Antibodies as probes for ligand gating of single sarcoplasmic reticulum Ca2+-release channels

Abstract

A large (565 kDa) junctional sarcoplasmic reticulum (SR) protein, the ryanodine receptor (RYR), may play both a structural and a functional role in the mechanism of skeletal muscle excitation-contraction coupling. Recently, the primary amino acid sequence of the RYR has been elucidated. In this paper, we introduce an immunological approach to examine the functional (electrophysiological) properties of the RYR when it is incorporated into planar lipid bilayers. The effects of two polyclonal antibodies against the SR junctional face membrane (JFM) and the RYR (anti-JFM and anti-RYR) were tested on the single-channel gating properties of the RYR SR Ca2(+)-release channel. Junctional SR vesicles were fused into planar lipid bilayers in solutions containing caesium salts. Solutions were designed to minimize the background conductances of the SR K+ and Cl- channels. Three actions of the anti-JFM antibody were distinguished on the basis of single-channel gating and conductance. The anti-RYR antibody had a single action, a simultaneous decrease in single-channel open probability (Po) and conductance. Both antibodies appear to alter single-channel gating by disrupting the Ca2(+)-activation mechanism of the channel. Anti-RYR-antibody-induced gating abnormalities were reversed by ATP, although the ATP-re-activated channels had altered gating kinetics. Two antigenic regions, recognizing the anti-RYR antibody, in the C-terminal end of the RYR primary amino acid sequence contain or are closely associated with putative ligand (Ca2+ and ATP)-binding sites identified previously. Our results demonstrate (1) that the antibodies induced abnormal gating (decreased open probability and stabilization of subconducting states) of SR release channels, and (2) that abnormal gating is not associated with physical obstruction or alteration of the conduction pathway. Thus antibodies directed at specific regions of the RYR (e.g. putative ligand-binding sites) can be used as effective probes with which to study the structural and functional properties of the SR Ca2(+)-release channel gating at the single-channel level.

Properties of Ryr3 Ryanodine Receptor Isoform in Mammalian Brain

“…Thus, the binding of the antibody may not affect the ryanodine binding activity of RyR. We could not exclude the possibility that the antibody might alter the properties of RyR channels as previously reported (38,39), because we could not obtain Ryr3 without complex formation with the antibody. However, well-consistent properties of ryanodine binding with those of bullfrog ␤-RyR (Fig.…”

Molecular structure and tissue distribution of ryanodine receptors calcium channels

“…25 In addition, Fill et al have indicated that polyclonal antibodies directed against the region 4445-4586, in RyRl, could reduce the open probability of the channe1. 26 More recently, an affinity purified antibody directed against epitopes present between residues 4478-4512 (sequence 13C2), was shown to increase the calcium sensitivity of RyRl incorporated into planar lipid bilayers, without moddying the Mg2+, ATP, Ry, and ruthenium red modulation.l9 When the 13C2 antibody was further affinity purified on the peptide sequence PEPEPEPEPE (13C2pl) included in the 13C2 fragment, it was shown, in single channel recording experiments, to specifically inhibit calcium binding and to inhibit calcium or caffeine channel activation, without affecting ATP dependent channel activity.20 It appears that the PE repeated motif is a high affinity calcium binding domain, which can be blocked by a specific antibody. When other epitopes near the PE repeat are targeted by the anti-13C2 antisera a possible local conformational change could be responsible for an increased calcium binding affinity and calcium-sensitivity.20 A different polyclonal antibody, reacting against residues 4380-4625, which contain the PE repeat, was also shown to decrease dexorubicin and calciuminduced calcium release.27 It will be interesting to test whether the PE repeat is one of the major epitopes recognized by this antibody.…”

A brain‐specific transcript from the 3′‐terminal region of the skeletal muscle ryanodine receptor gene

“…According to our molecular model of the ryanodine receptor [3], this expressed protein has the channel region containing four putative transmembrane segments (Ml-M4), but lacks the 'foot' region. Furthermore this protein is thought to hold the region around amino acid residue 4500 which is suggested to be involved in Ca2' binding and calcium-induced calcium release in the skeletal muscle ryanodine receptor [26,27]. A possible scheme for the expression of the skeletal muscle ryanodine receptor gene is shown in Fig.…”