Divergent Functional Properties of Ryanodine Receptor Types 1 and 3 Expressed in a Myogenic Cell Line

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 [(3)H]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.

Functional characterization of orbicularis oculi and extraocular muscles

“…The difference in sensitivity to 4-cmc-induced Ca 2+ release but not KCl-induced Ca 2+ release may reflect the different amounts of RyR1 and RyR3 expressed in EOM-derived myotubes. Such a hypothesis is compatible with previous findings (Fessenden et al, 2000;Matyash et al, 2002;Sekulic-Jablanovic et al, 2015) who showed that (a) EOM biopsies contain ∼100-times more RYR3 transcript than triceps biopsies, whereas orbicularis oculi express only ∼4-times more; (b) RyR3 have a significantly lower sensitivity to 4-cmc; and (c) in reconstituted 1B5 myotubes, RyR3 cannot form functional contacts with the DHPR and therefore would not contribute directly to the KCl-induced Ca 2+ release.…”

Type 3 ryanodine receptors of skeletal muscle are segregated in a parajunctional position

“…Regardless of whether RyR3 and RyR1 are totally segregated, as we have suggested, or partly mixed in the junction, the activation of RyR3 is necessarily indirect, because RyR3 is not capable of being activated by skeletal DHPR (20). Thus, it is expected that RyR3 activation is a secondary event in e-c coupling, and that it follows the primary activation of RyR1 by DHPR (34), resulting in its amplification.…”

“…Muscles developing in the absence of RyR1 as a result of a lethal genetic mutation show strong developmental defects and lack of e-c coupling, despite the presence of RyR3 (18,19). Even when expressed at high levels, RyR3 alone does not induce restoration of e-c coupling (20) and tetrad formation by DHPRs (17). RyR3 seems to play a minor role in e-c coupling of mouse skeletal muscle, because changes resulting from an engineered RyR 1 null mutation are subtle (16,21).…”

Allosterically coupled calcium and magnesium binding sites are unmasked by ryanodine receptor chimeras

“…1A. Indeed, at 5-10μM Ca 2+ , WT RyR3 was shown to exhibit increased subconductance behavior relative to WT RyR1 [28] and [33]. This correlation is additionally supported by increased subconductance behavior of brain WT RyRs at specific Ca 2+ and ATP concentrations [34].…”