CA Isoforms Research Articles

Carbonic Anhydrase Inhibitors: Design of Membrane‐Impermeant Copper(II) Complexes of DTPA‐, DOTA‐, and TETA‐Tailed Sulfonamides Targeting the Tumor‐Associated Transmembrane Isoform IX

The synthesis and carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity of two series of aromatic sulfonamides and their Cu(II) derivatives, incorporating metal-complexing moieties of the DTPA, DOTA, and TETA type are reported. The new compounds were designed in such a way as to possess high affinity for Cu(II) ions, exploiting four pendant carboxylate moieties in the DTPA derivatives, as well as the cyclen/cyclam macrocyles, and three pendant acetate moieties in the DOTA and TETA derivatives. The new derivatives showed modest inhibition of the cytosolic isoform CA I (K(I) values in the range of 66-2130 nM), were better CA II inhibitors (K(I) values in the range of 21-360 nM), and excellent inhibitors of the tumor-associated isoform CA IX (K(I) values in the range of 4.1-110 nM), with selectivity ratios for the inhibition of the tumor (CA IX) over the cytosolic (CA II) isozyme in the range of 10.74-20.88 for the best derivatives. Copper complexes were more inhibitory than the corresponding ligand sulfonamides, and showed membrane impermeability, thus, having the possibility to specifically target the transmembrane CA IX that has an extracellular active site. Incorporation of radioactive copper isotopes in this type of CA inhibitor may lead to interesting diagnostic/therapeutic applications for such compounds.

Effect of egg turning and incubation time on carbonic anhydrase gene expression in the blastoderm of the Japanese quail ( Coturnix c. japonica )

1. The gene expression of carbonic anhydrase, a key enzyme for the production of sub-embryonic fluid (SEF), was assessed in turned and unturned eggs of the Japanese quail. The plasma membrane-associated isoforms CA IV, CA IX, CA XII, CA XIV, and the cytoplasmic isoform CA II, were investigated in the extra-embryonic tissue of the blastoderm and in embryonic blood. 2. Eggs were incubated at 37·6°C, c.60% RH, and turned hourly (90°) or left unturned. From 48 to 96 h of incubation mRNA was extracted from blastoderm tissue, reverse-transcribed to cDNA and quantified by real-time qPCR using gene-specific primers. Blood collected at 96 h was processed identically. 3. Blastoderm CA IV gene expression increased with the period of incubation only in turned eggs, with maxima at 84 and 96 h of incubation. Only very low levels were found in blood. 4. Blastoderm CA II gene expression was greatest at 48 and 54 h of incubation, subsequently declining to much lower levels and unaffected by turning. Blood CA II gene expression was about 25-fold greater than in the blastoderm. 5. The expression of CA IX in the blastoderm was the highest of all isoforms, yet unaffected by turning. CA XII did not amplify and CA XIV was present at unquantifiable low levels. 6. It is concluded that only gene expression for CA IV is sensitive to egg turning, and that increased CA IV gene expression could account for the additional SEF mass found at 84 to 96 h of incubation in embryos of turned eggs.

Identification and characterization of a novel, shorter isoform of the small conductance Ca2+‐activated K+ channel SK2

Throughout the CNS, small conductance Ca(2+)-activated potassium (SK) channels modulate firing frequency and neuronal excitability. We have identified a novel, shorter isoform of standard SK2 (SK2-std) in mouse brain which we named SK2-sh. SK2-sh is alternatively spliced at exon 3 and therefore lacks 140 amino acids, which include transmembrane domains S3, S4 and S5, compared with SK2-std. Western blot analysis of mouse hippocampal tissue revealed a 47 kDa protein product as predicted for SK2-sh along with a 64 kDa band representing the standard SK2 isoform. Electrophysiological recordings from transiently expressed SK2-sh revealed no functional channel activity or interaction with SK2-std. With the help of real-time PCR, we found significantly higher expression levels of SK2-sh mRNA in cortical tissue from AD cases when compared with age-matched controls. A similar increase in SK2-sh expression was induced in cortical neurons from mice by cytokine exposure. Substantial clinical evidence suggests that excess cytokines are centrally involved in the pathogenesis of Alzheimer's disease. Thus, SK2-sh as a downstream target of cytokines, provide a promising target for additional investigation regarding potential therapeutic intervention.

Domain structure responsible for the different properties between α and β Ca 2+/calmodulin-dependent protein kinase II analyzed by their chimera enzymes

To understand the domain structure responsible for different enzymatic properties, we constructed chimera cDNAs of the α and β isoforms of Ca 2+/calmodulin-dependent protein kinase II (CaM kinase II). The chimera DNAs were expressed in neuroblastoma cells, and the affinity for calmodulin and the subcellular localization of chimera enzymes were investigated. Here, we found that the region in immediately N-terminal of the calmodulin-binding site (exons 10 and 11), including the autophosphorylation site, mainly affected the affinity of each isoform for calmodulin and that the N-terminal region (exons 1 and 2), including the ATP-binding site, modified the affinity for calmodulin of each isoform. It was confirmed that the association of β CaM kinase II with the particulate fraction was determined by β-specific insertion, and also found that the association with the particulate fraction was modified by exons 1 and 2 of each isoform. Kinases without β-specific insertion and chimera kinases consisting of exons 1 and 2 of β and other regions of α appeared reduced in the transport of kinases to neurite. These results indicated that the structure of exons 10 and 11 and exons 1 and 2 modified the properties of CaM kinase II holoenzyme.

Crystal structure of human carbonic anhydrase XIII and its complex with the inhibitor acetazolamide

The cytosolic isoform XIII is a recently discovered member of the human carbonic anhydrase (hCA, EC 4.2.1.1) family. It is selectively expressed among other tissues in the reproductive organs, where it may control pH and ion balance regulation, ensuring thus proper fertilization conditions. The authors report here the X-ray crystallographic structure of this isozyme in the unbound state and in complex with a classical sulfonamide inhibitor, namely acetazolamide. A detailed comparison of the obtained structural data with those already reported for other CA isozymes provides novel insights into the catalytic properties of the members of this protein family. On the basis of the inhibitory properties of acetazolamide against various cytosolic/transmembrane isoforms and the structural differences detected within the active site of the various CA isoforms, further prospects for the design of isozyme-specific CA inhibitors are here proposed.

Dysregulation of large-conductance Ca2+-activated K+ channel expression in nonsyndromal mental retardation due to a cereblon p.R419X mutation

A nonsense mutation (R419X) in the human cereblon gene [mutation (mut) CRBN] causes a mild type of autosomal recessive nonsyndromal mental retardation (ARNSMR). CRBN, a cytosolic protein, regulates the assembly and neuronal surface expression of large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) in brain regions involved in memory and learning. Using the real-time quantitative polymerase chain reaction, we show that mut CRBN disturbs the development of adult brain BK(Ca) isoforms. These changes are predicted to result in BK(Ca) channels with a higher intracellular Ca(2+) sensitivity, faster activation, and slower deactivation kinetics. Such alterations may contribute to cognitive impairments in patients with mild ARNSMR.

Carbonic anhydrase activators: Activation of the human cytosolic isozyme III and membrane-associated isoform IV with amino acids and amines

An activation study of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA III (cytosolic) and IV (membrane-associated) with a series of natural and non-natural amino acids and aromatic/heterocyclic amines is reported. hCA III was efficiently activated by d-His, serotonin, pyridyl-alkylamines, and aminoethyl-piperazine/morpholine ( K As of 91nM–1.12 μM), whereas the best hCA IV activators were 4-amino-phenylalanine, serotonin, and 4-(2-aminoethyl)-morpholine ( K As of 79 nM–3.14 μM). Precise steric and electronic requirements are needed to be present in the molecules of effective CA III/IV activators, in order to assure an adequate fit within the enzyme active site for the formation of the enzyme-activator complex, and for efficient proton transfer processes between the active site and the reaction medium. The activation profiles of CA III and IV are distinct from those of all other mammalian CA isoforms investigated so far for their interaction with amino acids and amines.

Carbonic anhydrase inhibitors: Inhibition of the new membrane-associated isoform XV with phenols

Inhibition of the newest isoform of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), CA XV, with a series of phenols was investigated. Murine CA XV showed an inhibition profile by phenols distinct of those of the cytosolic human isoforms CA I and II. Phenol and some of its 2-, 3-, and 4-substituted derivatives incorporating hydroxy, fluoro, carboxy, and acetamido moieties were effective CA XV inhibitors, with inhibition constants in the range of 7.20–11.30 μM, whereas compounds incorporating 4-amino-, 4-cyano, or 3-hydroxy groups were less effective ( K Is of 335–434 μM). The best phenol inhibitor was clioquinol ( K I of 2.33 μM). Phenols show a different inhibition mechanism as compared to sulfonamides and their isosteres, and may lead to the design of compounds with selectivity for inhibiting different CA isozymes with medicinal chemistry applications.

The Caenorhabditis elegans AMP-activated Protein Kinase AAK-2 Is Phosphorylated by LKB1 and Is Required for Resistance to Oxidative Stress and for Normal Motility and Foraging Behavior

AAK-2 is one of two alpha isoforms of the AMP-activated protein kinase in Caenorhabditis elegans and is involved in life span maintenance, stress responses, and germ cell cycle arrest upon dauer entry. We found that AAK-2 was phosphorylated at threonine 243 in response to paraquat treatment and that this phosphorylation depends on PAR-4, the C. elegans LKB1 homologue. Both aak-2 mutation and par-4 knockdown increased the sensitivity of C. elegans worms to paraquat, and the double deficiency did not further increase sensitivity, indicating that aak-2 and par-4 act in a linear pathway. Both mutations also slowed body bending during locomotion and failed to reduce head oscillation in response to anterior touch. Consistent with this abnormal motility and behavioral response, expression of the AAK-2::green fluorescent protein fusion protein was observed in the ventral cord, some neurons, body wall muscle, pharynx, vulva, somatic gonad, and excretory cell. Our study suggests that AMPK can influence the behavior of C. elegans worms in addition to its well known function in metabolic control.

Selective blockade of T-type Ca 2+ channels suppresses human breast cancer cell proliferation

We have measured the expression of T-type Ca 2+ channel mRNA in breast cancer cell lines (MCF-7 (ERα+) using Western blot and quantitative real-time PCR (Q-RT-PCR). These results revealed that the MCF-7 cells express both α 1G and α 1H isoforms of T-type Ca 2+ channels. In order to further clarify the role of T-type Ca 2+ channels in proliferation, we tested the effects of a selective T-type Ca 2+ channel inhibitor NNC-55-0396 on cellular proliferation. MCF-7 (ERα+) cellular proliferation was inhibited by the compound. In contrast, NNC-55-0396 at same concentration had no effect on the proliferation of MCF-10A cells, a non-cancer breast epithelial cell line. We also found that message expression of the T-type Ca 2+ channels were only expressed in rapidly growing non-confluent cells but not in the cytostatic confluent cells. Knocking down the expression of T-type Ca 2+ channels with siRNA targeting both α 1G and α 1H resulted in growth inhibition as much as 45% ± 5.0 in MCF-7 cells as compared to controls. In conclusion, our results suggest that T-type Ca 2+ channel antagonism/silencing may reduce cellular proliferation in mitogenic breast cells.

Synthesis and biological evaluation of a new family of anti-benzylanilinosulfonamides as CA IX inhibitors

We report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors prepared regio- and stereoselectively by reacting sulfanilamide with ethyl trans-phenylglycidate in the presence of cobalt(II) chloride. Various derivatizations of the ester moiety in the parent compound led to a small library of derivatives (2 R,3 R and 2 S,3 S) which displayed interesting inhibitory activities towards the human tumor-associated isoform CA IX. One of the new compounds shows high selectivity in inhibiting hCA IX compared to the two physiologically relevant, cytosolic isozymes hCA I and hCA II. A molecular modeling study was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the active sites of hCA IX and hCA II.

Identification of an alternatively spliced isoform of carbonic anhydrase XII in diffusely infiltrating astrocytic gliomas

Carbonic anhydrase XII (CA XII) is a transmembrane enzyme that is associated with neoplastic growth. CA XII has been proposed to be involved in acidification of the extracellular milieu, creating an appropriate microenvironment for rapid tumor growth. Because RNA sequence databases have indicated that two isoforms of CA XII might exist in human tissues, and because alternatively spliced protein forms have been linked to aggressive behavior of cancer cells, we designed a study to evaluate the presence of the two forms of CA XII in diffuse astrocytomas, a tumor type known for its aggressive and often noncurable behavior. Reverse transcription PCR of tumor samples surprisingly revealed that CA XII present in diffuse astrocytomas is mainly encoded by a shorter mRNA variant. We further showed by Western blotting that anti-CA XII antibody recognized both isoforms in the glioblastoma cell lines, and we then evaluated the expression of CA XII in astrocytomas using immunohistochemistry and correlated the results with various clinicopathological and molecular factors. Of 370 diffusely infiltrating astrocytomas, 363 cases (98%) showed immunoreactions for CA XII. Importantly, CA XII expression correlated with poorer patient prognosis in univariate (p = 0.010, log-rank test) and multivariate survival analyses (p = 0.039, Cox analysis). From these results, we conclude that CA XII is commonly expressed in diffuse astrocytomas and that it might be used as a biomarker of poor prognosis. The absence of 11 amino acids in the shorter isoform, which seems to be common in astrocytomas, may affect the normal quaternary structure and biological function of CA XII.

Carbonic anhydrase inhibitors: The X-ray crystal structure of ethoxzolamide complexed to human isoform II reveals the importance of thr200 and gln92 for obtaining tight-binding inhibitors

Ethoxzolamide, an almost forgotten inhibitor of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), is the only classical inhibitor whose structure in adduct with any isoform was not reported yet. We report here the inhibition data of this molecule with the 12 catalytically active mammalian isozymes (CA I-CA XIV) and the X-ray crystal structure with the cytosolic, ubiquitous isoform CA II. These data are presumably useful for the design of novel CA inhibitors, targeting various CA isozymes, considering that ethoxzolamide was already the lead molecule to obtain the second generation inhibitors, dorzolamide and brinzolamide, clinically used antiglaucoma agents with topical action, as well as various other investigational agents.

Carbonic Anhydrases An Overview

Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread metalloenzymes all over the phylogenetic tree, with at least 4 distinct gene families encoding for them. At least 16 different alpha-CA isoforms were isolated in mammals, where these enzymes play crucial physiological roles. Representatives of the beta-delta-CA family are highly abundant in plants, diatoms, eubacteria and archaea. These enzymes are efficient catalysts for the reversible hydration of carbon dioxide to bicarbonate, but at least the alpha-CAs possess a high versatility, being able to catalyze different other hydrolytic processes The catalytic mechanism of the alpha-CAs is understood in detail: the active site consists of a Zn(II) ion co-ordinated by three histidine residues and a water molecule/hydroxide ion. The latter is the active species, acting as a potent nucleophile. For beta- and gamma-CAs, the zinc hydroxide mechanism is valid too, although at least some beta-class enzymes do not have water directly coordinated to the metal ion. CAs are inhibited by two classes of compounds: the metal complexing anions and the sulfonamides and their isosteres (sulfamates, sulfamides etc.) possessing the general formula RXSO(2)NH(2) (R = aryl; hetaryl; perhaloalkyl; X = nothing, O or NH). At least 25 clinically used drugs/agents in clinical development show applications as diuretics and antiglaucoma drugs, anticonvulsants, with some compounds being developed as anticancer agents/diagnostic tools for tumors, antiobesity agents, and antimicrobials/antifungals (inhibitors targeting CAs from pathogenic organisms such as Helicobacter pylori, Mycobacterium tuberculosis, Plasmodium falciparum, Candida albicans, etc). Several important physiological and physio-pathological functions are played by CA isozymes present in organisms all over the phylogenetic tree, related to respiration and transport of CO(2)/bicarbonate between metabolizing tissues and the lungs, pH and CO(2) homeostasis, electrolyte secretion in a variety of tissues/organs, biosynthetic reactions, such as the gluconeogenesis and ureagenesis among others (in animals), CO(2) fixation (in plants and algae), etc. The presence of these ubiquitous enzymes in so many tissues and in so different isoforms, represents an attractive goal for the design of inhibitors or activators with biomedical applications.

The Development of Topically Acting Carbonic Anhydrase Inhibitors as Antiglaucoma Agents

Inhibition of carbonic anhydrase (CA, EC 4.2.1.1) isoforms present in the eyes (CA I, II, IV and XII), with sulfonamides such as acetazolamide, methazolamide, ethoxzolamide and dichlorophenamide, is still widely used for the systemic treatment of glaucoma. The mechanism of action of these drugs consists in inhibition of CA isozymes present in ciliary processes of the eye, with the consequent reduction of bicarbonate and aqueous humour secretion, and of elevated intraocular pressure (IOP) characteristic of this disease. As isoforms CA II/IV/XII are present in many other tissues/organs, generally, systemic CAIs possess undesired side effects such as numbness and tingling of extremities; metallic taste; depression; fatigue; malaise; weight loss; decreased libido; gastrointestinal irritation; metabolic acidosis; renal calculi and transient myopia. For avoiding these side effects, recently, topically effective CAIs have been developed in the last 10 years, with two drugs available clinically: dorzolamide and brinzolamide. Both these drugs are applied topically as water solutions/suspensions, alone or in combination with other agents (beta-blockers, prostaglandin derivatives, etc) and produce a consistent and prolonged reduction of IOP. Furthermore, recent reports show both the systemically as well as topically acting sulfonamide CAIs to be effective in the treatment of macular edema, macular degeneration disease, or diabetic retinopathy, for which pharmacological treatment is unavailable up to now. Much research is in act in the search of more effective topically acting CAIs, free of the inconveniences and side effects of the presently available drugs. For achieving this goal, two recently reported strategy, the tail approach and its variant, the sugar-tail approach, were extensively applied for the synthesis of large numbers of derivatives possessing desired physico-chemical properties. Many such new sulfonamides showed promising antiglaucoma activity in animal models of the disease.

Diuretics: From Classical Carbonic Anhydrase Inhibitors to Novel Applications of the Sulfonamides

The widely clinically used benzothiadiazines and high ceiling diuretics, such as hydrochlorothiazide, hydroflumethiazide, quinethazone, metolazone, chlorthalidone, indapamide, furosemide and bumetanide, contain SO(2)NH(2) moieties acting as an effective zinc-binding function in carbonic anhydrases (CAs, EC 4.2.1.1) inhibitors. These drugs were launched in a period when only isoform CA II was known and considered physiologically/pharmacologically relevant. Although acting as moderate-weak inhibitors of CA II, all these drugs considerably inhibit other CA isozymes known nowadays to be involved in critical physiologic processes, among the 16 CAs present in vertebrates. Some low nanomolar (or even subnanomolar) inhibitors against such isoforms were recently detected, such as metholazone against CA VII, XII and XIII, chlorthalidone against CA VB, VII, IX, XII and XIII, indapamide against CA VII, IX, XII and XIII, furosemide against CA I, II and XIV, and bumethanide against CA IX and XII. The X-ray crystal structure of the CA II-indapamide adduct was also reported recently, revealing interesting aspects useful for the drug design of CA inhibitors. It has also been proposed that the recently observed beneficial effect of indapamide for the treatment of patients with hypertension and type 2 diabetes might be due to its potent inhibition of CA isoforms present in kidneys and blood vessels, which would thus explain both the blood pressure lowering effects as well as organ-protective activity of the drug. Thus, these old drugs may be useful as leads for new applications.

Indanesulfonamides as carbonic anhydrase inhibitors and anticonvulsant agents: Structure–activity relationship and pharmacological evaluation

A small library of indanesulfonamides was screened for the inhibition of the human carbonic anhydrase (CA, EC 4.2.1.1) isoforms involved in neuronal excitation, that is, isoforms VII, XII and XIV. These CA isoforms are becoming interesting target for the design of agents useful for the treatment of epilepsy. The inhibition pattern of these indanesulfonamide compounds towards these three isoforms was excellent, with many nanomolar inhibitors detected ( K Is in the range of 0.78–10 nM against hCA VII; 0.32–56 nM against hCA XII, and 0.47–1030 nM against hCA XIV, respectively). The maximal electroshock seizure (MES) test performed on mice showed a good anticonvulsant activity for some compounds which protected the mice against convulsions in the 50–62.5% range at a dose of 50 mg/kg. In parallel, the blood–brain barrier passive permeation of these sulfonamides was also estimated by using a computational approach.

Carbonic anhydrase activators: Activation of the human tumor-associated isozymes IX and XII with amino acids and amines

The first activation study of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms associated to tumors, hCA IX and XII, with a small library of natural and non-natural amino acids as well as aromatic/heterocyclic amines is reported. hCA IX was activated efficiently by dopamine, adrenaline and heterocyclic amines possessing aminoethyl-/aminomethyl-moieties ( K As of 9 nM–1.07 μM), whereas the best hCA XII activators were serotonin, l-adrenaline, 4-(2-aminoethyl)-morpholine and d-Phe ( K A of 0.24–0.41 μM). Precise steric and electronic requirements are needed to be present in the molecules of effective hCA IX/hCA XII activators, in order to assure an adequate fit within the enzyme active site cavity for the formation of the enzyme–activator complex, and for an efficient proton transfer process within this complex, leading to the release of a proton and formation of the catalytically active, zinc-hydroxide species of the enzyme. Selective activation of these CA isoforms might be useful to develop pharmacologic tools or to understand whether some of these biogenic amines/amino acids may influence the progression of tumors overexpressing CA IX and/or CA XII.

Carbonic anhydrase inhibitors. Sulfonamide diuretics revisited—old leads for new applications?

Sulfonamide diuretics such as hydrochlorothiazide, hydroflumethiazide, quinethazone, metolazone, chlorthalidone, indapamide, furosemide and bumetanide were tested as inhibitors of the zinc enzyme carbonic anhydrases (CAs, EC 4.2.1.1). These drugs were discovered in a period when only isoform CA II was known and considered physiologically/pharmacologically relevant. We prove here that although acting as moderate to weak inhibitors of CA II, all these drugs considerably inhibit other isozymes known nowadays to be involved in critical physiologic processes, among the 16 CAs present in vertebrates. Some low nanomolar/subnanomolar inhibitors against such isoforms were detected, such as among others metolazone against CA VII, XII and XIII, chlorthalidone against CA VB, VII, IX, XII and XIII, indapamide against CA VII, IX, XII and XIII, furosemide against CA I, II and XIV, and bumethanide against CA IX and XII. The X-ray crystal structure of the CA II-indapamide adduct was also resolved at high resolution, and the binding of this sulfonamide to the enzyme was compared to that of dichlorophenamide, sulpiride and a pyridinium containing sulfonamide. Indapamide binds to CA II in a manner not seen earlier for any other CA inhibitor, which might be important for the design of compounds with a different inhibition profile.

A Smooth Muscle Cav1.2 Calcium Channel Splice Variant Underlies Hyperpolarized Window Current and Enhanced State-dependent Inhibition by Nifedipine

Native smooth muscle L-type Ca(v)1.2 calcium channels have been shown to support a fraction of Ca(2+) currents with a window current that is close to resting potential. The smooth muscle L-type Ca(2+) channels are also more susceptible to inhibition by dihydropyridines (DHPs) than the cardiac channels. It was hypothesized that smooth muscle Ca(v)1.2 channels exhibiting hyperpolarized shift in steady-state inactivation would contribute to larger inhibition by DHP, in addition to structural differences of the channels generated by alternative splicing that modulate DHP sensitivities. In addition, it has also been shown that alternative splicing modulates DHP sensitivities by generating structural differences in the Ca(v)1.2 channels. Here, we report a smooth muscle L-type Ca(v)1.2 calcium channel splice variant, Ca(v)1.2SM (1/8/9(*)/32/Delta33), that when expressed in HEK 293 cells display hyperpolarized shifts for steady-state inactivation and activation potentials when compared with the established Ca(v)1.2b clone (1/8/9(*)/32/33). This variant activates from more negative potentials and generates a window current closer to resting membrane potential. We also identified the predominant cardiac isoform Ca(v)1.2CM clone (1a/8a/Delta9(*)/32/33) that is different from the established Ca(v)1.2a (1a/8a/Delta9(*)/31/33). Importantly, Ca(v)1.2SM channels were shown to be more sensitive to nifedipine blockade than Ca(v)1.2b and cardiac Ca(v)1.2CM channels when currents were recorded in either 5 mM Ba(2+) or 1.8 mM Ca(2+) external solutions. This is the first time that a smooth muscle Ca(v)1.2 splice variant has been identified functionally to possess biophysical property that can be linked to enhanced state-dependent block by DHP.