Roque El-Hayek

Identification of Calcium Release-triggering and Blocking Regions of the II-III Loop of the Skeletal Muscle Dihydropyridine Receptor

In an attempt to identify and characterize functional domains of the rabbit skeletal muscle dihydropyridine receptor α subunit II-III loop, we synthetized several peptides corresponding to different regions of the loop: peptides A, B, C, C1, C2, D (cf. Fig. 1). Peptide A (Thr-Leu) activated ryanodine binding to, and induced Ca release from, rabbit skeletal muscle triads, but none of the other peptides had such effects. Peptide A-induced Ca release and activation of ryanodine binding were partially suppressed by an equimolar concentration of peptide C (Glu-Pro) but were not affected by the other peptides. These results suggest that the short stretch in the II-III loop, Thr-Leu, is responsible for triggering SR Ca release, while the other region, Glu-Pro, functions as a blocker of the release trigger. A hypothesis is proposed to account for how these subdomains interact with the sarcoplasmic reticulum Ca release channel protein during excitation-contraction coupling.

A POSTULATED ROLE OF THE NEAR AMINO-TERMINAL DOMAIN OF THE RYANODINE RECEPTOR IN THE REGULATION OF THE SARCOPLASMIC RETICULUM CA2+ CHANNEL

To test the hypothesis that interactions among several putative domains of the ryanodine receptor (RyR) are involved in the regulation of its Ca2+ release channel, we synthesized several peptides corresponding to selected NH2-terminal regions of the RyR. We then examined their effects on ryanodine binding and Ca2+ release activities of the sarcoplasmic reticulum isolated from skeletal and cardiac muscle. Peptides 1–2s, 1–2c, and 1 enhanced ryanodine binding to cardiac RyR and induced a rapid Ca2+ release from cardiac SR in a dose-dependent manner. The order of the potency for the activation of the Ca2+ release channel was 1–2c > 1 > 1–2s. Interestingly, these peptides produced significant activation of the cardiac RyR at near zero or subactivating [Ca2+], indicating that the peptides enhanced the Ca2+ sensitivity of the channel. Peptides 1–2c, 1–2s, and 1 had virtually no effect on skeletal RyR, although occasional and variable extents of activation were observed in ryanodine binding assays performed at 36 °C. Peptide 3 affected neither cardiac nor skeletal RyR. We propose that domains 1 and 1–2 of the RyR, to which these activating peptides correspond, would interact with one or more other domains within the RyR (including presumably the Ca2+-binding domain) to regulate the Ca2+channel.

Involvement of the Glu724–Pro760 Region of the Dihydropyridine Receptor II-III Loop in Skeletal Muscle-type Excitation-Contraction Coupling

Our previous study (El-Hayek, R., Antoniu, B., Wang, J. P., Hamilton, S. L., and Ikemoto, N. (1995)J. Biol. Chem. 270, 22116–22118) suggested the hypothesis that skeletal muscle-type excitation-contraction coupling is regulated by two domains (activating and blocking) of the II-III loop of the dihydropyridine receptor α1 subunit. We investigated this hypothesis by examining conformational changes in the ryanodine receptor induced by synthetic peptides and by transverse tubular system (T-tubule) depolarization. Peptide A, corresponding to the Thr671–Leu690 region, rapidly changed the ryanodine receptor conformation from a blocked state (low fluorescence of the conformational probe, methyl coumarin acetamide, attached specifically to the ryanodine receptor) to an activated state (high methyl coumarin acetamide fluorescence) as T-tubule depolarization did. Peptide C, corresponding to the Glu724–Pro760region, blocked both conformational changes induced by peptide A and T-tubule depolarization. Its ability to block peptide A-induced and depolarization-induced activation was considerably impaired by replacing the portion of peptide C corresponding to the Phe725–Pro742 region of the loop with cardiac muscle-type sequence. These results are consistent with the model that depolarization-induced activation of excitation-contraction coupling and blocking/repriming are mediated by the peptide A region and the peptide C region (containing the critical Phe725–Pro742 sequence) of the II-III loop, respectively.

Neomycin: A novel potent blocker of communication between T-tubule and sarcoplasmic reticulum

Ca2+ release from the sarcoplasmic reticulum (SR) was induced in isolated triads by direct stimulation of the SR moiety by polylysine, or stimulation via chemical depolarization of the transverse tubule (T‐tubule) moiety. Polylysine‐induced release was blocked by neomycin with an IC50 (the concentration for half‐maximal inhibition) of O.3, μM. However, the IC50 for neomycin block of depolarization‐induced Ca2+ release sharply decreased in a voltage‐dependent fashion, and it was 5.3 nM at a maximal extent of T‐tubule depolarization. These results suggest that the high affinity binding of neomycin to the triad leads to the specific blocking of the signal transmission from T‐tubule to SR.

Signal transmission and transduction in excitation-contraction coupling.

For the better understanding of the molecular mechanism of E-C coupling, two key questions remain to be resolved: (a) how the excitation signal elicited in the T-tubule membrane is transmitted to the ryanodine receptor, RyR (signal transmission), and (b) how the signal transmitted from the T-tubule to the RyR is translated into the action of opening the sarcoplasmic reticulum Ca2+ channel to induce Ca2+ release and muscle contraction (signal transduction). Our recent studies on E-C coupling with the use of the isolated triads and synthetic peptides have provided several pieces of new information. It appears that the signal transmission is mediated by the voltage-controlled binding of the Thr671-Leu690 region (Trigger) of the cytoplasmic II-III loop of the dihydropyridine receptor alpha 1 a subunit to the putative activator site on the RyR. The transmitted signal is translated to the action of channel opening by mediation of rapid conformational changes occurring in the RyR. Upon T-tubule polarization the Glu724-Pro760 region of the loop (Blocker) replaces the RyR-bound Trigger. This reprimes the RyR to the original conformational state.

Reciprocal control of the conformational state of the sarcoplasmic reticulum calcium channel protein by polarization and depolarization in the transverse tubule

We attached the conformational probe methylcoumarin acetate (MCA) specifically to the junctional foot protein (JFP) moiety of triads, and monitored conformational changes in the JFP during polarization and depolarization of the T‐tubule moiety. The MCA fluorescence decreased upon T‐tubule polarization, and the fluorescence changes were blocked by preventing T‐tubule polarization or by a nimodipine block of the T‐tubule‐to‐sarcoplasmic reticulum communication. Depolarization of the T‐tubule reversed the MCA fluorescence decrease which had been produced by T‐tubule polarization. These results suggest that the conformational and functional states of the JFP are regulated by T‐tubule polarization and depolarization in a reciprocal fashion.