Gabor Mikala

The L-type calcium channel in the heart: The beat goes on

Sydney Ringer would be overwhelmed today by the implications of his simple experiment performed over 120 years ago showing that the heart would not beat in the absence of Ca2+. Fascination with the role of Ca2+ has proliferated into all aspects of our understanding of normal cardiac function and the progression of heart disease, including induction of cardiac hypertrophy, heart failure, and sudden death. This review examines the role of Ca2+ and the L-type voltage-dependent Ca2+ channels in cardiac disease.

Molecular determinants of Ca2+ channel function and drug action

Molecular cloning has revealed the existence of six high-voltage activated Ca2+ channel types. Expression studies have shown that basic high-voltage activated channel function, which is typical for the L-(skeletal muscle, cardiac muscle and neuroendocrine tissue), N-, P-, Q- and R-type channels is carried by the corresponding alpha 1 subunits. Auxiliary subunits, such as alpha 2/delta and beta, modulate the kinetics of activation, inactivation, current density and drug binding, thereby creating considerable potential for multiple Ca2+ channel functions. Glutamic acid residues in the pore (P) loops are molecular components that impart high selectivity for Ca+. Binding or pharmacologically active sites for Ca2+ channel drugs have been localized on various segments of the alpha 1 subunit in close proximity to the pore lining. In this article, Gyula Varadi and colleagues review the roles of the different subunits in Ca2+ channel function and suggest that Ca2+ channel drugs act by blocking or, in some cases, activating channel function via binding directly or indirectly to the pore structure of the channel.

Targeted disruption of the voltage-dependent calcium channel α2/δ-1-subunit

Cardiac L-type voltage-dependent Ca(2+) channels are heteromultimeric polypeptide complexes of alpha(1)-, alpha(2)/delta-, and beta-subunits. The alpha(2)/delta-1-subunit possesses a stereoselective, high-affinity binding site for gabapentin, widely used to treat epilepsy and postherpetic neuralgic pain as well as sleep disorders. Mutations in alpha(2)/delta-subunits of voltage-dependent Ca(2+) channels have been associated with different diseases, including epilepsy. Multiple heterologous coexpression systems have been used to study the effects of the deletion of the alpha(2)/delta-1-subunit, but attempts at a conventional knockout animal model have been ineffective. We report the development of a viable conventional knockout mouse using a construct targeting exon 2 of alpha(2)/delta-1. While the deletion of the subunit is not lethal, these animals lack high-affinity gabapentin binding sites and demonstrate a significantly decreased basal myocardial contractility and relaxation and a decreased L-type Ca(2+) current peak current amplitude. This is a novel model for studying the function of the alpha(2)/delta-1-subunit and will be of importance in the development of new pharmacological therapies.

Cardiac-specific Overexpression of the α1 Subunit of the L-type Voltage-dependent Ca2+ Channel in Transgenic Mice LOSS OF ISOPROTERENOL-INDUCED CONTRACTION

The l-type voltage-dependent calcium channel (l-VDCC) regulates calcium influx in cardiac myocytes. Activation of the β-adrenergic receptor (βAR) pathway causes phosphorylation of thel-VDCC and that in turn increases Ca2+ influx. Targeted expression of the l-VDCC α1 subunit in transgenic (Tg) mouse ventricles resulted in marked blunting of the βAR pathway. Inotropic and lusitropic responses to isoproterenol and forskolin in Tg hearts were significantly reduced. Likewise, Ca2+ current augmentation induced by iso- proterenol and forskolin was markedly depressed in Tg cardiomyocytes. Despite no change in βAR number, isoproterenol-stimulated adenylyl cyclase activity was absent in Tg membranes and NaF and forskolin responses were reduced. We postulate an important pathway for regulation of the βAR by Ca2+ channels.

Auxiliary subunits operate as a molecular switch in determining gating behaviour of the unitary N‐type Ca2+ channel current in Xenopus oocytes

1 We systematically examined the biophysical properties of ω‐conotoxin GVIA‐sensitive neuronal N‐type channels composed of various combinations of the α1B, α2/δ and β1b subunits in Xenopus oocytes. 2 Whole‐cell recordings demonstrated that coexpression of the β1b subunit decelerated inactivation, whereas the α2/δ accelerated both activation and inactivation, and cancelled the kinetic effects of the β1b. The α2/δ and the β1b controlled voltage dependence of activation differently: the β1b significantly shifted the current‐voltage relationship towards the hyperpolarizing direction; however, the α2/δ shifted the relationship only slightly in the depolarizing direction. The extent of voltage‐dependent inactivation was modified solely by the β1b. 3 Unitary currents measured using a cell‐attached patch showed stable patterns of opening that were markedly different among subunit combinations in their kinetic parameters. The α2/δ and the β1b subunits also acted antagonistically in regulating gating patterns of unitary N‐type channels. Open time was shortened by the α2/δ, while the fraction of long opening was enhanced by the β1b. The α2/δ decreased opening probability (Po), while the β1b increased Po. α1Bα2/δβ1b produced unitary activity with an open time distribution value in between those of α1Bα2/δ and α1Bβ1b. However, both the α2/δ and the β1b subunits reduced the number of null traces. 4 These results suggest that the auxiliary subunits alone and in combination contribute differently in forming gating apparatuses in the N‐type channel, raising the possibility that subunit interaction contributes to the generation of functional diversity of N‐type channels in native neuronal preparations also.

Differential contribution by conserved glutamate residues to an ion-selectivity site in the L-type Ca2+ channel pore.

In voltage‐gated cation channels, it is thought that residues responsible for ion‐selectivity are located within the pore‐lining SS1‐SS2 segments. In this study, we compared the ion permeation properties of mutant calcium channels in which highly conserved glutamate residues, located at analogous positions in the SS2 regions of all four motifs, were individually replaced. All of the mutants exhibited a loss of selectivity for divalent over monovalent cations. However, the permeation properties of the individual mutants varied in a position dependent manner. The results provide strong evidence that these glutamate residues, positioned at equivalent locations in the aligned sequences, play significantly different roles in forming the selectivity barrier of the calcium channel, and are probably arranged in an asymmetrical manner inside the ion‐conducting pore.

Inhibition of DNA binding by the phosphorylation of poly ADP-ribose polymerase protein catalysed by protein kinase C

Purified type II (beta) and type III (alpha) protein kinase C phosphorylates highly purified polyADP-ribose polymerase in vitro whereby 2 mols of phosphate are transferred from ATP to serine and threonine residues present in the 36 and 56 kDa polypeptide domains of the polymerase protein. Calf thymus DNA was a non-competitive inhibitor of the protein kinase C catalyzed phosphorylation of polyADP-ribose polymerase. Coincidental with the phosphorylation of the protein the polymerase activity and DNA binding capacity of polyADP-ribose polymerase were inhibited. These in vitro findings may have possible cell biological significance in cellular signal transduction.

Effects of 2,3-butanedione monoxime (BDM) on calcium channels expressed in Xenopus oocytes

1 We examine the actions of a chemical phosphatase, 2,3‐butanedione monoxime (BDM), on endogenous and expressed Ca2+ channel currents in Xenopus oocytes. In previous studies on L‐type Ca2+ channel currents in cardiomyocytes and dorsal root ganglia, the inhibitory effects of BDM were attenuated by activation of protein kinase A. 2 Ba2+ currents (IBa) through a human wild‐type L‐type Ca2+ channel complex (i.e. hα1C, α2‐δa and hβ1b) are inhibited by BDM with an IC50 of 16 mM, with 10 mM producing a 36.1 ± 2.2 % inhibition. IBa through endogenous oocyte N‐type Ca2+ channels, upregulated by exogenous α2‐δa and hβ1b subunits, are inhibited to a similar degree by BDM. 3 To examine whether the action of BDM is dependent on PKA‐dependent phosphorylation, a clone of hα1C deficient in all five serine PKA consensus sites (hα1C‐SA5) was co‐expressed with α2‐δa and the human cardiac hβ3 subunit, which naturally lacks PKA consensus sites. This complex exhibited a sensitivity to BDM that was similar to the wild‐type complex, with 10 mM BDM producing 31.6 ± 1.5 % inhibition. 4 As limited proteolysis upregulates Ca2+ channels in cardiomyocytes and renders them less sensitive to BDM, experiments were performed with a carboxyl terminus deletion mutant, hα1C‐Δ1633. IBa through this subunit showed a sensitivity to BDM that was similar to the wild‐type complex, with 10 mM BDM producing 31.3 ± 1.4 % inhibition. However, co‐expression with α2‐δa and hβ3 subunits reduced potency, and is reflected by an increased IC50 of 22.7 mM. 5 The actions of BDM were examined on a rat brain rbA‐1 Ca2+ channel clone, α1A, co‐expressed with α2‐δb and β1b subunit homologues from rat brain. BDM inhibited the current through this channel complex to a similar degree to that seen for cardiac wild‐type channels, with 10 mM BDM causing a 33.1 ± 3.5 % inhibition. 6 The effects of BDM were compared at two holding potentials, ‐80 and ‐30 mV, using the hα1C‐Δ1633, α2‐δa and hβ3 subunit combination. At ‐30 mV BDM is more potent with 10 mM BDM reducing IBa by 39.8 ± 2.7 %, compared with 20.8 ± 2.2 % at ‐80 mV. 7 The data suggest that BDM may not exert its inhibitory action by means of a chemical phosphatase effect, but by channel block. The similar potency observed between α1C, α1A and endogenous (N‐type) channels may help point towards a possible site of action; differences with the carboxyl deletion mutant may help further to define a locus of interaction.

Single amino acid substitutions within the ion permeation pathway alter single-channel conductance of the human L-type cardiac Ca2+ channel.

Voltage-dependent L-type Ca2+ channels select for Ca2+ and other divalent cations by high-affinity Ca2+ binding and ion-ion interactions in the permeation pathway. We have recently identified a series of highly conserved glutamate residues, located within the SS2 segments of each of the four repeats of the human heart Ca2+ channel alpha 1 subunit, as major determinants of ion selectivity of the channel. To further investigate the functional role of these glutamate residues in ion permeation, we have individually neutralized the glutamic acids in repeats II and IV by substitution with alanine or glutamine. Wild-type and mutant Ca2+ channels were expressed in Xenopus oocytes. Apparent affinity for external Ca2+ was assessed by measuring the block by Ca2+ of inward Li+ currents through the Ca2+ channels. Mutations reducing net negative charge at these positions resulted in a 10-fold reduction in the affinity of the channel for external Ca2+. Single-channel conductance was measured with either divalent or monovalent cations as the charge carriers. Substitution of glutamic acid at position 677 with either alanine or glutamine increased single-channel conductance, whereas the same substitutions at position 1387 resulted in a decrease in single-channel conductance with Ba2+ as the charge carrier. In contrast, the unitary current amplitude carried by Na+, in the absence of external divalent cations, was not altered by these mutations. The results suggest that these conserved glutamate residues participate in high-affinity Ca2+ binding within the pore. The different effects on Ba2+ permeation indicate an asymmetric arrangement of the glutamate residues between repeats II and IV.(ABSTRACT TRUNCATED AT 250 WORDS)

cAMP-dependent phosphorylation sites and macroscopic activity of recombinant cardiac L-type calcium channels

The involvement of cAMP-dependent phosphorylation sites in establishing the basal activity of cardiac L-type Ca2+ channels was studied in HEK 293 cells transiently cotransfected with mutants of the human cardiac α1 and accessory subunits. Systematic individual or combined elimination of high consensus protein kinase A (PKA) sites, by serine to alanine substitutions at the amino and carboxyl termini of the α1 subunit, resulted in Ca2+ channel currents indistinguishable from those of wild type channels. Dihydropyridine (DHP)-binding characteristics were also unaltered. To explore the possible involvement of nonconsensus sites, deletion mutants were used. Carboxyl-terminal truncations of the α1 subunit distal to residue 1597 resulted in increased channel expression and current amplitudes. Modulation of PKA activity in cells transfected with the wild type channel or any of the mutants did not alter Ca2+ channel functions suggesting that cardiac Ca2+ channels expressed in these cells behave, in terms of lack of PKA control, like Ca2+ channels of smooth muscle cells.