Divergent functional properties of ryanodine receptor types 1 and 3 expressed in a myogenic cell line

James D. Fessenden, Yarning Wang, Rennee A. Moore, S. R Wayne Chen, Paul D. Allen, Isaac N Pessah

Research output: Contribution to journalArticle

107 Scopus citations

Abstract

Of the three known ryanodine receptor (RyR) isoforms expressed in muscle, RyR1 and RyR2 have well-defined roles in contraction. However, studies on mammalian RyR3 have been difficult because of low expression levels relative to RyR1 or RyR2. Using the herpes simplex virus 1 (HSV-1) helper-free amplicon system, we expressed either RyR1 or RyR3 in 1B5 RyR-deficient myotubes. Western blot analysis revealed that RyR1- or RyR3-transduced cells expressed the appropriate RyR isoform of the correct molecular mass. Although RyR1 channels exhibited the expected unitary conductance for Cs+ in bilayer lipid membranes, 74 of 88 RyR3 channels exhibited pronounced subconductance behavior. Western blot analysis with an FKBP12/12.6-selective antibody reveals that differences in gating behavior exhibited by RyR1 and RyR3 may be, in part, the result of lower affinity of RyR3 for FKBP12. In calcium imaging studies, RyR1 restored skeletal-type excitation-contraction coupling, whereas RyR3 did not. Although RyR3-expressing myotubes were more sensitive to caffeine than those expressing RyR1, they were much less sensitive to 4-chloro-m-cresol (CMC). In RyR1-expressing cells, regenerative calcium oscillations were observed in response to caffeine and CMC but were never seen in RyR3-expressing, 1B5 cells. In [3H]ryanodine binding studies, only RyR1 exhibited sensitivity to CMC, but both RyR isoforms responded to caffeine. These functional differences between RyR1 and RyR3 expressed in a mammalian muscle context may reflect differences in association with accessory proteins, especially FKBP12, as well as structural differences in modulator binding sites.

Original languageEnglish (US)
Pages (from-to)2509-2525
Number of pages17
JournalBiophysical Journal
Volume79
Issue number5
StatePublished - 2000

ASJC Scopus subject areas

  • Biophysics

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    Fessenden, J. D., Wang, Y., Moore, R. A., Chen, S. R. W., Allen, P. D., & Pessah, I. N. (2000). Divergent functional properties of ryanodine receptor types 1 and 3 expressed in a myogenic cell line. Biophysical Journal, 79(5), 2509-2525.