Current Molecular Pharmacology - Volume 8, Issue 1, 2015

Voltage-gated calcium channels are not only mediators of cell signalling events, but also are recipients of signalling inputs from G protein coupled receptors (GPCRs) and their associated second messenger pathways. The coupling of GPCRs to calcium channels is optimized through the formation of receptor-channel complexes. In addition, this provides a mechanism for receptorchannel co-trafficking to and from the plasma membrane. On the other hand, voltage-gated calcium channel activity affects other types of ion channels such as voltage-and calcium-activated potassium channels. Coupling efficiency between these two families of channels is also enhanced through the formation of channel-channel complexes. This review provides a concise overview of the current state of knowledge on the physical interactions between voltage-gated calcium channels and members of the GPCR family, and with other types of ion channels.

Intracellular calcium transients generated by activation of voltage-gated calcium (CaV) channels generate local signals, which initiate physiological processes such as secretion, synaptic transmission, and excitation-contraction coupling. Regulation of calcium entry through CaV channels is crucial for control of these physiological processes. In this article, I review experimental results that have emerged over several years showing that cardiac CaV1.2 channels form a local signaling complex, in which their proteolytically processed distal C-terminal domain, an A-Kinase Anchoring Protein, and cyclic AMP-dependent protein kinase (PKA) interact directly with the transmembrane core of the ion channel through the proximal C-terminal domain. This signaling complex is the substrate for β-adrenergic up-regulation of the CaV1.2 channel in the heart during the fight-or-flight response. Protein phosphorylation of two sites at the interface between the distal and proximal C-terminal domains contributes importantly to control of basal CaV1.2 channel activity, and phosphorylation of Ser1700 by PKA at that interface up-regulates CaV1.2 activity in response to β-adrenergic signaling. Thus, the intracellular C-terminal domain of CaV1.2 channels serves as a signaling platform, mediating beat-to-beat physiological regulation of channel activity and up-regulation by β-adrenergic signaling in the fight-or-flight response.

Voltage-gated calcium ion channels are essential for numerous biological functions of excitable cells and there is wide spread appreciation of their importance as drug targets in the treatment of many disorders including those of cardiovascular and nervous systems. Each Cacna1 gene has the potential to generate a number of structurally, functionally, and in some cases pharmacologically unique CaVα1 subunits through alternative pre-mRNA splicing and the use of alternate promoters. Analyses of rapidly emerging deep sequencing data for a range of human tissue transcriptomes contain information to quantify tissue-specific and alternative exon usage patterns for Cacna1 genes. Cellspecific actions of nuclear DNA and RNA binding proteins control the use of alternate promoters and the selection of alternate exons during pre-mRNA splicing, and they determine the spectrum of protein isoforms expressed within different types of cells. Amino acid compositions within discrete protein domains can differ substantially among CaV isoforms expressed in different tissues, and such differences may be greater than those that exist across CaV channel homologs of closely related species. Here we highlight examples of CaV isoforms that have unique expression patterns and that exhibit different pharmacological sensitivities. Knowledge of expression patterns of CaV isoforms in different human tissues, cell populations, ages, and disease states should inform strategies aimed at developing the next generation of CaV channel inhibitors and agonists with improved tissue-specificity.

The voltage-gated Cav1.2 calcium channels respond to membrane depolarization by increasing the membrane permeability to Ca2+, a major signal for cardiac muscle contraction, regulation of vascular tone and CREB-dependent transcriptional activation. CACNB2 is one of the four homologous genes coding for the auxiliary Cavβ subunits, which are important modulators of the Ca2+ channel activity. Five serious mental disorders - autism spectrum disorder, attention deficit-hyperactivity disorder, bipolar disorder, major depressive disorder, and schizophrenia, - and three major cardiovascular diseases - hypertension, heart failure and sudden cardiac death, - have recently been linked to the CACNB2 gene coding for the Cavβ2 subunits. Here I will focus on the Cavβ2-specific molecular determinant β2-CED as an emerging pharmacological target.

Ca2+ influx via L-type Ca2+ channel (L-VDCC; CaV1.2) is required for cardiac and smooth muscle contraction. These channels are located in the plasma membrane and along the T-tubules (in cardiomyocytes), along with various scaffold and trafficking proteins. CaV1.2 is modulated by different hormones and transmitters and was implicated in a variety of cardiovascular pathologies, many of which also involve protein kinase C (PKC). One of the prominent pathways of PKC activation in cardiac and smooth muscle cells is via activation of Gq-coupled receptors and subsequent activation of protein lipase C (PLC). CaV1.2 was shown to be modulated, phosphorylated by, and associated with PKC both in vitro and in vivo. Despite the well documented enhancing effect of vasoconstrictors operating via Gq on CaV1.2 channels, the molecular mechanism by which PKC affects the channel has not yet been resolved. Furthermore, the nature of PKC modulation of CaV1.2 appears to be species-, age- and tissue-dependent. Results from experiments in heterologous expression systems are often contradicting and are difficult to coalesce. The choice of both the heterologous expression system and the CaV1.2 isoform expressed are at the core of this conundrum. Complete reconstitution of the enhancing effect of PKC was successful only in Xenopus oocytes and only when the long N-terminus (NT) isoform of the channel was expressed. This review summarizes past and new findings regarding the mechanism by which activated PKC modulates CaV1.2 channels in native tissues and heterologous expression systems, and suggests perspectives for future research.

The major function of the voltage-gated calcium channels is to provide the Ca2+ flux into the cell. L-type voltage-gated calcium channels (Cav1) serve as voltage sensors that couple membrane depolarization to many intracellular processes. Electrical activity in excitable cells affects gene expression through signaling pathways involved in the excitation-transcription (E-T) coupling. E-T coupling starts with activation of the Cav1 channel and results in initiation of the cAMP-response element binding protein (CREB)-dependent transcription. In this review we discuss the new quantitative approaches to measuring E-T signaling events. We describe the use of wavelet transform to detect heterogeneity of transcriptional activation in nuclei. Furthermore, we discuss the properties of discovered microdomains of nuclear signaling associated with the E-T coupling and the basis of the frequency-dependent transcriptional regulation.

The cardiac L-type Ca2+ channel plays a key role in cardiac excitation-contraction coupling, action potential duration, and gene expression. Abnormalities in CaV1.2 function, including increased long-opening-mode gating and blunted adrenergic responsiveness, are associated with heart failure and hypertrophy. The increased activation of CaV1.2, in turn, triggers Ca2+ -responsive signaling pathways, which contribute to the pathogenesis of heart failure and hypertrophy. CaV1.2 in heart is associated with large supramolecular complexes that impact on channel trafficking, localization, turnover, and function. Much of the prevailing dogma relating to mechanisms underlying CaV1.2 trafficking and modulation is derived from studies using recombinant channels reconstituted in heterologous expression systems. However, recent results using knock-in mice indicate that several long-standing “facts” about CaV1.2 regulation derived from heterologous expression studies are not replicated in native heart, emphasizing the critical need for mechanistic studies in the context of actual cardiomyocytes. In this review, we discuss the use of the use of knockin and knockout mice as well as new tools, including doxycycline-induced expression of informative α1C mutants within cardiomyocytes to probe adrenergic regulation of CaV1.2.

Voltage-gated calcium (CaV) channels conduct Ca2+ ions into cells in response to depolarization and thereby contribute to regulate diverse biological events in a wide variety of tissues including nerves, glands and muscles. They are responsible for initiation of excitation-contraction and excitation-secretion coupling, and are involved in the regulation of protein phosphorylation and gene transcription, among many other intracellular events. The activity of CaV channels may be regulated by a number of cell surface receptors acting through G proteins as well as by protein phosphorylation and other post-translational modifications. Likewise, it is acknowledged that CaV channels are organized into active signaling platforms depending upon interactions with other molecules including cytoskeletal proteins. Diverse studies have shown that several cytoskeletal components may act as binding partners that help regulate, localize and determine cell surface expression of CaV channel in response to extracellular events. In this review, we survey the interaction of CaV channels with the cytoskeleton and its potential physiological implications.

Here we review the contribution of the various subtypes of voltage-activated calcium channels (VACCs) to the regulation of catecholamine release from chromaffin cells (CCs) at early life. Patch-clamp recording of inward currents through VACCs has revealed the expression of highthreshold VACCs (high-VACCs) of the L, N, and PQ subtypes in rat embryo CCs and ovine embryo CCs. Low-threshold VACC (low-VACC) currents (T-type) have also been recorded in rat embryo CCs and rat neonatal slices of adrenal medullae. Near full blockade by nifedipine and nimodipine of the K+-elicited secretion as well as the hypoxia induced secretion (HIS) supports the dominant role of L-VACC subtypes to the regulation of exocytosis at early life. Partial blockade by ω-conotoxin GVIA and ω-agatoxin IVA suggests a transient participation of N and PQ high-VACCs to the regulation of the HIS response at early stages of CC exposure to hypoxia. T-type low-VACC current did not elicit exocytosis triggered by electrical depolarising pulses applied to rat embryo CCs in one study, but largely contributed to the HIS response in neonatal rat adrenal slices in another. In spite of scarce available data, the sequence of events driving the HIS response in CCs at early life could be established as follows: (i) hypoxia blocks one or more K+ channels; (ii) as a consequence, mild membrane depolarisation occurs; (iii) T-type low-VACCs open at membrane potentials more hyperpolarised than those required to recruit the high-VACCs; (iv) firing of action potentials then occurs; (v) fast-inactivating N and PQ high-VACCs transiently open and low-inactivating L high-VACCs remain open along the hypoxia stimulus; (vi) increase of cytosolic Ca2+ takes place; and (vii) the exocytotic release of catecholamine occurs in two phases, an explosive initial phase, driven by Ca2+ entry through L, N and PQ channels, followed by a more sustained catecholamine release at a slower rate driven by L-type channels.

Calcium agonists such as FPL 64174 increase macroscopic calcium channel currents and induce substantial changes in current kinetics. Their molecular mechanism of action is currently unknown. Here we propose a technique enabling the estimation of FPL 64174 effects on rate constants of the voltage sensing machinery and pore transitions from macroscopic CaV1.2 current kinetics making use of a hybrid stochastic-deterministic optimization procedure. Current traces of wild type CaV1.2, a channel construct with neutralized segment IIS4 (IIS4N) and a pore mutant (A780T) were fitted to a circular four-state (rest, activated, open, deactivated) channel model in control and FPL 64174 (1 µM). The estimated rate constants provided novel insights in how structural elements of the voltage sensing unit and the channel pore influence the action of FPL 64174. The new approach may be applicable for the analysis of drug effects on other ion channels as well as for quantification of VS transitions and rate constants of pore gating from macroscopic current kinetics.

Alterations in dendritic spine morphology and postsynaptic structure are a hallmark of neurological disorders. Particularly spine pruning of striatal medium spiny neurons and aberrant rewiring of corticostriatal synapses have been associated with the pathology of Parkinson’s disease and LDOPA induced dyskinesia, respectively. Owing to its low activation threshold the neuronal L-type calcium channel CaV1.3 is particularly critical in the control of neuronal excitability and thus in the calcium-dependent regulation of neuronal functions. CaV1.3 channels are located in dendritic spines and contain a C-terminal class 1 PDZ domain-binding sequence. Until today the postsynaptic PDZ domain proteins shank, densin-180, and erbin have been shown to interact with CaV1.3 channels and to modulate their current properties. Interestingly experimental evidence suggests an involvement of all three PDZ proteins as well as CaV1.3 itself in regulating dendritic and postsynaptic morphology. Here we briefly review the importance of CaV1.3 and its proposed interactions with PDZ proteins for the stability of dendritic spines. With a special focus on the pathology associated with Parkinson’s disease, we discuss the hypothesis that CaV1.3 L-type calcium channels may be critical modulators of dendritic spine stability.

During the past decades, an increasing number of ion channel and transporter types have been identified acting together to produce cardiac and neuronal pacemaker action potentials. The basis of pacemaker activity was understood in more detail by using single-microelectrode recordings on cells isolated from pacemaker regions. Meanwhile, this powerful technique was complemented by computer modeling and recombinant technologies, including gene inactivation of ion channels and transporters, which may be involved in the generation of the electrical activity of pacemaker cells. Several genes of the voltage-gated Ca2+ channel (VGCC) family have been ablated, and their role in cardiac and neuronal pacemaking is compared in the present summary, focusing on the role of murine R-type voltage-gated Ca2+ channels encoded by cacna1e and expressing the ion conducting subunit Cav2.3.