Localization of cardiac L-type Ca(2+) channels to a caveolar macromolecular signaling complex is required for beta(2)-adrenergic regulation

L-type Ca(2+) channels play a critical role in regulating Ca(2+)-dependent signaling in cardiac myocytes, including excitation-contraction coupling; however, the subcellular localization of cardiac L-type Ca(2+) channels and their regulation are incompletely understood. Caveolae are specialized microdomains of the plasmalemma rich in signaling molecules and supported by the structural protein caveolin-3 in muscle. Here we demonstrate that a subpopulation of L-type Ca(2+) channels is localized to caveolae in ventricular myocytes as part of a macromolecular signaling complex necessary for beta(2)-adrenergic receptor (AR) regulation of I(Ca,L). Immunofluorescence studies of isolated ventricular myocytes using confocal microscopy detected extensive colocalization of caveolin-3 and the major pore-forming subunit of the L-type Ca channel (Ca(v)1.2). Immunogold electron microscopy revealed that these proteins colocalize in caveolae. Immunoprecipitation from ventricular myocytes using anti-Ca(v)1.2 or anti-caveolin-3 followed by Western blot analysis showed that caveolin-3, Ca(v)1.2, beta(2)-AR (not beta(1)-AR), G protein alpha(s), adenylyl cyclase, protein kinase A, and protein phosphatase 2a are closely associated. To determine the functional impact of the caveolar-localized beta(2)-AR/Ca(v)1.2 signaling complex, beta(2)-AR stimulation (salbutamol plus atenolol) of I(Ca,L) was examined in pertussis toxin-treated neonatal mouse ventricular myocytes. The stimulation of I(Ca,L) in response to beta(2)-AR activation was eliminated by disruption of caveolae with 10 mM methyl beta-cyclodextrin or by small interfering RNA directed against caveolin-3, whereas beta(1)-AR stimulation (norepinephrine plus prazosin) of I(Ca,L) was not altered. These findings demonstrate that subcellular localization of L-type Ca(2+) channels to caveolar macromolecular signaling complexes is essential for regulation of the channels by specific signaling pathways.     

Localization of cardiac sodium channels in caveolin-rich membrane domains: regulation of sodium current amplitude

This study demonstrates that caveolae, omega-shaped membrane invaginations, are involved in cardiac sodium channel regulation by a mechanism involving the alpha subunit of the stimulatory heterotrimeric G-protein, Galpha(s), via stimulation of the cell surface beta-adrenergic receptor. Stimulation of beta-adrenergic receptors with 10 micromol/L isoproterenol in the presence of a protein kinase A inhibitor increased the whole-cell sodium current by a "direct" cAMP-independent G-protein mechanism. The addition of antibodies against caveolin-3 to the cell's cytoplasm via the pipette solution abrogated this direct G protein-induced increase in sodium current, whereas antibodies to caveolin-1 or caveolin-2 did not. Voltage-gated sodium channel proteins were found to associate with caveolin-rich membranes obtained by detergent-free buoyant density separation. The purity of the caveolar membrane fraction was verified by Western blot analyses, which indicated that endoplasmic/sarcoplasmic reticulum, endosomal compartments, Golgi apparatus, clathrin-coated vesicles, and sarcolemmal membranes were excluded from the caveolin-rich membrane fraction. Additionally, the sodium channel was found to colocalize with caveolar membranes by immunoprecipitation, indirect immunofluorescence, and immunogold transmission electron microscopy. These results suggest that stimulation of beta-adrenergic receptors, and thereby Galpha(s), promotes the presentation of cardiac sodium channels associated with caveolar membranes to the sarcolemma.     

Function, Regulation, Pharmacology, and Molecular Structure of ATP-Sensitive K+ Channels in the Cardiovascular System

K_ATP Channels in Cardiovascular System. ATP-sensitive K⁺ (K_ATP) channels are inhibited by intracellular ATP and activated by intracellular nucleoside diphosphates, and thus provide a link between cellular metabolism and excitability. K_ATP channels are widely distributed in various tissues and may be associated with diverse cellular functions. In the heart, the K_ATP channel appears to be activated during ischemic or hypoxic conditions and may be responsible for the increase of K⁺ efflux and shortening of the action potential duration. Therefore, opening of this channel may result in cardioprotective as well as proarrhythmic effects. In the vascular smooth muscle, the K_ATP channel is believed to mediate the relaxation of vascular tone. Thus, K_ATP channels play important regulatory roles in the cardiovascular system. Furthermore, K_ATP channels are the targets of two important classes of drugs, i.e., the antidiabetic sulfonylureas, which block the channels, and a series of vasorelaxants called "K⁺ channel openers," which tend to maintain the channels in an open conformation. Recently, the molecular structure of K_ATP channels has been clarified. The K_ATP channel in pancreatic β-cells is a complex composed of at least two subunits, a member of inwardly rectifying K⁺ channels and a sulfonylurea receptor. Subsequently, two additional homologs of the sulfonylurea receptor, which form cardiac and smooth muscle type K_ATP channels, respectively, have been reported. Further works are now in progress to understand the molecular mechanisms of K_ATP channel function.     

On the Mechanism of Inhibition of KATP Channels by Glibenclamide in Rat Ventricular Myocytes

Glibenclamide Block of K_ATP Channels. Introduction: The mechanism by which glibenclamide inhibits K_ATP channel activity has been examined in membrane patches from isolated rat ventricular cells. Methods and Results: Inside-out patches were exposed to zero, or low, [ATP] to activate K_ATP channels. Glibenclamide did not affect single channel conductance, but reversibly reduced channel open probability from either side of the membrane. Internal (cytoplasmic) glibenclamide inhibited with half-maximal inhibitory [glibenclamide] = 6 μM, Hill coefficient = 0.35. Complete channel inhibition was not observed, even at 300 μM [glibenclamide]. The response to step increases of internal [glibenclamide] could be resolved into two phases of channel inhibition (t_{1/2, fast}, < 1 sec, t_{1/2, slow}= 10.5 ± 0.9 sec, n = 8). Step decrease of [glibenclamide] caused a single resolvable phase of reactivation (t_1/2= 20.4 ± 0.7 sec, n = 16). Channel inhibition by internal glibenclamide could be relieved by ADP, but only in the presence of Mg²⁺.
Conclusion: Glibenclamide can inhibit K_ATP channels from either side of the membrane, with block from one side being competitive with block from the other. Internal MgADP antagonizes the blocking action of glibenclamide. Glibenclamide inhibition of cardiac K_ATP channels differs quantitatively and qualitatively from the inhibition of pancreatic K_ATP channels.     

Ankyrins and human disease: what the electrophysiologist should know

The coordinate activity of ion channels and transporters in cardiac muscle is critical for normal excitation-contraction coupling and cardiac rhythm. In the past decade, human gene variants, which alter ion channel biophysical properties, have been linked with fatal cardiac arrhythmias. Ankyrins are a family of "adaptor" proteins, which play critical roles in the proper expression and membrane localization of ion channels and transporters in excitable and nonexcitable cells. Recent findings demonstrate a new paradigm for human cardiac arrhythmia based not on gene mutations that affect channel biophysical properties, but instead on mutations that affect ion channel/transporter localization at excitable membranes in heart. Human ANK2 mutations are associated with "ankyrin-B syndrome" (an atypical arrhythmia syndrome with risk of sudden cardiac death). Human gene mutations, which affect ankyrin-G-based pathways for voltage-gated Na_v channel localization, are associated with Brugada syndrome, a second potentially fatal arrhythmia. Together, these data demonstrate the importance of the molecular events involved in the cellular organization of membrane domains in excitable cells. Moreover, these data define an exciting new field of cardiac "channelopathies" due to defects in proper channel targeting/localization.     

Sodium Channel Inactivation in Heart: A Novel Role of the Carboxy-Terminal Domain

Electrical activity in the heart depends critically on the interactions of multiple ion channels to coordinate the timing of excitation and contraction of the ventricles. Voltage-gated sodium channels underlie the rapid spread of impulses through the atria and ventricles, but the importance of sodium (Na⁺) channels to the control of the ventricular action potential has only most recently become apparent through the investigation of the relationship between mutation-induced clinical phenotypes and the altered function of mutant Na⁺ channels linked to inherited arrhythmias. Investigation into the structural basis of disease-associated mutations of the cardiac Na⁺ channel has led to the discovery of novel role of the Na⁺ channel carboxy-terminal (CT) domain in controlling channel inactivation. Intramolecular interactions between the carboxy-terminal domain and an intracellular peptide loop that forms the inactivation gate are required to minimize channel reopening during prolonged depolarization. Disruption of this interaction leads to persistent sodium channel current, action potential prolongation, and elevated risk of cardiac arrhythmia.     

Molecular Biology of the Voltage-Gated Potassium Channels of the Cardiovascular System

Cardiovascular K⁺ Channel Molecular Biology. K⁺ channels represent the most diverse class of voltage-gated ion channels in terms of function and structure. Voltage-gated K⁺ channels in the heart establish the resting membrane K permeability, modulate the frequency and duration of action potentials, and are targets of several antiarrhythmic drugs. Consequently, an understanding of K⁺ channel structure-function relationships and pharmacology is of great practical interest. However, the presence of multiple overlapping currents in native cardiac myocytes complicates the study of basic K⁺ channel function and drug-channel interactions in these cells. The application of molecular cloning technology to cardiovascular K⁺ channels has identified the primary structure of these proteins, and heterologous expression systems have allowed a detailed analysis of channel function and pharmacology without contaminating currents. To date six different K⁺ channels have been cloned from rat and human heart, and all have been functionally characterized in either Xenopus oocytes or mammalian tissue culture systems. This initial research is an important step toward understanding the molecular basis of the action potential in the heart. An important challenge for the future is to determine the cell-specific expression and relative contribution of these cloned channels to cardiac excitability.     

The phosphorylation of caveolin-2 on serines 23 and 36 modulates caveolin-1-dependent caveolae formation

Caveolin-1 and -2 are the two major coat proteins found in plasma membrane caveolae of most of cell types. Here, by using adenoviral transduction of either caveolin-1 or caveolin-2 or both isoforms into cells lacking both caveolins, we demonstrate that caveolin-2 positively regulates caveolin-1-dependent caveolae formation. More importantly, we show that caveolin-2 is phosphorylated in vivo at two serine residues and that the phosphorylation of caveolin-2 is necessary for its actions as a positive regulator of caveolin-1 during organelle biogenesis in prostate cancer cells. Mutation of the primary phosphorylation sites on caveolin-2, serine 23 and 36, reduces the number of plasmalemma-attached caveolae and increases the accumulation of noncoated vesicles, but does not affect trafficking of caveolin-2, interaction with caveolin-1 or its biophysical properties. Thus, the phosphorylation of caveolin-2 is a novel mechanism to regulate the dynamics of caveolae assembly.     

Na+ Current in Human Ventricle: Implications for Sodium Loading and Homeostasis

The Na current (I_Na) in human ventricle is carried through a specific isoform of the voltage gated Na channel in heart. The pore forming α-subunit is encoded by the gene SCN5A . Up to four β-subunits may be associated, and the larger macromolecular complex may include attachments to cytoskeleton and scaffolding proteins, all of which may affect the gating kinetics of the current. I_Na underlies initiation and propagation of action potentials in the heart and plays a prominent role in cardiac electrophysiology and arrhythmia. In addition, I_Na also loads the ventricular cell with Na⁺ ions and plays an important role in intracellular Na homeostasis. This review considers the structure and function of the human cardiac Na channel that carries I_Na with a particular consideration of the implications of alterations in I_Na in acquired cardiac diseases such as hypertrophy, failure, and ischemia, which affect Na loading.     

The Role of Late INa and Antiarrhythmic Drugs in EAD Formation and Termination in Purkinje Fibers

Multiple components of cardiac Na current play a role in determining electrical excitation in the heart. Recently, the role of nonequilibrium components in controlling cardiac action potential plateau duration, and their importance in regulating the occurrence of afterdepolarizations and arrhythmias have garnered more attention. In particular, late Na current (late I _Na) has been shown to be important in LQT2 and LQT3 arrhythmias. Class III agents like dofetilide, clofilium, and sotalol, which can all cause a drug-induced form of LQT2, significantly lengthen action potential duration at 50% and 90% repolarization in isolated rabbit Purkinje fibers, and can initiate the formation of early afterdepolarizations, and extra beats. These actions can lead to the development of a serious ventricular tachycardia, torsades de pointes, in animal models and patients. However, pretreatment with agents that block late I _Na, like lidocaine, mexiletine, and RSD1235, a novel mixed ion channel blocker for the rapid pharmacologic conversion of atrial fibrillation, significantly attenuates the prolonging effects of Class III agents or those induced by ATX-II, a specific toxin that delays Na channel inactivation and amplifies late I _Na greatly, mimicking LQT3. The Na channel block caused by lidocaine and RSD1235 can be through the open or inactivated states of the channel, but both equivalently inhibit a late component of Na current ( I _Na), recorded at 22°C using whole-cell patch clamp of Nav1.5 expressed in HEK cells. These protective actions of lidocaine, mexiletine, and RSD1235 may result, at least in part, from their ability to inhibit late I _Na during action potential repolarization, and inhibition of the inward currents contributing to EAD and arrhythmia formation.     

A sodium channel pore mutation causing Brugada syndrome

BACKGROUND: Brugada and long QT type 3 syndromes are linked to sodium channel mutations and clinically cause arrhythmias that lead to sudden death. We have identified a novel threonine-to-isoleucine missense mutation at position 353 (T353I) adjacent to the pore-lining region of domain I of the cardiac sodium channel (SCN5A) in a family with Brugada syndrome. Both male and female carriers are symptomatic at young ages, have typical Brugada-type electrocardiogram changes, and have relatively normal corrected QT intervals. OBJECTIVES: To characterize the properties of the newly identified cardiac sodium channel (SCN5A) mutation at the cellular level. RESULTS: Using whole-cell voltage clamp, we found that heterologous expression of SCN5A containing the T353I mutation resulted in 74% +/- 6% less peak macroscopic sodium current when compared with wild-type channels. A construct of the T353I mutant channel fused with green fluorescent protein failed to traffic properly to the sarcolemma, with a large proportion of channels sequestered intracellularly. Overnight exposure to 0.1 mM mexiletine, a Na(+) channel blocking agent, increased T353I channel trafficking to the membrane to near normal levels, but the mutant channels showed a significant late current that was 1.6% +/- 0.2% of peak sodium current at 200 ms, a finding seen with long QT mutations. CONCLUSIONS: The clinical presentation of patients carrying the T353I mutation is that of Brugada syndrome and could be explained by a cardiac Na(+) channel trafficking defect. However, when the defect was ameliorated, the mutated channels had biophysical properties consistent with long QT syndrome. The lack of phenotypic changes associated with the long QT syndrome could be explained by a T353I-induced trafficking defect reducing the number of mutant channels with persistent currents present at the sarcolemma.     

Caveolin-1 and -3 dissociations from caveolae to cytosol in the heart during aging and after myocardial infarction in rat

OBJECTIVE: Caveolins, the structural proteins of caveolae, modulate numerous signaling pathways including Nitric Oxide (NO) production. Among the caveolin family, caveolin-1 and -3 are mainly expressed in endothelial and muscle cells, respectively. In this study, we investigate whether (i) changes in caveolin abundance and/or distribution occur during cardiac aging and failure in rat, and (ii) the process could influence NO synthase (NOS) activity. METHODS: Using immunohistolabelling and Western blot approaches, expression and distribution of caveolins were analysed in adult (Ad), senescent (S-Sh) and myocardial infarction-induced failing (S-MI) hearts. NOS3/caveolin-1 interactions were evaluated by immunoprecipitation assays. RESULTS: At the microscope level, caveolin-1 distribution in the endothelial cells was unchanged between the groups. Conversely the typical distribution of caveolin-3 in myocyte sarcolemma was dramatically altered in S-MI rats, resulting in a heterogeneous pattern throughout the septum. Total abundance of caveolin-1 and -3 remained stable whatever the group. In the fractions free of caveolae (Triton X-100 soluble), the levels of caveolin-1 alpha and -3 increased with aging (+20%, and +104%, P<0.05 versus Ad, respectively) and were further enhanced in S-MI (+25%, +30%, P<0.05, P<0.001 versus S-Sh respectively). In these fractions, NOS3/caveolin-1 alpha complexes increased as well. In addition, NOS activity was negatively correlated to caveolin-1 level in the cytosolic fractions. CONCLUSIONS: We demonstrate that dissociation of caveolin from caveolae is associated with aging and heart failure, the process being related to the decreased NOS activity.     

Cholesterol modulates the recruitment of Kv1.5 channels from Rab11-associated recycling endosome in native atrial myocytes

Cholesterol is an important determinant of cardiac electrical properties. However, underlying mechanisms are still poorly understood. Here, we examine the hypothesis that cholesterol modulates the turnover of voltage-gated potassium channels based on previous observations showing that depletion of membrane cholesterol increases the atrial repolarizing current I_Kur. Whole-cell currents and single-channel activity were recorded in rat adult atrial myocytes (AAM) or after transduction with hKv1.5-EGFP. Channel mobility and expression were studied using fluorescence recovery after photobleaching (FRAP) and 3-dimensional microscopy. In both native and transduced-AAMs, the cholesterol-depleting agent MβCD induced a delayed (≈7 min) increase in I_Kur; the cholesterol donor LDL had an opposite effect. Single-channel recordings revealed an increased number of active Kv1.5 channels upon MβCD application. Whole-cell recordings indicated that this increase was not dependent on new synthesis but on trafficking of existing pools of intracellular channels whose exocytosis could be blocked by both N-ethylmaleimide and nonhydrolyzable GTP analogues. Rab11 was found to coimmunoprecipitate with hKv1.5-EGFP channels and transfection with Rab11 dominant negative (DN) but not Rab4 DN prevented the MβCD-induced I_Kur increase. Three-dimensional microscopy showed a decrease in colocalization of Kv1.5 and Rab11 in MβCD-treated AAM. These results suggest that cholesterol regulates Kv1.5 channel expression by modulating its trafficking through the Rab11-associated recycling endosome. Therefore, this compartment provides a submembrane pool of channels readily available for recruitment into the sarcolemma of myocytes. This process could be a major mechanism for the tuning of cardiac electrical properties and might contribute to the understanding of cardiac effects of lipid-lowering drugs.     

Structure of Connexin43 and its Regulation by pHi

pH_i Regulation of Connexin43. Electrical coupling in the heart provides an effective mechanism fur propagating the cardiac action potential efficiently throughout the entire heart. Cells within the heart are electrically coupled through specialized membrane channels called gap junctions. Studies have shown that gap junctions are dynamic, carefully regulated channels that are important for normal cardiogenesis. We have recently been interested in the molecular mechanisms by which intracellular acidification leads to gap junction channel closure. Previous results in this lab have shown that truncation of the carboxyl terminal (CT) of connexin43 (Cx43) does not interfere with functional channel expression. Further, the pH-dependent closure of C×43 channels is significantly impaired by removal of this region of the protein. Other studies have shown that the CT is capable of interacting with its receptor even when not covalently attached to the channel protein. From these data we have proposed a particle-receptor model to explain the pH-dependent closure of Cx43 gap junction channels. Detailed analysis of the CT has revealed interesting new information regarding its possible structure. Here we review the most recent studies that have contributed to our understanding of the molecular mechanisms of regulation of the cardiac gap protein C×43.     

Sphingolipid-cholesterol domains (lipid rafts) in normal human and dog thyroid follicular cells are not involved in thyrotropin receptor signaling

Partition of signaling molecules in sphingolipid-cholesterol-enriched membrane domains, among which are the caveolae, may contribute to signal transduction efficiency. In normal thyroid, nothing is known about a putative TSH/cAMP cascade compartmentation in caveolae or other sphingolipid-cholesterol-enriched membrane domains. In this study we show for the first time that caveolae are present in the apical membrane of dog and human thyrocytes: caveolin-1 mRNA presence is demonstrated by Northern blotting in primary cultures and that of the caveolin-1 protein by immunohistochemistry performed on human thyroid tissue. The TSH receptor located in the basal membrane can therefore not be located in caveolae. We demonstrate for the first time by biochemical methods the existence of sphingolipid-cholesterol-enriched domains in human and dog thyroid follicular cells that contain caveolin, flotillin-2, and the insulin receptor. We assessed a possible sphingolipid-cholesterol-enriched domains compartmentation of the TSH receptor and the alpha- subunit of the heterotrimeric G(s) and G(q) proteins using two approaches: Western blotting on detergent-resistant membranes isolated from thyrocytes in primary cultures and the influence of 10 mm methyl-beta-cyclodextrin, a cholesterol chelator, on basal and stimulated cAMP accumulation in intact thyrocytes. The results from both types of experiments strongly suggest that the TSH/cAMP cascade in thyroid cells is not associated with sphingolipid-cholesterol-enriched membrane domains.     

Serotonin inhibits voltage-gated K+ currents in pulmonary artery smooth muscle cells: role of 5-HT2A receptors, caveolin-1, and KV1.5 channel internalization

Multiple lines of evidence indicate that serotonin (5-hydroxytryptamine [5-HT]) and voltage-gated K+ (KV) channels play a central role in the pathogenesis of pulmonary hypertension (PH). We hypothesized that 5-HT might modulate the activity of KV channels, therefore establishing a link between these pathogenetic factors in PH. Here, we studied the effects of 5-HT on KV channels present in rat pulmonary artery smooth muscle cells (PASMC) and on hKV1.5 channels stably expressed in Ltk- cells. 5-HT reduced native KV and hKV1.5 currents, depolarized cell membrane, and caused a contraction of isolated pulmonary arteries. The effects of 5-HT on KV currents and contraction were markedly prevented by the 5-HT2A receptor antagonist ketanserin. Incubation with inhibitors of phospholipase C (U73122), classic protein kinase Cs (G?6976), or tyrosine kinases (genistein and tyrphostin 23), the cholesterol depletion agent beta-cyclodextrin or concanavalin A, an inhibitor of endocytotic processes, also prevented the effects of 5-HT. In homogenates from pulmonary arteries, 5-HT2A receptors and caveolin-1 coimmunoprecipitated with KV1.5 channels, and this was increased on stimulation with 5-HT. Moreover, KV1.5 channels were internalized when cells were stimulated with 5-HT, and this was prevented by concanavalin A. These findings indicate that activation of 5-HT2A receptors inhibits native KV and hKV1.5 currents via phospholipase C, protein kinase C, tyrosine kinase, and a caveolae pathway. KV channel inhibition accounts, at least partly, for 5-HT-induced pulmonary vasoconstriction and might play a role in PH.     

BK channel subunit composition modulates molecular tolerance to ethanol

Background:  The large conductance calcium-activated potassium channel (also called BK channel or Slo channels) is a well-studied target of alcohol action, and plays an important role in behavioral tolerance.
Methods:  Using patch clamp electrophysiology, we examined human BK channels expressed in HEK293 cells to test whether tolerance to ethanol occurs in excised patches and whether it is influenced by subunit composition. Three combinations were examined: hSlo, hSlo + β₁, and hSlo + β₄.
Results:  The 2 components of BK alcohol adaptation (Component 1: rapid tolerance to acute potentiation, and Component 2: a more slowly developing decrease in current density) were observed, and varied according to subunit combination. Using a 2-exposure protocol, Component 1 tolerance was evident in 2 of the 3 combinations, because it was more pronounced for hSlo and hSlo + β₄.
Conclusions:  Thus, rapid tolerance in human BK occurs in cell-free membrane patches, independent of cytosolic second messengers, nucleotides or changes in free calcium. Alcohol pretreatment for 24 hours altered subsequent short-term plasticity of hSlo + β₄ channels, suggesting a relationship between classes of tolerance. Finally, Component 2 reduction in current density showed a striking dependency on channel composition. Twenty-four hour exposure to 25 mM ethanol resulted in a down-regulation of BK current in hSlo and hSlo + β₄ channels, but not in hSlo + β₁ channels. The fact that hSlo + β₁ channels show less sensitivity to acute challenge, in conjunction with less Component 1 and Component 2 tolerance, suggests subunit composition is an important factor for these elements of alcohol response.     

Oestrogen-mediated tyrosine phosphorylation of caveolin-1 and its effect on the oestrogen receptor localisation: an in vivo study

Recently, it has been shown that 17beta estradiol (E2) induces a rapid and transient activation of the Src ERK phosphorylation cascade: a clear indication that the alpha oestrogen receptor (ERalpha) is able to associate with the plasma membrane. Increasing evidence suggests that caveolae, which are caveolin-1 containing, highly hydrophobic membrane domains, play an important role in E2 induced signal transduction. Caveolae can accumulate signalling molecules preferentially; thus, they may have a regulatory role in signalling processes. Results from previous experiments have shown that E2 treatment decreased the number of surface connected caveolae significantly in uterine smooth muscle cells and also downregulated the expression of caveolin-1. In addition to providing further evidence that ERalpha interacts with caveolin/caveolae in uterine smooth muscle cells, this study also shows that the interaction between caveolin-1 and ERalpha is actually facilitated by E2. One of the signal transduction components found to accumulate in caveolae is Src kinase in an amount that increases simultaneously with increases in the amount of ERalpha. Upon E2 treatment, Src kinase is tyrosine phosphorylated, which, in turn, stimulates Src kinase to phosphorylate caveolin-1. Phosphorylation of caveolin-1 can drive caveolae to pinch off from the plasma membrane, thereby decreasing the amount of plasma membrane-associated caveolin-1. This loss of caveolin/caveolae activates the signal cascade that triggers cell proliferation.     

β-Adrenergic control of sarcolemmal CaV1.2 abundance by small GTPase Rab proteins

The number and activity of Ca_v1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca²⁺-induced Ca²⁺ release and myocardial contraction. β-Adrenergic receptor (βAR) activation stimulates sarcolemmal insertion of Ca_V1.2. This supplements the preexisting sarcolemmal Ca_V1.2 population, forming large “superclusters” wherein neighboring channels undergo enhanced cooperative-gating behavior, amplifying Ca²⁺ influx and myocardial contractility. Here, we determine this stimulated insertion is fueled by an internal reserve of early and recycling endosome-localized, presynthesized Ca_V1.2 channels. βAR-activation decreased Ca_V1.2/endosome colocalization in ventricular myocytes, as it triggered “emptying” of endosomal Ca_V1.2 cargo into the t-tubule sarcolemma. We examined the rapid dynamics of this stimulated insertion process with live-myocyte imaging of channel trafficking, and discovered that Ca_V1.2 are often inserted into the sarcolemma as preformed, multichannel clusters. Similarly, entire clusters were removed from the sarcolemma during endocytosis, while in other cases, a more incremental process suggested removal of individual channels. The amplitude of the stimulated insertion response was doubled by coexpression of constitutively active Rab4a, halved by coexpression of dominant-negative Rab11a, and abolished by coexpression of dominant-negative mutant Rab4a. In ventricular myocytes, βAR-stimulated recycling of Ca_V1.2 was diminished by both nocodazole and latrunculin-A, suggesting an essential role of the cytoskeleton in this process. Functionally, cytoskeletal disruptors prevented βAR-activated Ca²⁺ current augmentation. Moreover, βAR-regulation of Ca_V1.2 was abolished when recycling was halted by coapplication of nocodazole and latrunculin-A. These findings reveal that βAR-stimulation triggers an on-demand boost in sarcolemmal Ca_V1.2 abundance via targeted Rab4a- and Rab11a-dependent insertion of channels that is essential for βAR-regulation of cardiac Ca_V1.2.

The influx of Ca²⁺ through L-type Ca²⁺ channels (Ca_V1.2) is indispensable for cardiac excitation–contraction coupling (EC-coupling). These multimeric proteins consist of a pore-forming and voltage-sensing α_1c subunit, and auxiliary β- and α₂δ-subunits. In ventricular myocytes, Ca_V1.2 mainly localize to the t-tubule sarcolemma and open briefly, allowing a small amount of Ca²⁺ influx, in response to the wave of depolarization that travels through the conduction system of the heart from its point of origin, usually in the SA-node. This initial influx is amplified manifold though Ca²⁺-induced Ca²⁺ release (CICR) from juxtaposed type 2 ryanodine receptors (RyR2) on the junctional sarcoplasmic reticulum, a short ∼12 nm across the dyadic cleft. The synchronous opening of thousands of RyR2 generates a transient, global elevation in intracellular calcium concentration ([Ca²⁺]_i), resulting in contraction. Reducing Ca_V1.2 channel current (I_Ca) results in less CICR, smaller [Ca²⁺]_i transients, and less forceful contractions. Conversely, larger amplitude I_Ca elicits greater Ca²⁺ release from the sarcoplasmic reticulum, producing more forceful contractions. The level of Ca²⁺ influx through Ca_V1.2 channels therefore tunes EC-coupling.I_Ca is a product of the number of channels in the sarcolemma and their open probability (P_o). Consequently, there are two possible, nonmutually exclusive strategies that may be adopted to alter I_Ca and consequently the magnitude of EC-coupling: 1) Adjust Ca_V1.2 channel activity (P_o) and 2) modify sarcolemmal Ca_V1.2 channel expression (N). The first strategy of increasing channel P_o has long been associated with β-adrenergic receptor (βAR)-mediated signaling in the heart (1–3). During acute physical or emotional stress, norepinephrine spills from sympathetic varicosities onto cardiomyocytes, activating βARs. The ensuing G_αs/adenylyl cyclase/cAMP/PKA signaling cascade culminates in PKA phosphorylation of several effector proteins, including Ca_V1.2 [or an element of their interactome (4)], enhancing their activity to generate this positive inotropic response.As to the second strategy to increase I_Ca, there remains a paucity of information regarding the mechanisms regulating Ca_V1.2 channel abundance in the cardiomyocyte sarcolemma. Classic secretory transport literature suggests that Ca_V1.2 channels are trafficked from the endoplasmic reticulum to the trans-Golgi-network and onward to their dyadic position in the sarcolemma. Underscoring the importance of faithful Ca_V1.2 channel trafficking, altered Ca_V1.2 channel density has been reported in both failing (5) and aging (6) ventricular myocytes, and impaired anterograde trafficking of Ca_V1.2 channels to the t-tubules of human ventricular myocytes has been linked to dilated cardiomyopathy (7). Yet, despite the importance of tight homeostatic control of Ca_V1.2 channel trafficking to prevent Ca²⁺ dysregulation, the molecular steps defining Ca_V1.2 channel sorting and insertion remain poorly understood. Therefore, elucidation of the trafficking pathways that regulate Ca_V1.2 channel abundance is critical for our understanding of the pathophysiology of heart failure and myocardial aging, and could potentially reveal new therapeutic or rejuvenation targets. Along that vein, in the treatment of cystic fibrosis, multiple drugs are in various stages of use or development to improve trafficking to, or to amplify or stabilize, CFTR channels at the apical membrane of airway epithelial cells (8).There exist no measurements of Ca_V1.2 channel lifetimes in cardiomyocytes, but pulse-chase experiments in immortalized cell lines support a lifetime of plasma membrane (PM)-localized Ca_V1.2 of ∼3 h (9), while total cellular Ca_V1.2 lifetime is >20 h (10). This disparity suggests membrane-Ca_V1.2 turns over much more dynamically than the total cellular channel content and implies ongoing local control by endosomal trafficking. Disturbance of the equilibrium between channel insertion/recycling and internalization would be predicted to lead to alterations in sarcolemmal Ca_V1.2 channel abundance. Trafficking of vesicular cargo through the endosomal pathway is regulated by Rab-GTPases, a >60-member family within the larger Ras superfamily of small GTPases (11, 12). Rab5 is involved in endocytosis and control of vesicular cargo influx into early endosomes (EEs; also called sorting endosomes), while Rab4 controls efflux of cargo out of EEs and fast recycling (t_1/2 ∼1 to 2 min) back to the PM (13). Rab11, expressed on recycling endosomes (RE; also called the endocytic recycling compartment or ERC), regulates slow recycling (t_1/2 ∼12 min) of cargo from this compartment back to the PM (13). In cortical neurons and pancreatic β-cells, activity-dependent Ca_V1.2 channel internalization has been postulated to play important roles in Ca²⁺ homeostasis, with implications for homeostatic synaptic plasticity and insulin production, respectively (11, 14). In mouse neonatal cardiomyocytes, Rab11b has been reported to limit Ca_V1.2 PM expression (15), while recent studies performed in HEK and HL-1 cells reported that endocytic recycling of cardiac Ca_V1.2 channels, regulates their surface abundance (10, 16). Despite this crucial information from other cell-types, there has been a lack of rigorous investigations, at the molecular level, into how Ca_V1.2 channel recycling is regulated in cardiac myocytes.Here, we identify a dynamic, subsarcolemmal pool of Ca_V1.2-cargo–carrying endosomes that are rapidly mobilized to the ventricular myocyte sarcolemma along targeted Rab4a and Rab11a GTPase-regulated recycling pathways in response to βAR-stimulation. Using electrophysiology, cell biology, total internal reflection fluorescence (TIRF), and superresolution microscopy, we report that enhanced t-tubule sarcolemmal Ca_V1.2 abundance via targeted, isoproterenol (ISO)-stimulated recycling of these channels is essential for βAR-regulation of cardiac Ca_V1.2.