Gene Therapy to Inhibit the Calcium Channel β Subunit

Cingolani, Eugenio; Correa, Genaro A Ramirez; Kizana, Eddy; Murata, Mitsushige; Cho, Hee Cheol; Marbán, Eduardo

doi:10.1161/circresaha.107.155721

Cited by 61 publications

(21 citation statements)

References 39 publications

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“…Several evidences suggest that alterations in Ca V ␤ 2 underlie some forms of myocardial dysfunction (61)(62)(63)(64), and so far, gene therapy to inhibit Ca V ␤ 2 has been considered only in correlation to an electrophysiological phenotype (65). …”

Section: Discussionmentioning

confidence: 99%

Homodimerization of the Src Homology 3 Domain of the Calcium Channel β-Subunit Drives Dynamin-dependent Endocytosis

Miranda-Laferte

González-Gutiérrez

Schmidt

et al. 2011

Journal of Biological Chemistry

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Voltage-dependent calcium channels constitute the main entry pathway for calcium into excitable cells. They are heteromultimers formed by an ␣ 1 pore-forming subunit (Ca V ␣ 1 ) and accessory subunits. To achieve a precise coordination of calcium signals, the expression and activity of these channels is tightly controlled. The accessory ␤-subunit (Ca V ␤), a membrane associated guanylate kinase containing one guanylate kinase (␤-GK) and one Src homology 3 (␤-SH3) domain, has antagonistic effects on calcium currents by regulating different aspects of channel function. Although ␤-GK binds to a conserved site within the ␣ 1 -pore-forming subunit and facilitates channel opening, ␤-SH3 binds to dynamin and promotes endocytosis. Here, we investigated the molecular switch underlying the functional duality of this modular protein. We show that ␤-SH3 homodimerizes through a single disulfide bond. Substitution of the only cysteine residue abolishes dimerization and impairs internalization of L-type Ca V 1.2 channels expressed in Xenopus oocytes while preserving dynamin binding. Covalent linkage of the ␤-SH3 dimerization-deficient mutant yields a concatamer that binds to dynamin and restores endocytosis. Moreover, using FRET analysis, we show in living cells that Ca V ␤ form oligomers and that this interaction is reduced by Ca V ␣ 1 . Association of Ca V ␤ with a polypeptide encoding the binding motif in Ca V ␣ 1 inhibited endocytosis. Together, these findings reveal that ␤-SH3 dimerization is crucial for endocytosis and suggest that channel activation and internalization are two mutually exclusive functions of Ca V ␤. We propose that a change in the oligomeric state of Ca V ␤ is the functional switch between channel activator and channel internalizer.Voltage-dependent calcium channels link membrane depolarization to transient increases in cytosolic calcium concentration, which in turn mediate a variety of cellular processes, including gene expression, contraction, neurotransmission, and exocytosis (1). To direct the signal to a specific subset of an extensive family of effectors, cells are endowed with a complex network of regulators that constrain the timing and spreading of the calcium increase. The subset of voltage-dependent calcium channels that are activated by strong depolarization, also called high voltage-activated (HVA) 3 channels, are heteromultimers consisting of a central pore-forming subunit (Ca V ␣ 1 ) that associates with one or more accessory subunits. Among them the accessory ␤-subunit (Ca V ␤) has traditionally been recognized as one of the most important modulator of HVA channels (1-3). More recently, it has been shown that the same subunit mediates several other cellular processes (for review, see Ref. 4), including regulation of insulin secretion (5), gene transcription (6) and endocytosis (7). Four Ca V ␤ isoforms (Ca V ␤ 1 to Ca V ␤ 4 ) have been cloned from different nonallelic genes. Crystallographic studies of three of these provided the molecular basis for its functional versatility by identify...

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Section: Discussionmentioning

confidence: 99%

Homodimerization of the Src Homology 3 Domain of the Calcium Channel β-Subunit Drives Dynamin-dependent Endocytosis

Miranda-Laferte

González-Gutiérrez

Schmidt

et al. 2011

Journal of Biological Chemistry

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“…The L-type Ca 2ϩ current (L-current) density was similar in sham-operated WT myocytes compared Figure 7C). It has been reported that the reduction of the L-current by continuous infusion of L-channel blocker 35 or the knockdown of L-channel accessory ␤ subunit 36 channels that open at the resting membrane potential of a HEK293 cell (ie, window current). 37,38 As shown in Figure 8A, 10 mmol/L Ca 2ϩ significantly increased the NFAT-luciferase reporter activity relative to that of cells transfected with control vehicle and T-channel blocker mibefradil effectively blocked NFAT-luciferase activity triggered by calcium.…”

Section: Currents From Acutely Isolated Left Ventricular Myocytesmentioning

confidence: 99%

The Ca _V 3.2 T-Type Ca ²⁺ Channel Is Required for Pressure Overload–Induced Cardiac Hypertrophy in Mice

Chiang

Huang

Chieng

et al. 2009

Circulation Research

145

123

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Abstract-Voltage-gated T-type Ca 2ϩ channels (T-channels) are normally expressed during embryonic development in ventricular myocytes but are undetectable in adult ventricular myocytes. Interestingly, T-channels are reexpressed in hypertrophied or failing hearts. It is unclear whether T-channels play a role in the pathogenesis of cardiomyopathy and what the mechanism might be. Here we show that the ␣ 1H voltage-gated T-type Ca 2ϩ channel (Ca v 3.2) is involved in the pathogenesis of cardiac hypertrophy via the activation of calcineurin/nuclear factor of activated T cells (NFAT) pathway. Specifically, pressure overload-induced hypertrophy was severely suppressed in mice deficient for Ca v 3.2 (Ca v 3.2 Ϫ/Ϫ ) but not in mice deficient for Ca v 3.1 (Ca v 3.1 Ϫ/Ϫ ). Angiotensin II-induced cardiac hypertrophy was also suppressed in Ca v 3.2 Ϫ/Ϫ mice. Consistent with these findings, cultured neonatal myocytes isolated from Ca v 3.2 Ϫ/Ϫ mice fail to respond hypertrophic stimulation by treatment with angiotensin II. Together, these results demonstrate the importance of Ca v 3.2 in the development of cardiac hypertrophy both in vitro and in vivo. To test whether Ca v 3.2 mediates the hypertrophic response through the calcineurin/NFAT pathway, we generated Ca v 3.2 Ϫ/Ϫ , NFAT-luciferase reporter mice and showed that NFAT-luciferase reporter activity failed to increase after pressure overload in the Ca v 3.2 Ϫ/Ϫ /NFAT-Luc mice. Our results provide strong genetic evidence that Ca v 3.2 indeed plays a pivotal role in the induction of calcineurin/NFAT hypertrophic signaling and is crucial for the activation of pathological cardiac hypertrophy. H ypertrophic growth and remodeling of the adult heart begin as normal compensatory responses to a variety of physiological and pathological stimuli including exercise, pregnancy, pressure overload, hypertension, myocardial infarction, and primary genetic abnormalities. [1][2][3] Prolonged hypertrophic response may eventually lead to heart failure and death. Pathological hypertrophy is often associated with structural and functional remodeling of the heart. Profound changes in gene expression during pathological cardiac hypertrophy are common, particularly in the case of genes that code for proteins involved in regulating contraction ion channels, for example. 4 Alterations of intracellular Ca 2ϩ handling could lead to abnormal Ca 2ϩ signaling cascades, a phenomenon that has been shown to contribute to the pathogenesis of cardiac hypertrophy and heart failure. 5,6 However, the detailed mechanism of how the cardiac myocytes distinguish Ca 2ϩ transients that occur at every heartbeat from those meant to trigger intracellular hypertrophic signaling remains largely unknown. Intracellular Ca 2ϩ levels could be altered because of either changes in Ca 2ϩ release from intracellular organelles or the influx of extracellular Ca 2ϩ , and in both cases, this could be attributable to the activities of either ligand-or voltage-activated Ca 2ϩ channels. Indeed, ligand-activated Ca 2ϩ channels, such as ...

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“…In addition, mice with cardiac myocyte-specific NOS3 overexpression had limited hypertrophy and contractile dysfunction after pressure overload and myocardial infarction (10,23). Furthermore, it is known that I Ca plays a role in hypertrophy (14). Ca 2ϩ influx via I Ca activates calcineurin, leading to nuclear factor of activated T cells dephosphorylation and the activation of hypertrophic signaling.…”

Section: Nos3 and Arrhythmogenesismentioning

confidence: 99%

Endothelial nitric oxide synthase decreases β-adrenergic responsiveness via inhibition of the L-type Ca²⁺ current

Wang

Kohr

Wheeler

et al. 2008

American Journal of Physiology-Heart and Circulatory Physiology

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Gene Therapy to Inhibit the Calcium Channel β Subunit

Cited by 61 publications

References 39 publications

Homodimerization of the Src Homology 3 Domain of the Calcium Channel β-Subunit Drives Dynamin-dependent Endocytosis

Homodimerization of the Src Homology 3 Domain of the Calcium Channel β-Subunit Drives Dynamin-dependent Endocytosis

The Ca _V 3.2 T-Type Ca ²⁺ Channel Is Required for Pressure Overload–Induced Cardiac Hypertrophy in Mice

Endothelial nitric oxide synthase decreases β-adrenergic responsiveness via inhibition of the L-type Ca²⁺ current

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Gene Therapy to Inhibit the Calcium Channel β Subunit

Cited by 61 publications

References 39 publications

Homodimerization of the Src Homology 3 Domain of the Calcium Channel β-Subunit Drives Dynamin-dependent Endocytosis

Homodimerization of the Src Homology 3 Domain of the Calcium Channel β-Subunit Drives Dynamin-dependent Endocytosis

The Ca V 3.2 T-Type Ca 2+ Channel Is Required for Pressure Overload–Induced Cardiac Hypertrophy in Mice

Endothelial nitric oxide synthase decreases β-adrenergic responsiveness via inhibition of the L-type Ca2+ current

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The Ca _V 3.2 T-Type Ca ²⁺ Channel Is Required for Pressure Overload–Induced Cardiac Hypertrophy in Mice

Endothelial nitric oxide synthase decreases β-adrenergic responsiveness via inhibition of the L-type Ca²⁺ current