Balwant S. Tuana

The muscle-specific calmodulin-dependent protein kinase assembles with the glycolytic enzyme complex at the sarcoplasmic reticulum and modulates the activity of glyceraldehyde-3-phosphate dehydrogenase in a Ca2+/calmodulin-dependent manner

The skeletal muscle specific Ca2+/calmodulin-dependent protein kinase (CaMKIIβM) is localized to the sarcoplasmic reticulum (SR) by an anchoring protein, αKAP, but its function remains to be defined. Protein interactions of CaMKIIβM indicated that it exists in complex with enzymes involved in glycolysis at the SR membrane. The kinase was found to complex with glycogen phosphorylase, glycogen debranching enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and creatine kinase in the SR membrane. CaMKIIβM was also found to assemble with aldolase A, GAPDH, enolase, lactate dehydrogenase, creatine kinase, pyruvate kinase, and phosphorylase b kinase from the cytosolic fraction. The interacting proteins were substrates of CaMKIIβM, and their phosphorylation was enhanced in a Ca2+- and calmodulin (CaM)-dependent manner. The CaMKIIβM could directly phosphorylate GAPDH and markedly increase (∼3.4-fold) its activity in a Ca2+/CaM-dependent manner. These data suggest that the muscle CaMKIIβM isoform may serve to assemble the glycogen-mobilizing and glycolytic enzymes at the SR membrane and specifically modulate the activity of GAPDH in response to calcium signaling. Thus, the activation of CaMKIIβM in response to calcium signaling would serve to modulate GAPDH and thereby ATP and NADH levels at the SR membrane, which in turn will regulate calcium transport processes.

Modification of a discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide gel system for minigel electrophoresis

A highly porous and efficient discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system was recently described by J. P. Doucet and J. M. Trifaró [1988) Anal. Biochem. 168, 265-271). The system was developed to separate with high and broad resolution the components from large volume samples after an overnight electrophoresis. This system was found to have many advantages. However, when used directly as a minigel system, this method cannot sustain the high voltage inherent to minigel electrophoresis and produces artefacts, namely a double front and a loss of resolution in the low molecular weight range. These problems were eliminated using the buffer system of M. A. Porzio and A.M. Pearson [1977) Biochem. Biophys. Acta 490, 27-34) in the separating gel and in the electrode chambers. The resulting modified discontinuous minigel system has the same advantages as the technique described for large slab gel electrophoresis, including the effective and rapid transfer of high molecular weight proteins to nitrocellulose membranes, as well as the advantages of the minigel format.

The association of cardiac dystrophin with myofibrils/Z-disc regions in cardiac muscle suggests a novel role in the contractile apparatus.

Dystrophin serves a variety of roles at the cell membrane through its associations, and defects in the dystrophin gene can give rise to muscular dystrophy and genetic cardiomyopathy. We investigated localization of cardiac dystrophin to determine potential intracellular sites of association. Subcellular fractionation revealed that while the majority of dystrophin was associated with the sarcolemma, about 35% of the 427-kDa form of dystrophin was present in the myofibrils. The dystrophin homolog utrophin was detectable only in the sarcolemmal membrane and was absent from the myofibrils as were other sarcolemmal glycoproteins such as adhalin and the sodium-calcium exchanger. Extraction of myofibrils with KCl and detergents could not solubilize dystrophin. Dystrophin could only be dissociated from the myofibrillar protein complex in 5 M urea followed by sucrose density gradient centrifugation where it co-fractionated with one of two distinctly sedimenting peaks of actin. Immunoelectron microscopy of intracellular regions of cardiac muscle revealed a selective labeling of Z-discs by dystrophin antibodies. In the genetically determined cardiomyopathic hamster, strain CHF 147, the time course of development of cardiac insufficiency correlated with an overall 75% loss of myofibrillar dystrophin. These findings collectively show that a significant pool of the 427-kDa form of cardiac dystrophin was specifically associated with the contractile apparatus at the Z-discs, and its loss correlated with progression to cardiac insufficiency in genetic cardiomyopathy. The loss of distinct cellular pools of dystrophin may contribute to the tissue-specific pathophysiology in muscular dystrophy.

Effects of central sodium on epithelial sodium channels in rat brain

We evaluated the effects of intracerebroventricular (icv) infusion of Na(+)-rich artificial cerebrospinal fluid (aCSF), with or without the mineralocorticoid receptor (MR) blocker spironolactone, on epithelial Na(+) channel (ENaC) subunits and regulators, such as MR, serum/glucocorticoid-inducible kinase 1, neural precursor cells expressed developmentally downregulated 4-like gene, 11beta-hydroxylase, and aldosterone synthase, in brain regions of Wistar rats. The effects of icv infusion of the amiloride analog benzamil on brain tissue and CSF Na(+) concentration ([Na(+)]) were also assessed. In the choroid plexus and ependyma of the anteroventral third ventricle, ENaC subunits are present in apical and basal membranes. Na(+)-rich aCSF increased beta-ENaC mRNA and immunoreactivity in the choroid plexus and increased alpha- and beta-ENaC immunoreactivities in the ependyma. Na(+)-rich aCSF increased alpha- and beta-ENaC-gold-labeled particles in the microvilli of the choroid plexus and in basolateral membranes of the ependyma. Spironolactone only prevented the increase in beta-ENaC immunoreactivity in the choroid plexus and ependyma. In the supraoptic nucleus, paraventricular nucleus, and subfornical organ, Na(+)-rich aCSF did not affect mRNA expression levels of the studied genes. Benzamil significantly increased CSF [Na(+)] in the control, but not Na(+)-rich, aCSF group. In contrast, benzamil prevented the increase in hypothalamic tissue [Na(+)] by Na(+)-rich aCSF. These results suggest that CSF Na(+) upregulates ENaC expression in the brain epithelia, but not in the neurons of hypothalamic nuclei. ENaC in the choroid plexus and ependyma appear to contribute to regulation of Na(+) homeostasis in the brain.

Dihydropyridine binding to the L-type Ca2+ channel in rabbit heart sarcolemma and skeletal muscle transverse-tubules: role of disulfide, sulfhydryl and phosphate groups.

The dihydropyridine receptor is associated with the L-type Ca2+ channel in the cell membrane. In this study we have examined the effects of group-specific modification on dihydropyridine binding in heart sarcolemmal membranes isolated from the rabbit. Specifically, dithiothreitol and glutathione were employed to assess the possible role of disulfide (-SS-) bonds in the binding of [3H]dihydropyridines. NEM, PCMS and iodoacetamide were employed to examine the effect of blocking free sulfhydryl groups (-SH) on the binding of [3H]dihydropyridines to their receptor in heart sarcolemma. Glutathione inhibited [3H]PN200-110 binding to sarcolemmal membranes 100%, with an IC50 value of 50 microM, while DTT inhibited maximally by 75% with an IC50 value in the millimolar range. Alkylation of free sulfhydryl groups by NEM or iodoacetamide inhibited binding of [3H]PN200-110 binding in cardiac sarcolemma approx. 40-60%. Blocking of free sulfhydryl groups by PCMS completely inhibited [3H]PN200-110 binding to their receptor in sarcolemmal membranes in a dose-dependent manner with an IC50 value of 20 microM. These results suggest the involvement of disulfide bonds and free sulfhydryl groups in DHP binding to the L-type Ca2+ channel in heart muscle. We also examined the effect of membrane phosphorylation on the specific binding of the dihydropyridine [3H]nitrendipine to its receptor. Phosphorylation was studied in cardiac sarcolemmal as well as skeletal muscle transverse-tubule membranes. Phosphorylation due to endogenous protein kinase and cAMP-dependent protein kinase was without effect on [3H]nitrendipine binding in both cardiac sarcolemmal and skeletal muscle membranes. Addition of exogenous calmodulin under conditions known to promote Ca2+/calmodulin-dependent phosphorylation increased [3H]nitrendipine binding 20% with no alteration in KD in both types of membrane preparation. These results suggest a role for calmodylin in dihydropyridine binding to L-type Ca2+ channels.

REGULATION OF DIHYDROPYRIDINE AND RYANODINE RECEPTOR GENE EXPRESSION IN SKELETAL MUSCLE : ROLE OF NERVE, PROTEIN KINASE C, AND CAMP PATHWAYS

The dihydropyridine (DHP) and ryanodine (RY) receptors play a critical role in depolarization-induced calcium release in skeletal muscle, yet the factors which govern their expression remain unknown. We investigated the roles of electrical activity and trophic factors in the regulation of the genes encoding the α, α, and β subunits of the DHP receptor as well as the RY receptor in rat skeletal muscle in vivo. Muscle paralysis, induced by denervation, had no effect on the DHP receptor mRNA levels while the RY receptor mRNA was decreased. In contrast, chronic superfusion of tetrodotoxin onto the sciatic nerve resulted in a marked increase in mRNA levels and transcriptional activity of both DHP and RY receptor genes. Since nerve can induce changes in second messenger pathways which modulate muscle gene expression, we attempted to identify factors which regulate DHP and RY receptor expression using cultured myotubes. Elevated cAMP levels specifically inhibited the expression of RY receptor mRNA while 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, increased the transcripts encoding the RY receptor and the α subunit of the DHP receptor. Changes in the level of mRNAs were paralleled by altered receptor numbers. Neither cAMP nor protein kinase C altered transcriptional activity of the DHP and RY receptor genes. These results demonstrate that neural factor(s) regulate DHP and RY receptor mRNA levels in vivo via transcriptional mechanisms while protein kinase C and cAMP can modulate DHP and RY receptor transcript levels by a transcription-independent process.

A novel isoform of sarcolemmal membrane-associated protein (SLMAP) is a component of the microtubule organizing centre.

The microtubule organizing centre (MTOC) or the centrosome serves a crucial role in the establishment of cellular polarity, organization of interphase microtubules and the formation of the bipolar mitotic spindle. We have elucidated the genomic structure of a gene encoding the sarcolemmal membrane-associated protein (SLMAP), which encodes a 91 kDa polypeptide with a previously uncharacterized N-terminal sequence encompassing a forkhead-associated (FHA) domain that resides at the centrosome. Anti-peptide antibodies directed against SLMAP N-terminal sequences showed colocalization with γ-tubulin at the centrosomes at all phases of the cell cycle. Agents that specifically disrupt microtubules did not affect SLMAP association with centrosomes. Furthermore, SLMAP sequences directed a reporter green fluorescent protein (GFP) to the centrosome, and deletions of the newly identified N-terminal sequence from SLMAP prevented the centrosomal targeting. Deletion-mutant analysis concluded that overall, structural determinants in SLMAP were responsible for centrosomal targeting. Elevated levels of centrosomal SLMAP were found to be lethal, whereas mutants that lacked centrosomal targeting inhibited cell growth accompanied by an accumulation of cells at the G2/M phase of the cell cycle.

MOLECULAR CLONING, EXPRESSION, AND CHROMOSOMAL ASSIGNMENT OF SARCOLEMMAL-ASSOCIATED PROTEINS : A FAMILY OF ACIDIC AMPHIPATHIC ALPHA -HELICAL PROTEINS ASSOCIATED WITH THE MEMBRANE

Two overlapping cDNAs encoding a novel sarcolemmal associated protein (SLAP) were isolated from a cardiac cDNA expression library by immunoscreening with anti-sarcolemmal antibodies. Further characterization of these clones showed that they belonged to a family of related cDNAs that potentially encode polypeptides of 37, 46, and 74 kDa designated SLAP1, SLAP2, and SLAP3, respectively. The SLAP3 transcript was ubiquitously expressed, whereas SLAP1 and SLAP2 transcripts were predominantly expressed in cardiac, soleus, and smooth muscle. SLAP was encoded by a single gene that mapped to chromosome 3p14.3–21.2, and the various transcripts are likely generated by alternative splicing. The primary structure of SLAP predicted that it would have large regions of coiled-coil structure including an 11-heptad acidic amphipathic α-helical segment. The carboxyl-terminal region of the SLAP proteins was predicted to have a transmembrane domain, although there was no discernible signal sequence. SLAPs could only be solubilized from cardiac membrane with detergents suggesting that they were integral membrane proteins. Subcellular distribution studies showed that MYC epitope-tagged SLAP localized to regions of juxtaposition between neighboring cell membranes although an intracellular pool of the protein was also present in cells undergoing apparent cleavage. Immunohistochemical localization of SLAP in cardiac muscle revealed that SLAP associated with the sarcolemma and also displayed a reticular pattern of staining that resembled the transverse tubules and the sarcoplasmic reticulum. The SLAPs define a new family of tail-anchored membrane proteins that exhibit tissue-specific expression and are uniquely situated to serve a variety of roles through their coiled-coil motifs.

Hydrophobic profiles of the tail anchors in SLMAP dictate subcellular targeting

BackgroundTail anchored (TA) membrane proteins target subcellular structures via a C-terminal transmembrane domain and serve prominent roles in membrane fusion and vesicle transport. Sarcolemmal Membrane Associated Protein (SLMAP) possesses two alternatively spliced tail anchors (TA1 or TA2) but their specificity of subcellular targeting remains unknown.ResultsTA1 or TA2 can direct SLMAP to reticular structures including the endoplasmic reticulum (ER), whilst TA2 directs SLMAP additionally to the mitochondria. Despite the general structural similarity of SLMAP to other vesicle trafficking proteins, we found no evidence for its localization with the vesicle transport machinery or a role in vesicle transport. The predicted transmembrane region of TA2 is flanked on either side by a positively charged amino acid and is itself less hydrophobic than the transmembrane helix present in TA1. Substitution of the positively charged amino acids, in the regions flanking the transmembrane helix of TA2, with leucine did not alter its subcellular targeting. The targeting of SLMAP to the mitochondria was dependent on the hydrophobic nature of TA2 since targeting of SLMAP-TA2 was prevented by the substitution of leucine (L) for moderately hydrophobic amino acid residues within the transmembrane region. The SLMAP-TA2-4L mutant had a hydrophobic profile that was comparable to that of SLMAP-TA1 and had identical targeting properties to SLMAP-TA1.ConclusionThus the overall hydrophobicity of the two alternatively spliced TAs in SLMAP determines its subcellular targeting and TA2 predominantly directs SLMAP to the mitochondira where it may serve roles in the function of this organelle.

Regulated expression and temporal induction of the tail-anchored sarcolemmal-membrane-associated protein is critical for myoblast fusion

Sarcolemmal-membrane-associated proteins (SLMAPs) define a new class of coiled-coil tail-anchored membrane proteins generated by alternative splicing mechanisms. An in vivo expression analysis indicated that SLMAPs are present in somites (11 days post-coitum) as well as in fusing myotubes and reside at the level of the sarcoplasmic reticulum and transverse tubules in adult skeletal muscles. Skeletal-muscle myoblasts were found to express a single 5.9 kb transcript, which encodes the full-length approximately 91 kDa SLMAP3 isoform. Myoblast differentiation was accompanied by the stable expression of the approximately 91 kDa SLMAP protein as well as the appearance of an approximately 80 kDa isoform. Deregulation of SLMAPs by ectopic expression in myoblasts resulted in a potent inhibition of fusion without affecting the expression of muscle-specific genes. Membrane targeting of the de-regulated SLMAPs was not critical for the inhibition of myotube development. Protein-protein interaction assays indicated that SLMAPs are capable of self-assembling, and the de-regulated expression of mutants that were not capable of forming SLMAP homodimers also inhibited myotube formation. These results imply that regulated levels and the temporal induction of SLMAP isoforms are important for normal muscle development.