Use of continuous-elution gel electrophoresis as a preparative tool for blot overlay analysis

Blot overlay techniques have long been used to directly visualize protein-protein interactions within membrane complexes. However, this approach is often hampered by the limited quantities of purified membrane proteins available for conjugation with marker molecules. Here we applied continuous-elution gel electrophoresis as a preparative alternative to isolate sufficient amounts of a homogeneous protein sample to be used as a peroxidase-labeled probe in blot overlays. Microsomal muscle proteins ranging from approximately 20 to 600 kDa were electrophoretically separated and various marker proteins present in eluted fractions were identified by immunoblotting. Since the supramolecular structure of calsequestrin has recently been determined, this terminal cisternae protein was isolated as a model protein for studying protein-protein interactions. In blot overlay assays, peroxidase-conjugated calsequestrin specifically bound to the ryanodine receptor, triadin, calsequestrin itself, and junctin, illustrating that the biological binding affinities are retained in electrophoretically prepared muscle proteins. Potential applications for differential blot overlay approaches and for analyzing pathophysiological preparations from dystrophic muscle were evaluated. Since continuous-elution gel electrophoresis can separate a wide range of differently sized proteins from subcellular fractions, our report indicates that this technique can be utilized for the rapid identification of protein-protein interactions in future high-throughput analyses of subproteomes.     

The Catecholaminergic Polymorphic Ventricular Tachycardia Mutation R33Q Disrupts the N-terminal Structural Motif That Regulates Reversible Calsequestrin Polymerization

Calsequestrin undergoes dynamic polymerization with increasing calcium concentration by front-to-front dimerization and back-to-back packing, forming wire-shaped structures. A recent finding that point mutation R33Q leads to lethal catecholaminergic polymorphic ventricular tachycardia (CPVT) implies a crucial role for the N terminus. In this study, we demonstrate that this mutation resides in a highly conserved alternately charged residue cluster (DGKDR; cluster 1) in the N-terminal end of calsequestrin. We further show that this cluster configures itself as a ring system and that the dipolar arrangement within the cluster brings about a critical conformational flip of Lys³¹-Asp³² essential for dimer stabilization by formation of a H-bond network. We additionally show that Ca²⁺-induced calsequestrin aggregation is nonlinear and reversible and can regain the native conformation by Ca²⁺ chelation with EGTA. This study suggests that cluster 1 works as a molecular switch and governs the bidirectional transition between the CASQ2 monomer and dimer. We further demonstrate that mutations disrupting the alternating charge pattern of the cluster, including R33Q, impair Ca²⁺-CASQ2 interaction, leading to altered polymerization-depolymerization dynamics. This study provides new mechanistic insight into the functional effects of the R33Q mutation and its potential role in CPVT.     

Increased calcium leak associated with reduced calsequestrin expression in hyperthyroid cardiomyocytes

IntroductionCalcium (Ca²⁺) leak during cardiac diastole is chiefly mediated by intracellular Ca²⁺ channel/Ryanodine Receptors. Increased diastolic Ca²⁺ leak has been proposed as the mechanism underlying the appearance of hereditary arrhythmias. However, little is known about alterations in diastolic Ca²⁺ leak and the specific roles played by key intracellular Ca²⁺-handling proteins in hyperthyroidism, a known arrhythmogenic condition.AimWe sought to determine whether there were modifications in diastolic Ca²⁺ leak, based on the recording of Ca²⁺ sparks and Ca²⁺ waves; we also investigated changes in the expression and activity of key Ca²⁺ handling proteins, including ryanodine receptors, Sarco-Endoplasmic Reticulum Ca²⁺ ATPase pump and calsequestrin in isolated left-ventricular cardiomyocytes isolated from hyperthyroid rats.Materials and methodsElectrocardiography (ECG) recordings were performed in control and hyperthyroid rats. Ca²⁺ sparks, Ca²⁺ waves, and electrically-stimulated Ca²⁺ transients were recorded in Fluo-3-loaded cardiomyocytes from both experimental groups using confocal microscopy. In addition, left-ventricular homogenates and Ryanodine Receptor-enriched membrane fractions were prepared for assessing [³H]-ryanodine binding, hydrolytic ATPase activity of SERCA pump and expression levels of key proteins by Western blot, and cDNA for real-time qPCR.Results and conclusionsExtrasystoles were observed in hearts of hyperthyroid rats by ECG recordings. Arrhythmogenic activity, high incidence of Ca²⁺ waves, and de novo Ca²⁺ wavelets −in the absence of sarcoplasmic reticulum Ca²⁺ overload- were recorded in these cardiomyocytes. The exacerbated diastolic Ca²⁺ leak and arrhythmogenic activities were related to a diminished expression of calsequestrin along with increased SERCA pump activity, which, in effect, promoted a gain-of-function in RyRs without alterations in SR Ca²⁺ load, RyR expression or its Ca²⁺ sensitivity.     

Junctin and triadin each activate skeletal ryanodine receptors but junctin alone mediates functional interactions with calsequestrin

Normal Ca²⁺ signalling in skeletal muscle depends on the membrane associated proteins triadin and junctin and their ability to mediate functional interactions between the Ca²⁺ binding protein calsequestrin and the type 1 ryanodine receptor in the lumen of the sarcoplasmic reticulum. This important mechanism conserves intracellular Ca²⁺ stores, but is poorly understood. Triadin and junctin share similar structures and are lumped together in models of interactions between skeletal muscle calsequestrin and ryanodine receptors, however their individual roles have not been examined at a molecular level. We show here that purified skeletal ryanodine receptors are similarly activated by purified triadin or purified junctin added to their luminal side, although a lack of competition indicated that the proteins act at independent sites. Surprisingly, triadin and junctin differed markedly in their ability to transmit information between skeletal calsequestrin and ryanodine receptors. Purified calsequestrin inhibited junctin/triadin-associated, or junctin-associated, ryanodine receptors and the calsequestrin re-associated channel complexes were further inhibited when luminal Ca²⁺ fell from 1 mM to ≤100 μM, as seen with native channels (containing endogenous calsequestrin/triadin/junctin). In contrast, skeletal calsequestrin had no effect on the triadin/ryanodine receptor complex and the channel activity of this complex increased when luminal Ca²⁺ fell, as seen with purified channels prior to triadin/calsequestrin re-association. Therefore in this cell free system, junctin alone mediates signals between luminal Ca²⁺, skeletal calsequestrin and skeletal ryanodine receptors and may curtail resting Ca²⁺ leak from the sarcoplasmic reticulum. We suggest that triadin serves a different function which may dominate during excitation–contraction coupling.     

Retrograde activation of store-operated calcium channel

Store-operated Ca2+ entry represents an important mechanism for refilling of a depleted intracellular-reticulum Ca2+ store following sustained activation of the IP3 receptor or ryanodine receptor RyR/Ca2+ release channel in the endoplasmic/sarcoplasmic reticulum (ER/SR). Recent studies have demonstrated the existence of store-operated Ca2+ channel (SOC) in muscle cells, whose activation process appears to be coupled to conformational changes of the RyR. Regulation of the plasma membrane (PM)-resided SOC by the SR-located RyR requires an integrity of the junctional membrane structure between SR and PM. Proteins that interact with RyR or influence the Ca2+ buffering capacity in the ER or SR lumen also participate in the activation process of SOC. Calsequestrin (CSQ) and calreticulin (CRT) are SR/ER-resident proteins, with highly negative charged regions at the carboxyl-terminal end that exhibit high buffering capacity for luminal Ca2+. CSQ and CRT not only modulate the intracellular Ca2+ release process but also might provide retrograde signals to regulate the function of SOC. The functional interplay between CSQ, RyR and SOC may serve essential roles of Ca2+ signaling in muscle contraction and development. A tight link between the expression of CRT and operation of SOC exist in certain cancer cells, where the reduced sensitivity to apoptosis may correlate with the altered function of SOC.     

Increased expression of the nicotinic acetylcholine receptor in stimulated muscle

Chronic low-frequency stimulation has been used as a model for investigating responses of skeletal muscle fibres to enhanced neuromuscular activity under conditions of maximum activation. Fast-to-slow isoform shifting of markers of the sarcoplasmic reticulum and the contractile apparatus demonstrated successful fibre transitions prior to studying the effect of chronic electro-stimulation on the expression of the nicotinic acetylcholine receptor. Comparative immunoblotting revealed that the alpha- and delta-subunits of the receptor were increased in 10-78 day stimulated specimens, while an associated component of the surface utrophin-glycoprotein complex, beta-dystroglycan, was not drastically changed in stimulated fast skeletal muscle. Previous studies have shown that electro-stimulation induces degeneration of fast glycolytic fibres, trans-differentiation leading to fast-to-slow fibre transitions and activation of muscle precursor cells. In analogy, our results indicate a molecular modification of the central functional unit of the post-synaptic muscle surface within existing neuromuscular junctions and/or during remodelling of nerve-muscle contacts.     

Immunological detection and localization of a calsequestrin-like protein in red beet and cucumber cells

Summary Calsequestrin is a calcium binding protein present in the sarcoplasmic reticulum (SR) of animal muscle cells and is thought to be essential for the rapid uptake and release of Ca²⁺, and thus for the regulation of Ca²⁺-dependent cellular functions. Higher plant cells of red beet (Beta vulgaris L.) and cucumber (Cucumis sativus L.) contain a polypeptide of about M_r 55000 that cross-reacts with a monoclonal antibody raised against calsequestrin from rabbit skeletal muscle SR. In beet this protein changes its apparent molecular weight with pH as indicated in Western immunoblotting. Although this protein bound calcium it was not the dominant calcium-binding protein in red beet. Washing of beet root tissue leads to a slight increase of this polypeptide in microsomal fractions as indicated by immunoblotting. After immunoblotting to partially purified cell membrane fractions this polypeptide appeared to be predominantly associated with endoplasmic reticulum-enriched fractions. Immunogold labelling of ultrathin sections of cucumber hypocotyl using the anti-calsequestrin antibody showed that gold particles were very largely confined to the cytosol and often in close proximity to the ER. Clusters of up to nine gold particles were observed, often over small vesicular areas, as observed in some animal tissues. These results indicate that red beet and cucumber cells contain a protein which may be related to animal calsequestrin. It appears to be associated with the ER and could be involved in cellular calcium regulation.     

Occurrence of atypical Ca2+ transients in triadin-binding deficient-RYR1 mutants

Triadin in the junctional sarcoplasmic reticulum (SR) of skeletal muscle cells has been suggested to interact with ryanodine receptor 1 (RYR1) via its KEKE motifs. Recently, we showed that amino acid residues D4878, D4907, and E4908 in RYR1 are critical for triadin-binding in vitro [J.M. Lee, S.H. Rho, D.W. Shin, C. Cho, W.J. Park, S.H. Eom, J. Ma, D.H. Kim, Negatively charged amino acids within the intraluminal loop of ryanodine receptor are involved in the interaction with triadin, J. Biol. Chem. 279 (2004) 6994-7000]. In order to test whether a disruption of the triadin-binding site(s) in RYR1 affects SR Ca(2+) release, alanine-substituted single (D4878A, D4907A, and E4908A) and triple (RYR1-TM) mutants of D4878, D4907, and E4908 were expressed in RYR1-null myotubes. Co-immunoprecipitation experiments showed a 50-60% decrease of triadin brought down in the D4907A and RYR1-TM complexes compared to the triadin-wtRYR1 complex. Ca(2+) imaging experiments using Fluo-4-AM showed atypical caffeine responses in myotubes expressing D4907A and RYR1-TM characterized by either a lack of or slower activation and faster inactivation of Ca(2+) transients. The results suggest that disruption of interaction between triadin and RYR1 impairs RYR1 function and SR Ca(2+) release.