A Role for Caveolin-3 in the Pathogenesis of Muscular Dystrophies.

Caveolae are the cholesterol-rich small invaginations of the plasma membrane present in many cell types including adipocytes, endothelial cells, epithelial cells, fibroblasts, smooth muscles, skeletal muscles and cardiac muscles. They serve as specialized platforms for many signaling molecules and regulate important cellular processes like energy metabolism, lipid metabolism, mitochondria homeostasis, and mechano-transduction. Caveolae can be internalized together with associated cargo. The caveolae-dependent endocytic pathway plays a role in the withdrawal of many plasma membrane components that can be sent for degradation or recycled back to the cell surface. Caveolae are formed by oligomerization of caveolin proteins. Caveolin-3 is a muscle-specific isoform, whose malfunction is associated with several diseases including diabetes, cancer, atherosclerosis, and cardiovascular diseases. Mutations in Caveolin-3 are known to cause muscular dystrophies that are collectively called caveolinopathies. Altered expression of Caveolin-3 is also observed in Duchenne's muscular dystrophy, which is likely a part of the pathological process leading to muscle weakness. This review summarizes the major functions of Caveolin-3 in skeletal muscles and discusses its involvement in the pathology of muscular dystrophies.

Unique cysteine-enriched, D2L5 and D4L6 extracellular loops in CaV3 T-type channels alter the passage and block of monovalent and divalent ions.

Invertebrate LCaV3 shares the quintessential features of vertebrate CaV3 T-type channels, with a low threshold of channel activation, rapid activation and inactivation kinetics and slow deactivation kinetics compared to other known Ca2+ channels, the CaV1 and CaV2 channels. Unlike the vertebrates though, CaV3 T-type channels in non-cnidarian invertebrates possess an alternative exon 12 spanning the D2L5 extracellular loop, which alters the invertebrate LCaV3 channel into a higher Na+ and lower Ca2+ current passing channel, more resembling a classical NaV1 Na+ channel. Cnidarian CaV3 T-type channels can possess genes with alternative cysteine-rich, D4L6 extracellular loops in a manner reminiscent of the alternative cysteine-rich, D2L5 extracellular loops of non-cnidarian invertebrates. We illustrate here that the preferences for greater Na+ or Ca2+ ion current passing through CaV3 T-type channels are contributed by paired cysteines within D2L5 and D4L6 extracellular loops looming above the pore selectivity filter. Swapping of invertebrate tri- and tetra-cysteine containing extracellular loops, generates higher Na+ current passing channels in human CaV3.2 channels, while corresponding mono- and di-cysteine loop pairs in human CaV3.2 generates greater Ca2+ current passing, invertebrate LCaV3 channels. Alanine substitutions of unique D2L5 loop cysteines of LCaV3 channels increases relative monovalent ion current sizes and increases the potency of Zn2+ and Ni2+ block by ~ 50× and ~ 10× in loop cysteine mutated channels respectively, acquiring characteristics of the high affinity block of CaV3.2 channels, including the loss of the slowing of inactivation kinetics during Zn2+ block. Charge neutralization of a ubiquitous aspartate residue of calcium passing CaV1, CaV2 and CaV3 channels, in the outer pore of the selectivity filter residues in Domain II generates higher Na+ current passing channels in a manner that may resemble how the unique D2L5 extracellular loops of invertebrate CaV3 channels may confer a relatively higher peak current size for Na+ ions over Ca2+ The extracellular loops of CaV3 channels are not engaged with accessory subunit binding, as the other Na+ (NaV1) and Ca2+ (CaV1/CaV2) channels, enabling diversity and expansion of cysteine-bonded extracellular loops, which appears to serve, amongst other possibilities, to alter to the preferences for passage of Ca2+ or Na+ ions through invertebrate CaV3 channels.

Caveolin proteins electrochemical oxidation and interaction with cholesterol.

Caveolae consist in lipid raft domains composed of caveolin proteins, cholesterol, glycosphingolipids, and GPI-anchored proteins. Caveolin proteins present three different types, caveolin 1 (CAV-1), caveolin 2 (CAV-2) and caveolin 3 (CAV-3), with a very similar structure and amino acid composition. The native caveolin proteins oxidation mechanism was investigated for the first time, at a glassy carbon electrode, using cyclic, square wave and differential pulse voltammetry. The three native caveolin proteins oxidation mechanism presented only one tyrosine and tryptophan amino acid residues oxidation peak. Denatured caveolin proteins presented also the tyrosine, tryptophan and cysteine amino acid residues oxidation peaks. The reverse cholesterol transport is related to caveolae and caveolin proteins, and CAV-1 is directly connected to cholesterol transport. The influence of cholesterol on the three caveolin proteins electrochemical behaviour was evaluated. In the absence and in the presence of cholesterol, significant differences in the CAV-1 oxidation peak current were observed.

T-type calcium channels functionally interact with spectrin (α/ß) and ankyrin B.

This study describes the functional interaction between the Cav3.1 and Cav3.2 T-type calcium channels and cytoskeletal spectrin (α/ß) and ankyrin B proteins. The interactions were identified utilizing a proteomic approach to identify proteins that interact with a conserved negatively charged cytosolic region present in the carboxy-terminus of T-type calcium channels. Deletion of this stretch of amino acids decreased binding of Cav3.1 and Cav3.2 calcium channels to spectrin (α/ß) and ankyrin B and notably also reduced T-type whole cell current densities in expression systems. Furthermore, fluorescence recovery after photobleaching analysis of mutant channels lacking the proximal C-terminus region revealed reduced recovery of both Cav3.1 and Cav3.2 mutant channels in hippocampal neurons. Knockdown of spectrin α and ankyrin B decreased the density of endogenous Cav3.2 in hippocampal neurons. These findings reveal spectrin (α/ß) / ankyrin B cytoskeletal and signaling proteins as key regulators of T-type calcium channels expressed in the nervous system.

Functional interaction of the two-pore domain potassium channel TASK-1 and caveolin-3.

The two-pore domain potassium channel TASK-1 is strongly expressed in the heart and has been shown to modulate the resting membrane potential and action potential. However, little is known about the regulation of TASK-1 channels. The present study was designed to determine whether TASK-1 is modulated by caveolin-3, a primary structural protein of cardiac caveolae. Functional studies with the whole-cell voltage clamp technique showed that the expression of caveolin-3 decreased recombinant TASK-1 currents significantly in HEK293T cells, and this effect was prevented by co-expressing the dominant negative mutant caveolin-3 P104L. Immunofluorescence imaging revealed the colocalization of TASK-1 and caveolin-3. Co-immunoprecipitation analysis indicated that caveolin-3 associated with TASK-1. When co-expressed with caveolin-3 P104L, the fluorescence intensity of caveolin-3 on the cell periphery was reduced. This agrees with the functional evidence that caveolin-3 P104L prevented the inhibitory effect of caveolin-3 on TASK-1 currents, possibly via reducing the plasma membrane targeting of caveolin-3. Further, our data from cardiomyocytes suggest that TASK-1 is associated with caveolin-3. In summary, our study indicates that TASK-1 is functionally regulated by caveolin-3, possibly via association with each other on the cell surface. These results point out a novel mechanism in the regulation of TASK-1.

Exploring Human Diseases and Biological Mechanisms by Protein Structure Prediction and Modeling.

Protein structure prediction and modeling provide a tool for understanding protein functions by computationally constructing protein structures from amino acid sequences and analyzing them. With help from protein prediction tools and web servers, users can obtain the three-dimensional protein structure models and gain knowledge of functions from the proteins. In this chapter, we will provide several examples of such studies. As an example, structure modeling methods were used to investigate the relation between mutation-caused misfolding of protein and human diseases including epilepsy and leukemia. Protein structure prediction and modeling were also applied in nucleotide-gated channels and their interaction interfaces to investigate their roles in brain and heart cells. In molecular mechanism studies of plants, rice salinity tolerance mechanism was studied via structure modeling on crucial proteins identified by systems biology analysis; trait-associated protein-protein interactions were modeled, which sheds some light on the roles of mutations in soybean oil/protein content. In the age of precision medicine, we believe protein structure prediction and modeling will play more and more important roles in investigating biomedical mechanism of diseases and drug design.

Topologically Diverse Human Membrane Proteins Partition to Liquid-Disordered Domains in Phase-Separated Lipid Vesicles.

The integration of membrane proteins into "lipid raft" membrane domains influences many biochemical processes. The intrinsic structural properties of membrane proteins are thought to mediate their partitioning between membrane domains. However, whether membrane topology influences the targeting of proteins to rafts remains unclear. To address this question, we examined the domain preference of three putative raft-associated membrane proteins with widely different topologies: human caveolin-3, C99 (the 99 residue C-terminal domain of the amyloid precursor protein), and peripheral myelin protein 22. We find that each of these proteins are excluded from the ordered domains of giant unilamellar vesicles containing coexisting liquid-ordered and liquid-disordered phases. Thus, the intrinsic structural properties of these three topologically distinct disease-linked proteins are insufficient to confer affinity for synthetic raft-like domains.

Modest effects of lipid modifications on the structure of caveolin-3.

Caveolin-3 (Cav3) is an unconventional membrane protein that serves as a critical scaffolding hub in caveolae and is genetically linked to various muscle disorders. In this work, we report the expression, purification, and characterization of full-length human Cav3. To mimic the palmitoylation of endogenous Cav3, we developed a generally applicable approach to covalently attached thioalkyl chains at natively modified cysteine residues. Nuclear magnetic resonance measurements indicate that lipidation exerts only a modest and local effect on the Cav3 structure, with little impact on the structures of the N-terminal domain, the scaffolding domain, and the extreme C-terminus.

Nanoscale distribution of ryanodine receptors and caveolin-3 in mouse ventricular myocytes: dilation of t-tubules near junctions.

We conducted super-resolution light microscopy (LM) imaging of the distribution of ryanodine receptors (RyRs) and caveolin-3 (CAV3) in mouse ventricular myocytes. Quantitative analysis of data at the surface sarcolemma showed that 4.8% of RyR labeling colocalized with CAV3 whereas 3.5% of CAV3 was in areas with RyR labeling. These values increased to 9.2 and 9.0%, respectively, in the interior of myocytes where CAV3 was widely expressed in the t-system but reduced in regions associated with junctional couplings. Electron microscopic (EM) tomography independently showed only few couplings with caveolae and little evidence for caveolar shapes on the t-system. Unexpectedly, both super-resolution LM and three-dimensional EM data (including serial block-face scanning EM) revealed significant increases in local t-system diameters in many regions associated with junctions. We suggest that this regional specialization helps reduce ionic accumulation and depletion in t-system lumen during excitation-contraction coupling to ensure effective local Ca²âº release. Our data demonstrate that super-resolution LM and volume EM techniques complementarily enhance information on subcellular structure at the nanoscale.

Characterization of the molecular architecture of human caveolin-3 and interaction with the skeletal muscle ryanodine receptor.

BACKGROUND: Caveolin-3 facilitates both caveolae formation and a range of cell signaling pathways, including Ca(2+) homeostasis. RESULTS: Caveolin-3 forms a disc-shaped nonamer that binds the Ca(2+)-release channel, RyR1. CONCLUSION: Multiple caveolin-3 nonamers bind to a single RyR1 homotetramer. SIGNIFICANCE: First three-dimensional structural insights into caveolin-3 assembly, interactions with RyR1 suggest a novel role in muscle contraction and/or for channel localization within the membrane. Caveolin-3 (cav-3), an integral membrane protein, is a building block of caveolae as well as a regulator of a number of physiological processes by facilitating the formation of multiprotein signaling complexes. We report that the expression of cav-3 in insect (Sf9) cells induces caveola formation, comparable in size with those observed in native tissue. We have also purified the recombinant cav-3 determining that it forms an oligomer of ∼220 kDa. We present the first three-dimensional structure for cav-3 (using transmission electron microscopy and single particle analysis methods) and show that nine cav-3 monomers assemble to form a complex that is toroidal in shape, ~16.5 nm in diameter and ~ 5.5 nm in height. Labeling experiments and reconstitution of the purified cav-3 into liposomes have allowed a proposal for the orientation of the protein with respect to the membrane. We have identified multiple caveolin-binding motifs within the ryanodine receptor (RyR1) sequence employing a bioinformatic analysis. We have then shown experimentally that there is a direct interaction between recombinant cav-3 nonamers and purified RyR1 homotetramers that would imply that at least one of the predicted cav-3-binding sites is exposed within the fully assembled RyR1 structure. The cav-3 three-dimensional model provides new insights as to how a cav-3 oligomer can bind multiple partners in close proximity to form signaling complexes. Furthermore, a direct interaction with RyR1 suggests a possible role for cav-3 as a modifier of muscle excitation-contraction coupling and/or for localization of the receptor to regions of the sarcoplasmic reticulum.

Modulation of Ca²+ activity in cardiomyocytes through caveolae-Gαq interactions.

Cardiomyocytes have a complex Ca(2+) behavior and changes in this behavior may underlie certain disease states. Intracellular Ca(2+) activity can be regulated by the phospholipase Cß-Gα(q) pathway localized on the plasma membrane. The plasma membranes of cardiomycoytes are rich in caveolae domains organized by caveolin proteins. Caveolae may indirectly affect cell signals by entrapping and localizing specific proteins. Recently, we found that caveolin may specifically interact with activated Gα(q), which could affect Ca(2+) signals. Here, using fluorescence imaging and correlation techniques we show that Gα(q)-Gßγ subunits localize to caveolae in adult ventricular canine cardiomyoctyes. Carbachol stimulation releases Gßγ subunits from caveolae with a concurrent stabilization of activated Gα(q) by caveolin-3 (Cav3). These cells show oscillating Ca(2+) waves that are not seen in neonatal cells that do not contain Cav3. Microinjection of a peptide that disrupts Cav3-Gα(q) association, but not a control peptide, extinguishes the waves. Furthermore, these waves are unchanged with rynaodine treatment, but not seen with treatment of a phospholipase C inhibitor, implying that Cav3-Gα(q) is responsible for this Ca(2+) activity. Taken together, these studies show that caveolae play a direct and active role in regulating basal Ca(2+) activity in cardiomyocytes.

Development of nanoparticle probes for multiplex SERS imaging of cell surface proteins.

Multiplexed SERS imaging of receptor proteins on the surface of mammalian cells has been carried out using functionalized silver nanoparticles. Deconvolution of four differently functionalized nanoparticles is readily achieved, and using this approach, receptor co-localization can be probed and protein-protein interactions can be elucidated at the surface of cells.

Caveolins sequester FA on the cytoplasmic leaflet of the plasma membrane, augment triglyceride formation, and protect cells from lipotoxicity.

Ectopic expression of caveolin-1 in HEK293 cells enhances FA sequestration in membranes as measured by a pH-sensitive fluorescent dye (1). We hypothesized that sequestration of FA is due to the enrichment of caveolin in the cytosolic leaflet and its ability to facilitate the formation of lipid rafts to buffer high FA levels. Here we show that ec-topic expression of caveolin-3 also results in enhanced FA sequestration. To further discriminate the effect that caveolins have on transmembrane FA movement and distribution, we labeled the outer membrane leaflet with fluorescein-phosphatidylethanolamine (FPE), whose emission is quenched by the presence of FA anions. Real-time measurements made with FPE and control experiments with positively charged fatty amines support our hypothesis that caveolins promote localization of FA anions through interactions with basic amino acid residues (lysines and arginines) present at the C termini of caveolins-1 and -3.

The genetic basis of long QT and short QT syndromes: a mutation update.

Long QT and short QT syndromes (LQTS and SQTS) are cardiac repolarization abnormalities that are characterized by length perturbations of the QT interval as measured on electrocardiogram (ECG). Prolonged QT interval and a propensity for ventricular tachycardia of the torsades de pointes (TdP) type are characteristic of LQTS, while SQTS is characterized by shortened QT interval with tall peaked T-waves and a propensity for atrial fibrillation. Both syndromes represent a high risk for syncope and sudden death. LQTS exists as a congenital genetic disease (cLQTS) with more than 700 mutations described in 12 genes (LQT1-12), but can also be acquired (aLQTS). The genetic forms of LQTS include Romano-Ward syndrome (RWS), which is characterized by isolated LQTS and an autosomal dominant pattern of inheritance, and syndromes with LQTS in association with other conditions. The latter includes Jervell and Lange-Nielsen syndrome (JLNS), Andersen syndrome (AS), and Timothy syndrome (TS). The genetics are further complicated by the occurrence of double and triple heterozygotes in LQTS and a considerable number of nonpathogenic rare polymorphisms in the involved genes. SQTS is a very rare condition, caused by mutations in five genes (SQTS1-5). The present mutation update is a comprehensive description of all known LQTS- and SQTS-associated mutations.

Caveolin regulates kv1.5 trafficking to cholesterol-rich membrane microdomains.

The targeting of ion channels to cholesterol-rich membrane microdomains has emerged as a novel mechanism of ion channel localization. Previously, we reported that Kv1.5, a prominent cardiovascular K(+) channel alpha-subunit, localizes to caveolar microdomains. However, the mechanisms regulating Kv1.5 targeting and the functional significance of this localization are largely unknown. In this study, we demonstrate a role for caveolin in the trafficking of Kv1.5 to lipid raft microdomains where cholesterol modulates channel function. In cells lacking endogenous caveolin-1 or -3, the association of Kv1.5 with low-density, detergent-resistant membrane fractions requires coexpression with exogenous caveolin, which can form channel-caveolin complexes. Caveolin is not required for cell surface expression, however, and caveolin-trafficking mutants sequester Kv1.5, but not Kv2.1, in intracellular compartments, resulting in a loss of functional cell surface channel. Coexpression with wild type caveolin-1 does not alter Kv1.5 current density; rather, it induces depolarizing shifts in steady-state activation and inactivation. These shifts are analogous to those produced by elevation of membrane cholesterol. Together, these results show that caveolin modulates channel function by regulating trafficking to cholesterol-rich membrane microdomains.

Interaction of synthetic peptides corresponding to the scaffolding domain of Caveolin-3 with model membranes.

Caveolin-1 and -3 are among the few proteins in which the functional domains are contiguous and modular. The interaction of synthetic peptides spanning the scaffolding domain of caveolin-3 with model membranes has been investigated. The peptides include the scaffolding domain, the aromatic and positively charged residues at the C-terminal end of this domain as well as deletion of three amino acids TFT, observed in certain patients with limb girdle muscular dystrophy. All of the peptides appear to be peripherally bound to the bilayer surface. However, no preferential binding to sphingomyelin and cholesterol-containing lipid vesicles was observed. Deletion of TFT appears to affect the association with lipid vesicles compared with the native sequence. Association with lipids decreases considerably when TFT as well as the aromatic-rich segment YWFYR, which occurs at the extreme C-terminus of the scaffolding domain, are deleted.

Caveolin-3 is adjacent to a group of extradyadic ryanodine receptors.

Caveolae are present in almost all cells and concentrate a wide variety of signaling molecules, receptors, transporters, and ion pumps. We have investigated the distribution of the ryanodine receptor, the Na(+)/Ca(2+) exchanger, the predominant Na(+) channel isoform rH1, and the L-type calcium channel, Ca(v)1.2, relative to the muscle-specific caveolin isoform, caveolin-3, in adult rat ventricular myocytes. Three-dimensional immunofluorescence images were deconvolved and analyzed. Caveolin-3 colocalizes with all of these molecules at the surface of the cell, but there is no significant colocalization between caveolin-3 and either the Na(+)/Ca(2+) exchanger or the Na(+) channel in the cell interior. The distribution of the surface colocalization indicates that the caveolae that colocalize with each molecule form distinct populations. This organization indicates that there are multiple populations of caveolae separable by location and occupants. In the interior of the cell, caveolin-3 shows a marked colocalization with a population of ryanodine receptors that are separate from those within the dyad. Because of their location, the signaling molecules contained within these caveolae may have preferred access to the neighboring nondyadic ryanodine receptors.