Novel Mechanism of Regulation of Protein 4.1G Binding Properties Through Ca2+/Calmodulin-Mediated Structural Changes

Protein 4.1G (4.1G) is a widely expressed member of the protein 4.1 family of membrane skeletal proteins. We have previously reported that Ca²⁺-saturated calmodulin (Ca²⁺/CaM) modulates 4.1G interactions with transmembrane and membrane-associated proteins through binding to Four.one-ezrin–radixin–moesin (4.1G FERM) domain and N-terminal headpiece region (GHP). Here we identify a novel mechanism of Ca²⁺/CaM-mediated regulation of 4.1G interactions using a combination of small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, and circular dichroism spectroscopy analyses. We document that GHP intrinsically disordered coiled structure switches to a stable compact structure upon binding of Ca²⁺/CaM. This dramatic conformational change of GHP inhibits in turn 4.1G FERM domain interactions due to steric hindrance. Based upon sequence homologies with the Ca²⁺/CaM-binding motif in protein 4.1R headpiece region, we establish that the 4.1G S⁷¹RGISRFIPPWLKKQKS peptide (pepG) mediates Ca²⁺/CaM binding. As observed for GHP, the random coiled structure of pepG changes to a relaxed globular shape upon complex formation with Ca²⁺/CaM. The resilient coiled structure of pepG, maintained even in the presence of trifluoroethanol, singles it out from any previously published CaM-binding peptide. Taken together, these results show that Ca²⁺/CaM binding to GHP, and more specifically to pepG, has profound effects on other functional domains of 4.1G.     

Decrease of Ca2+-ATPase activity in human keratinocytes during calcium-induced differentiation

Ca²⁺ regulates keratinocyte differentiation by increasing intracellular Ca²⁺ levels. Ca²⁺-ATPase in the Ca²⁺-induced differentiation of human keratinocytes was investigated by measuring Ca²-ATPase mRNA, protein, and activity levels. Human keratinocytes were grown in Keratinocyte Growth Medium containing 0.03, 0.1, or 1.2 mM Ca²⁺ and assayed on days 2, 5, 7, 14, and 21. Ca²⁺-ATPase mRNA levels were found to be modestly increased in 5-, 7-, and 14-day cultured cells as compared with 2-day cultured cells, but levels fell below that of the 2-day cultured cells in the 21-day cultured cells. The Ca²⁺-ATPase mRNA levels were not affected by Ca²⁺ levels. A 135-kDa protein in human keratinocytes cross reacted with the monoclonal antibody against human erythrocyte Ca²⁺-ATPase. The level of this protein was decreased by Ca²⁺ and lost during differentiation, in parallel with the loss of enzymatic activity. Ca²⁺ influx of postconfluent 1.2 mM Ca²-grown cells was higher than that of cells grown in lower Ca²⁺ concentrations. Ca²⁺ efflux from postconfluent cells grown in 0.03 mM Ca²⁺ was less than that from cells grown in stronger Ca²⁺ concentrations. These results suggest that the loss of the plasma membrane Ca²⁺-ATPase with time in culture contributes to the rise in intracelluar Ca²⁺, thus promoting keratinocyte differentiation. J. Cell. Physiol. 172:146–154, 1997. © 1997 Wiley-Liss, Inc.     

Immunolocalization of the sarcolemmal Ca2+/Mg2+ ecto-ATPase (myoglein) in rat myocardium

Cardiac plasma membrane Ca²⁺/Mg²⁺ ecto-ATPase (myoglein) requires millimolar concentrations of either Ca²⁺ or Mg²⁺ for maximal activity. In this paper, we report its localization by employing an antiserum raised against the purified rat cardiac Ca²⁺/Mg²⁺ ATPase. As assessed by Western blot analysis, the antiserum and the purified immunoglobulin were specific for Ca²⁺/Mg²⁺ ecto-ATPase; no cross reaction was observed towards other membrane bound enzymes such as cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or sarcolemmal Ca²⁺-pump ATPase. On the other hand, the cardiac Ca²⁺/Mg²⁺ ecto-ATPase was not recognized by antibodies specific for either cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or plasma membrane Ca²⁺-pump ATPase. Furthermore, the immune serum inhibited the Ca²⁺/Mg²⁺ ecto-ATPase activity of the purified enzyme preparation. Immunofluorescence of cardiac tissue sections and neonatal cultured cardiomyocytes with the Ca²⁺/Mg²⁺ ecto-ATPase antibodies indicated the localization of Ca²⁺/Mg²⁺ ecto-ATPase in association with the plasma membrane of myocytes, in areas of cell-matrix or cell-cell contact. Staining for the Ca²⁺/Mg²⁺ ecto-ATPase was not cardiac specific since the antibodies detected the presence of membrane proteins in sections from skeletal muscle, brain, liver and kidney. The results indicate that Ca²⁺/Mg²⁺ ecto-ATPase is localized to the plasma membranes of cardiomyocytes as well as other tissues such as brain, liver, kidney and skeletal muscle.     

Protein 4.2 Binds to the Carboxyl-terminal EF-hands of Erythroid ��-Spectrin in a Calcium- and Calmodulin-dependent Manner

Spectrin and protein 4.1 cross-link F-actin protofilaments into a network called the membrane skeleton. Actin and 4.1 bind to one end of β-spectrin. The adjacent end of α-spectrin, called the EF-domain, is calmodulin-like, with calcium-dependent and calcium-independent EF-hands. It has no known function. However, the sph^1J/sph^1J mouse has very fragile red cells and lacks the last 13 amino acids in the EF-domain, suggesting the domain is critical for skeletal integrity. Using pulldown binding assays, we find the α-spectrin EF-domain either alone or incorporated into a mini-spectrin binds native and recombinant protein 4.2 at a previously identified region of 4.2 (G₃ peptide). Native 4.2 binds with an affinity comparable with other membrane skeletal interactions (K_d = 0.30 μm). EF-domains bearing the sph^1J mutation are inactive. Binding of protein 4.2 to band 3 (K_d = 0.45 μm) does not interfere with the spectrin-4.2 interaction. Spectrin-4.2 binding is amplified by micromolar concentrations of Ca²⁺ (but not Mg²⁺) by three to five times. Calmodulin also binds to the EF-domain (K_d = 17 μm), and Ca²⁺-calmodulin blocks Ca²⁺-dependent binding of protein 4.2 but not Ca²⁺-independent binding. The data suggest that protein 4.2 is located near protein 4.1 at the spectrin-actin junctions. Because proteins 4.1 and 4.2 also bind to band 3, the erythrocyte anion channel, we suggest that one or both of these proteins cause a portion of band 3 to localize near the spectrin-actin junctions and provide another point of attachment between the membrane skeleton and the lipid bilayer.     

Chelation of intracellular Ca2+ inhibits murine keratinocyte differentiation in vitro

The role of intracellular Ca²⁺ in the regulation of Ca²⁺-induced terminal differentiation of mouse keratinocytes was investigated using the intracellular Ca²⁺ chelator 1,2-bis(o-aminophenoxy)-ethane-N, N, N′, N′-tetraacetic acid (BAPTA). A cell permeable acetoxymethyl (AM) ester derivative BAPTA (BAPTA/AM) was loaded into primary mouse keratinocytes in 0.05 mM Ca²⁺ medium, and then the cells were induced to differentiate by medium containing 0.12 or 0.5 mM Ca²⁺. Intracellular BAPTA loaded by BAPTA/AM (15–30 μM) inhibited the expression of epidermal differentiation-specific proteins keratin 1 (K1), keratin 10 (K10), filaggrin and loricrin as detected by immunoblotting. The differentiation-associated redistribution of E-cadherin on the cell membrane was delayed but not inhibited as determined by immunofluorescence. BAPTA also inhibited the expression of K1, K10 and Ioricrin mRNA. Furthermore, BAPTA prevented the decrease in DNA synthesis induced by 0.12 and 0.5 mM Ca²⁺, indicating the drug was inhibiting differentiation but was not toxic to keratinocytes. To evaluate the influence of BAPTA on intracellular Ca²⁺, the concentration of intracellular free Ca²⁺ (Ca_i) in BAPTA-loaded keratinocytes was examined by digital image analysis using the Ca²⁺-sensitive fluorescent probe fura-2, and Ca²⁺ influx was measured by ⁴⁵Ca²⁺ uptake studies. Increase in extracellular Ca²⁺ (Ca_o) in the culture medium of keratinocytes caused a sustained increase in both Ca_i and Ca²⁺ localized to ionomycin-sensitive intracellular stores in keratinocytes. BAPTA lowered basal Ca_i concentration and prevented the Ca_i increase. After 12 hours of BAPTA treatment, the basal level of Ca_i returned to the control value, but the Ca²⁺ localized in intracellular stores was substantially decreased. ⁴⁵Ca²⁺ uptake was initially (within 30 min) increased in BAPTA-loaded cells. However, the total ⁴⁵Ca²⁺ accumulation over 24 hours in BAPTA-loaded cells remained unchanged from control values. These results indicate that keratinocytes can maintain Ca_i and total cellular Ca²⁺ content in the presence of increased amount of intracellular Ca²⁺ buffer (e.g., BAPTA) by depleting intracellular Ca²⁺ stores over a long period. The inhibition by BAPTA of keratinocyte differentiation marker expression may result from depletion of the Ca²⁺-stores since this is the major change in intracellular Ca²⁺ detected at the time keratinocytes express the differentiation markers. In contrast, the redistribution of E-cadherin on the cell membrane may be more directly associated with Ca_i change. © 1995 Wiley-Liss, Inc.     

Resistance of Ca2+-ATPase to dilution by excess phospholipid in reconstituted vesicles

Ca²⁺-ATPase and other membrane proteins of the sarcoplasmic reticulum membrane from rabbit skeletal muscle have been reconstituted into lipid vesicles with increasing amounts of phosphatidylcholine. The protein composition and phospholipid concentration of these vesicles were analyzed by determining the density of the reconstituted membrane vesicles on linear H₂O-²H₂O gradients, in a constant concentration of sucrose. In all combinations of the Ca²⁺-ATPase with a weight excess of phosphatidylcholine, the reconstituted vesicles had a phospholipid-to-protein ratio similar to that of the native sarcoplasmic reticulum membrane, even though both solubilization and mixing had occurred. These vesicles of low phospholipid and high protein content exhibited all the original Ca²⁺-ATPase activity and ATP-stimulated calcium transport. The Ca²⁺-ATPase, and the calcium-binding proteins to a lesser extent, may order the lipid in such a manner so as to maintain the initial stoichiometry of lipid to protein observed in the native sarcoplasmic reticulum membrane.     

The TRPV4 Channel Contributes to Intercellular Junction Formation in Keratinocytes

Transient receptor potential vanilloid 4 (TRPV4) channel is a physiological sensor for hypo-osmolarity, mechanical deformation, and warm temperature. The channel activation leads to various cellular effects involving Ca²⁺ dynamics. We found that TRPV4 interacts with β-catenin, a crucial component linking adherens junctions and the actin cytoskeleton, thereby enhancing cell-cell junction development and formation of the tight barrier between skin keratinocytes. TRPV4-deficient mice displayed impairment of the intercellular junction-dependent barrier function in the skin. In TRPV4-deficient keratinocytes, extracellular Ca²⁺-induced actin rearrangement and stratification were delayed following significant reduction in cytosolic Ca²⁺ increase and small GTPase Rho activation. TRPV4 protein located where the cell-cell junctions are formed, and the channel deficiency caused abnormal cell-cell junction structures, resulting in higher intercellular permeability in vitro. Our results suggest a novel role for TRPV4 in the development and maturation of cell-cell junctions in epithelia of the skin.     

Phospholipid metabolism in calcium-regulated differentiation in cultured murine keratinocytes

The present findings associate phospholipid alteration, particularly the turnover of phosphatidylinositol, in Ca²⁺ induced differentiation of keratinocytes. These conclusions are based on the hydrolysis of ¹⁴C-AA from prelabeled PI and the accumulation ¹⁴C-DG and ¹⁴C-PA after cells are switched from low to normal concentrations of extracellular Ca²⁺. This novel finding implies that the biological changes which accompany keratinocyte differentiation after switch from low to normal extracellular medium may be due at least in part to increased accumulation of PA and DG which are major deacylation and reacylation products of phosphatidylinositol. A second interesting finding in these studies is the marked transformation of ¹⁴C-AA into lipoxygenase products by proliferating keratinocytes cultured in low Ca²⁺ medium when compared to differentiating cells cultured in normal Ca²⁺. The significance of decreased generation of lipoxygenase products in epidermal differentiation deserve further exploration.     

Protein kinase and cyclic AMP levels in differentiating myoblasts are altered by extracellular calcium

Protein kinase specific activities and cyclic AMP levels show a similar pattern of response, when the Ca²⁺ concentration is altered in the culture medium of differentiating chick skeletal muscle cells; an increase at intermediate Ca²⁺ concentrations (0.05–0.2mM), followed by a decrease at higher concentrations (2mM). Effects of Ca²⁺ on protein kinase appear to be on cyclic AMP-independent enzymes in both nucleus and cytoplasm, and are quite the reverse of Ca²⁺ effects on the muscle-specific enzyme, creatine kinase.     

Synthesis of the calcium transport ATPase of sarcoplasmic reticulum and other muscle proteins during development of muscle cells in vivo and in vitro

The effect of medium Ca²⁺ concentration upon the concentration and the rate of synthesis of muscle proteins was investigated in chicken pectoralis muscle cultures.There is an easily identifiable class of muscle protein which includes the Ca²⁺-ATPase of sarcoplasmic reticulum, myosin, troponin C, ATP : creatine phosphotransferase, muscle specific actin, tropomyosin 1 and 2, and muscle hemagglutinin, which show a large increase in concentration during normal development. The increased synthesis of these proteins was inhibited, without inhibition of cell proliferation, in culture media of relatively low Ca²⁺ concentration, 0.05–0.3 mM, where fusion was prevented. Similar medium Ca²⁺ concentration was required for the expression of all these proteins, suggesting their coordinate regulation. The proteins are denoted as ‘calcium-modulated proteins’. The increased Ca²⁺ transport activity of sarcoplasmic reticulum in cultured chicken pectoralis muscle cells during development at 1.8 mM medium calcium concentration represents de novo synthesis of the Ca²⁺ transport ATPase, as shown by immunoprecipitation, active site labeling and direct identification of the Ca²⁺ transport ATPase on two-dimensional gel electropherograms of whole muscle homogenates.The concentration and the turnover rate of the majority of the muscle proteins is not affected significantly by medium Ca²⁺ concentration between 0.06 and 1.8 mM.It is proposed that increase in cytoplasmic free Ca²⁺ concentration during fusion plays a central role in the regulation of the synthesis of calcium-modulated proteins.     

Phosphatidylinositol-4,5 bisphosphate (PIP2) inhibits apo-calmodulin binding to protein 4.1

Membrane skeletal protein 4.1R⁸⁰ plays a key role in regulation of erythrocyte plasticity. Protein 4.1R⁸⁰ interactions with transmembrane proteins, such as glycophorin C (GPC), are regulated by Ca²⁺-saturated calmodulin (Ca²⁺/CaM) through simultaneous binding to a short peptide (pep11; A²⁶⁴KKLWKVCVEHHTFFRL) and a serine residue (Ser¹⁸⁵), both located in the N-terminal 30 kDa FERM domain of 4.1R⁸⁰ (H·R30). We have previously demonstrated that CaM binding to H·R30 is Ca²⁺-independent and that CaM binding to H·R30 is responsible for the maintenance of H·R30 β-sheet structure. However, the mechanisms responsible for the regulation of CaM binding to H·R30 are still unknown. To investigate this, we took advantage of similarities and differences in the structure of Coracle, the Drosophila sp. homologue of human 4.1R⁸⁰, i.e. conservation of the pep11 sequence but substitution of the Ser¹⁸⁵ residue with an alanine residue. We show that the H·R30 homologue domain of Coracle, Cor30, also binds to CaM in a Ca²⁺-independent manner and that the Ca²⁺/CaM complex does not affect Cor30 binding to the transmembrane protein GPC. We also document that both H·R30 and Cor30 bind to phosphatidylinositol-4,5 bisphosphate (PIP₂) and other phospholipid species and that that PIP₂ inhibits Ca²⁺-free CaM but not Ca²⁺-saturated CaM binding to Cor30. We conclude that PIP₂ may play an important role as a modulator of apo-CaM binding to 4.1R⁸⁰ throughout evolution.     

Ca2+ and UVB Radiation Have No Effect on E-Cadherin-Mediated Melanocyte-Keratinocyte Adhesion

Direct cell-cell contact between melanocytes and keratinocytes has been shown to play an important role in the regulation of human melanocyte function and skin pigmentation. An important role for the calcium-dependent epithelium-specific cell adhesion molecule, E-cadherin, in melanocyte-keratinocyte adhesion was suggested previously. To further clarify regulation of E-cadherin-mediated melanocyte-keratinocyte interactions, we investigated the effects of physiological (Ca²⁺) and environmental (ultraviolet B [UVB] radiation) stimuli on the expression and functional activity of E-cadherin in melanocyte-keratinocyte adhesion. Expression of E-cadherin mRNA was detected by Northern blot analysis in cultured normal human melanocytes at levels similar to those in keratinocytes. Flow cytometry analysis with anti-human and anti-mouse-E-cadherin antibodies (anti-uvomorulin and ECCD-2) showed that cultured normal human keratinocytes, melanocytes, and two metastatic melanoma cell lines express E-cadherin strongly on the cell surfaces. Melanocyte adhesion, particularly to differentiating keratinocytes (cultured in 1.2 mM calcium) but not to proliferating keratinocytes or to fibroblasts, was decreased by 41.7 ± 4.5% in the absence of 1 mM Ca²⁺ during the binding assay. Addition of anti-mouse-E-cadherin antibody (ECCD-1) to the binding assay inhibited the adhesion of melanocytes to differentiating keratinocytes by 88.2 ± 1.1%, while addition of anti-P-cadherin antibody (PCD-1) had no effect. The levels of E-cadherin expression in melanocytes were not changed by the presence of calcium (1 mM) in the medium or by UVB irradiation (20 mJ/cm²) for one day before flow cytometry analysis. Moreover, these treatments had no effect on melanocyte-keratinocyte adhesion. These results demonstrate that E-cadherin is strongly involved in melanocyte adhesion to keratinocytes and suggest the implication of E-cadherin in the overall regulation of the skin pigmentary system.     

Regulation of the disassembly/assembly of the membrane skeleton in madin-darby canine kidney cells

The effects of pH, temperature, block of energy production, calcium/calmodulin, protein phosphorylation, and cytoskeleton-disrupting agents (cytochalasin D, nocodazole) on the integrity of the membrane skeleton were studied in polarized MDCK cells. The intracellular distributions of α-fodrin, actin, and ankyrin were monitored by immunofluorescence microscopy. The membrane skeleton, once assembled, seemed to be quite stable; the only factors releasing α-fodrin from the lateral walls were the acidification of the cytoplasm and the depletion of extracellular calcium ions. Upon cellular acidification, some actin was also released from its normal location along the lateral walls and was seen in colocalization with α-fodrin in the cytoplasm, whereas ankyrin remained associated with the lateral walls. No accumulation of plasma membrane lipids was observed in the cytoplasm of acidified cells, as visualized by TMA-DPH. These results suggest that the linkages between the fodrin-actin complex and its membrane association sites are broken upon acidification. The pH-induced change in α-fodrin localization was reversible upon restoring the normal pH. Reassembly of the membrane skeleton, however, required temperatures above +20°C, normal energy production, proper cell-cell contacts, and polymerized actin. Release of α-fodrin from the lateral walls to the cytoplasm was also observed upon depletion of extracellular calcium ions. This change was accompanied by the disruption of cell-cell contacts, supporting the role of proper cell-cell contacts in the maintenance of the membrane skeleton polarity. These results suggest that local alterations of the cytoplasmic pH and calcium ion concentration may be important in regulating the integrity of the membrane skeleton. © 1996 Wiley-Liss, Inc.     

Annexin II tetramer: structure and function

The annexins are a family of proteins that bind acidic phospholipids in the presence of Ca²⁺. The interaction of these proteins with biological membranes has led to the suggestion that these proteins may play a role in membrane trafficking events such as exocytosis, endocytosis and cell-cell adhesion. One member of the annexin family, annexin II, has been shown to exist as a monomer, heterodimer or heterotetramer. The ability of annexin II tetramer to bridge secretory granules to plasma membrane has suggested that this protein may play a role in Ca²⁺-dependent exocytosis. Annexin II tetramer has also been demonstrated on the extracellular face of some metastatic cells where it mediates the binding of certain metastatic cells to normal cells. Annexin II tetramer is a major cellular substrate of protein kinase C and pp60^src. Phosphorylation of annexin II tetramer is a negative modulator of protein function.Supported by a grant from the Medical Research Council of Canada     

Control of the onset of migration of neural crest cells in avian embryos

Summary To investigate the control of the timing in the epithelio-mesenchymal transformation of the neural crest into a migrating population, neural anlagen (neural tube plus crest) were isolated from 2-day quail embryos by proteases in the presence of Ca^{+ +} and explanted onto substrates favourable for neural crest cell migration. Explants isolated before normal migration had commenced required 3–8 h in vitro before neural crest cells started migration, but explants obtained at migratory stages showed an immediate onset of migration. The schedule was similar to that expected in vivo. When pre-migratory neural anlagen were isolated by protease in Ca^{+ +}- and Mg^{+ +}-free (CMF) medium, or when the protease was followed by a brief (5 min) exposure to CMF medium, neural crest cell migration commenced without delay, and the cohesion of the anlagen was impaired. Ca^{+ +}-free medium duplicated the effects of CMF, but neither Mg^{+ +}-free medium nor CMF treatment before treatment with protease stimulated migration and reduced cohesion. Precocious neural crest cell migration and reduced cohesion also followed when neural anlagen of pre-migratory stages were cultured with membrane. Ca^{+ +}-channel antagonists D600 and Nifedipine, without any exernal Ca^{+ +}-depletion.The decrease of cohesion of these tissues is consistent with results in other systems where protease/Ca^{+ +}-depletion inactivates Ca^{+ +}-dependent cell-cell adhesive mechanisms. Therefore, we suggest that Ca^{+ +}-dependent cell-cell adhesions play a part in preventing neural crest cells from migrating precociously and that the timed inactivation of this adhesion system normally helps trigger the onset of migration. The results with blockers of Ca^{+ +}-channels suggest that Ca^{+ +} levels may be involved in regulating this system.     

Unique Structural Changes in Calcium-Bound Calmodulin Upon Interaction with Protein 4.1R FERM Domain: Novel Insights into the Calcium-dependent Regulation of 4.1R FERM Domain Binding to Membrane Proteins by Calmodulin

Calmodulin (CaM) binds to the FERM domain of 80 kDa erythrocyte protein 4.1R (R30) independently of Ca²⁺ but, paradoxically, regulates R30 binding to transmembrane proteins in a Ca²⁺-dependent manner. We have previously mapped a Ca²⁺-independent CaM-binding site, pep11 (A²⁶⁴KKLWKVCVEHHTFFR), in 4.1R FERM domain and demonstrated that CaM, when saturated by Ca²⁺ (Ca²⁺/CaM), interacts simultaneously with pep11 and with Ser¹⁸⁵ in A¹⁸¹KKLSMYGVDLHKAKD (pep9), the binding affinity of Ca²⁺/CaM for pep9 increasing dramatically in the presence of pep11. Based on these findings, we hypothesized that pep11 induced key conformational changes in the Ca²⁺/CaM complex. By differential scanning calorimetry analysis, we established that the C-lobe of CaM was more stable when bound to pep11 either in the presence or absence of Ca²⁺. Using nuclear magnetic resonance spectroscopy, we identified 8 residues in the N-lobe and 14 residues in the C-lobe of pep11 involved in interaction with CaM in both of presence and absence of Ca²⁺. Lastly, Kratky plots, generated by small-angle X-ray scattering analysis, indicated that the pep11/Ca²⁺/CaM complex adopted a relaxed globular shape. We propose that these unique properties may account in part for the previously described Ca²⁺/CaM-dependent regulation of R30 binding to membrane proteins.     

The effect of cyclic nucleotides and protein phosphorylation on the permeability of human erythrocyte ghosts to certain cations

Summary Preparations of human erythrocyte membranes have been made which are in the form of sealed vesicles and which behave as osmometers on suspension in solutions of simple inorganic salts. Using these preparations the permeability of the membranes to Na⁺, K⁺, Mg²⁺ and Ca²⁺ was measured. Cyclic AMP (but not cyclic GMP) increased the permeability of the membranes to Ca²⁺ with a half maximal effect at a concentration of 25µm but did not affect the permeability to the other ions tested. Phosphorylation of proteins in the erthrocyte membrane lowered the permeability to Ca²⁺ without affecting the permeability to the other ions tested and there was a good correlation between the time course of protein phosphorylation and decrease in Ca²⁺ permeability.It is postulated that the system through which cyclic AMP causes an initial rapid rise in Ca²⁺ permeability followed by increased phosphorylation of membrane proteins and reduced Ca²⁺ permeability may have a widespread occurrence in biological systems and serve to control the concentration of Ca²⁺ in the cytoplasm.     

Calsequestrin. Structure,function, and evolution

Calsequestrin is the major Ca²⁺ binding protein in the sarcoplasmic reticulum (SR), serves as the main Ca²⁺ storage and buffering protein and is an important regulator of Ca²⁺ release channels in both skeletal and cardiac muscle. It is anchored at the junctional SR membrane through interactions with membrane proteins and undergoes reversible polymerization with increasing Ca²⁺ concentration. Calsequestrin provides high local Ca²⁺ at the junctional SR and communicates changes in luminal Ca²⁺ concentration to Ca²⁺ release channels, thus it is an essential component of excitation-contraction coupling. Recent studies reveal new insights on calsequestrin trafficking, Ca²⁺ binding, protein evolution, protein-protein interactions, stress responses and the molecular basis of related human muscle disease, including catecholaminergic polymorphic ventricular tachycardia (CPVT). Here we provide a comprehensive overview of calsequestrin, with recent advances in structure, diverse functions, phylogenetic analysis, and its role in muscle physiology, stress responses and human pathology.     

Formation of γ-glutamyl-ϵ-lysine bridges between membrane proteins by a Ca2+-regulated enzyme in intact erythrocytes

A rise in the intracellular concentration of Ca²⁺-ions in human erythrocytes causes the formation of high-molecular-weight membrane protein polymers, cross-linked by γ-glutamyl-?-lysine side chain bridges. Cross-linking involves proteins at the cytoplasmic side of the membrane (band 4.1, spectrin, and band 3 materials) and the reaction is catalyzed by the intrinsic transglutaminase. This enzyme is regulated by Ca²⁺-ions and it exists in a latent form in normal cells. The protein polymer, isolated from the membranes of Ca²⁺-loaded intact human red cells, is heterogeneous in size and may contain as many as 6 moles of γ-glutamyl-?-lysine cross-links per 100,000 gm of protein. Synthetic compounds, which either compete against the ?-lysine cross-linking functionalities of the protein substrates (eg, histamine, aminoacetonitrile, cystamine) or directly inactivate the transamidase (eg, cystamine), inhibit the membrane polymerization reaction in intact human erythrocytes. They also interfere with the Ca²⁺-induced irreversible shape change from discocyte to echinocyte and inhibit the irreversible loss of membrane deformability. Thus, the transamidase-catalyzed production of γ-glutamyl-?-lysine cross-links in the membrane may be a common denominator in these cellular manifestations.     

Two ryanodine receptor isoforms in nonmammalian vertebrate skeletal muscle: possible roles in excitation-contraction coupling and other processes

The ryanodine receptor (RyR) is a Ca²⁺ release channel in the sarcoplasmic reticulum in vertebrate skeletal muscle and plays an important role in excitation–contraction (E–C) coupling. Whereas mammalian skeletal muscle predominantly expresses a single RyR isoform, RyR1, skeletal muscle of many nonmammalian vertebrates expresses equal amounts of two distinct isoforms, α-RyR and β-RyR, which are homologues of mammalian RyR1 and RyR3, respectively. In this review we describe our current understanding of the functions of these two RyR isoforms in nonmammalian vertebrate skeletal muscle. The Ca²⁺ release via the RyR channel can be gated by two distinct modes: depolarization-induced Ca²⁺ release (DICR) and Ca²⁺-induced Ca²⁺ release (CICR). In frog muscle, α-RyR acts as the DICR channel, whereas β-RyR as the CICR channel. However, several lines of evidence suggest that CICR by β-RyR may make only a minor contribution to Ca²⁺ release during E–C coupling. Comparison of frog and mammalian RyR isoforms highlights the marked differences in the patterns of Ca²⁺ release mediated by RyR1 and RyR3 homologues. Interestingly, common features in the Ca²⁺ release patterns are noticed between β-RyR and RyR1. We will discuss possible roles and significance of the two RyR isoforms in E–C coupling and other processes in nonmammalian vertebrate skeletal muscle.