Functional Characterization of the Recombinant Type 3 Ca2+ Release Channel (Ryanodine Receptor) Expressed in HEK293 Cells

Abstract

To investigate the channel properties of the mammalian type 3 ryanodine receptor (RyR3), we have cloned the RyR3 cDNA from rabbit uterus by reverse transcriptase-polymerase chain reaction and expressed the cDNA in HEK293 cells. Immunoblotting studies showed that the cloned RyR3 was indistinguishable from the native mammalian RyR3 in molecular size and immunoreactivity. Ca 2؉ release measurements using the fluorescence Ca 2؉ indicator fluo 3 revealed that the cloned RyR3 functioned as a caffeine-and ryanodine-sensitive Ca 2؉ Ryanodine receptors are a family of intracellular Ca 2ϩ release channels that were originally identified in the sarcoplasmic reticulum (SR) 1 of striated muscles. To date, three members of this family have been identified in mammalian tissues, namely the skeletal muscle (RyR1), the cardiac muscle (RyR2), and the brain (RyR3) ryanodine receptor. These proteins are the products of different genes and share 66 -70% amino acid sequence identity (1-5). Earlier studies using RNA blot analysis revealed that the expression patterns of these isoforms were very different (6). RyR1 was predominantly expressed in skeletal muscle, whereas RyR2 was mainly expressed in heart and brain. The expression of RyR3 was detected in smooth muscle tissues and certain regions of the brain. However, results of recent ribonuclease protection assay demonstrate that all three RyR isoforms are widely and differentially expressed (7). These studies also indicate that most tissues express more than one RyR isoform. For example, skeletal muscles express both RyR1 and RyR3, although RyR3 is expressed at a much lower level than that of RyR1.

Evidence for novel caffeine and Ca2+ binding sites on the lobster skeletal ryanodine receptor

“…The e ects of ATP on lobster RyR are therefore highly dependent on cytosolic [Ca 2+ ]. The results suggest that the e ects of ATP on the lobster RyR are very similar to the reported e ects of ATP on the gating of mammalian isoforms of RyR (Smith et al, 1986;Kermode et al, 1995;Chen et al, 1997;Copello et al, 1997;Meissner et al, 1997). ATP appears to interact synergistically with Ca 2+ to increase Po to levels greater than the sum of the e ects of Ca 2+ alone and ATP alone.…”

“…The EC 50 values for the activation and inactivation of lobster skeletal RyR channels by Ca 2+ are 60 mM and 1 mM respectively. Ca 2+ activation of the lobster RyR therefore requires higher [Ca 2+ ] than any other isoform of RyR studied at the single channel level (Smith et al, 1986;Ashley & Williams, 1990;Bull & Marengo, 1993;Percival et al, 1994;Copello et al, 1997;Chen et al, 1997). The single channel studies demonstrate that inactivation of the lobster RyR by Ca 2+ occurs at only slightly higher levels than are generally required for inactivation of mammalian skeletal channels (Chu et al, 1993;Laver et al, 1995;Copello et al, 1997) but at lower levels to those required for inactivation of the mammalian cardiac RyR (Chu et al, 1993;Laver et al, 1995;Copello et al, 1997).…”

“…Ca eine, at concentrations (10 ± 40 mM) that will virtually fully open mammalian isoforms of RyR (Rousseau et al, 1988;Rousseau & Meissner, 1989;Sitsapesan & Williams, 1990;Chen et al, 1997) in the presence of activating cytosolic [Ca 2+ ], did not cause very large changes in the Po of lobster skeletal RyR (see Figures 7 and 8). It has been suggested that low concentrations (0.5 ± 2 mM) of agents acting via the ca eine binding sites on sheep cardiac RyR sensitize the channel to Ca 2+ (Sitsapesan & Williams, 1990;Williams & Holmberg, 1990) but that high concentrations of ca eine or related compounds can, in the presence of activating cytosolic [Ca 2+ ], fully activate the channel by invoking a di erent gating scheme (Sitsapesan & Williams, 1990;Williams & Holmberg, 1990;McGarry & Williams, 1994).…”

“…High levels of ca eine (410 mM) are required to cause slight activation of the lobster RyR. In comparison, 10 fold lower [ca eine] can increase the Po of mammalian cardiac (Rousseau & Meissner, 1989;Sitsapesan & Williams, 1990), skeletal (Rousseau et al, 1988) and recombinant rabbit uterus RyR3 (Chen et al, 1997) to 0.5 ± 1. The ca eine binding sites on the lobster RyR may be signi®cantly di erent to those on mammalian isoforms of RyR so far studied.…”

IP3 Receptors: Toward Understanding Their Activation

“…In summary, all IP 3 R subtypes are inhibited by Ca 2þ -CaM, but the molecular basis of this inhibition has not been established. It seems, on balance, that CaM is unlikely to be the accessory protein through which Ca 2þ universally inhibits IP 3 R. That need not preclude a role for CaM in modulating IP 3 R function (Taylor and Laude 2002), just as it does for RyR (Chen et al 1997;Fruen et al 2000;Rodney et al 2001), but we must look elsewhere for the site through which Ca 2þ inhibits IP 3 R.…”