forename: H
authors.initials: H
authors.lastname: Takeshima
authors.forename: R
authors.initials: R
authors.lastname: Penner
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D020746:Calcium Channels, L-Type; D006224:Cricetinae; D004594:Electrophysiology; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D009124:Muscle Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010957:Plasmids; D019837:Ryanodine Receptor Calcium Release Channel; D014162:Transfection / Q000502:physiology
chemical_list: D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium