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Voltage-operated Ca[sup 2+] channels are heteromultimeric proteins. Their structural diversity is caused by several genes encoding homologous subunits and by alternative splicing of single transcripts. Isoforms of α[sub 1] subunits, which contain the ion conducting pore, have been deduced from each of the six cDNA sequences cloned so far from different species. The isoforms predicted for the α[sub 1E] subunit are structurally related to the primary sequence of the amino terminus, the centre of the subunit (II–III loop), and the carboxy terminus. Mouse and human α[sub 1E] transcripts have been analysed by reverse transcription–polymerase chain reaction and by sequencing of amplified fragments. For the II–III loop three different α[sub 1E] cDNA fragments are amplified from mouse and human brain, showing that isoforms originally predicted from sequence alignment of different species are expressed in a single one. Both predicted α[sub 1E] cDNA fragments of the carboxy terminus are identified in vivo. Two different α[sub 1E] constructs, referring to the major structural difference in the carboxy terminus, were stably transfected in HEK293 cells. The biophysical properties of these cells were compared in order to evaluate the importance in vitro of the carboxy terminal insertion found in vivo. The wild-type α[sub 1E] subunit showed properties, typical for a high-voltage activated Ca[sup 2+] channel. The deletion of 43 amino acid residues at the carboxy terminus does not cause significant differences in the current density and the basic biophysical properties. However, a functional difference is suggested, as in embryonic stem cells, differentiated in vitro to neuronal cells, the pattern of transcripts indicative for different α[sub 1E] isoforms changes during development. In human cerebellum the longer α[sub 1E] isoform is expressed predominantly. Although, it has not been possible to ass... [ABSTRACT FROM AUTHOR]

We describe the development of an arenophile-mediated, nickel-catalyzed dearomative trans-1,2-carboamination protocol. A range of readily available aromatic compounds was converted to the corresponding dienes using Grignard reagents as nucleophiles. This strategy provided products with exclusive trans-selectivity and high enantioselectivity was observed in case of benzene and naphthalene. The utility of this methodology was showcased by controlled and stereoselective preparation of small, functionalized molecules.

A concise synthesis of (+)-pancratistatin and (+)-7-deoxypancratistatin from benzene using an enantioselective, dearomative carboamination strategy has been achieved. This approach, in combination with the judicious choice of subsequent olefin-type difunctionalization reactions, permits rapid and controlled access to a hexasubstituted core. Finally, minimal use of intermediary steps as well as direct, late stage C-7 hydroxylation provides both natural products in six and seven operations.

Nitric oxide (NO) modulates calcium transients and contraction of cardiomyocytes. However, it is largely unknown whether NO contributes also to alterations in the contractile function of cardiomyocytes during aging. Therefore, we analyzed the putative role of nitric oxide synthases and NO for the age-related alterations of cardiomyocyte contraction. We used C57BL/6 mice, nitric oxide synthase 1 (NOS1)-deficient mice (NOS1(-/-)) and mice with cardiomyocyte-specific NOS1-overexpression to analyze contractions, calcium transients (Indo-1 fluorescence), acto-myosin ATPase activity (malachite green assay), NADPH oxidase activity (lucigenin chemiluminescence) of isolated ventricular myocytes and cardiac gene expression (Western blots, qPCR). In C57BL/6 mice, cardiac expression of NOS1 was upregulated by aging. Since we found a negative regulation of NOS1 expression by cAMP in isolated cardiomyocytes, we suggest that reduced efficacy of β-adrenergic signaling that is evident in aged hearts promotes upregulation of NOS1. Shortening and relengthening of cardiomyocytes from aged C57BL/6 mice were decelerated, but were normalized by pharmacological inhibition of NOS1/NO. Cardiomyocytes from NOS1(-/-) mice displayed no age-related changes in contraction, calcium transients or acto-myosin ATPase activity. Aging increased cardiac expression of NADPH oxidase subunits NOX2 and NOX4 in C57BL/6 mice, but not in NOS1(-/-) mice. Similarly, cardiac expression of NOX2 and NOX4 was upregulated in a murine model with cardiomyocyte-specific overexpression of NOS1. We conclude that age-dependently upregulated NOS1, putatively via reduced efficacy of β-adrenergic signaling, induces NADPH oxidases. By increasing nitrosative and oxidative stress, both enzyme systems act synergistically to decelerate contraction of aged cardiomyocytes.

In critically ill patients regulation of heart-rate is often severely disturbed. Interaction of bacterial endotoxin (lipopolysaccharide, LPS) with hyperpolarization-activated cyclic nucleotide-gated cation-(HCN)-channels may interfere with heart-rate regulation. This study analyzes the effect of LPS, the HCN-channel blocker ivabradine or Ca(2+) -channel blockers (nifedipine, verapamil) on pacemaking in spontaneously beating neonatal rat cardiomyocytes (CM) in vitro. In vivo, the effect of LPS on the heart-rate of adult CD1-mice with and without autonomic blockade is analyzed telemetrically. LPS (100 ng/mL) and ivabradine (5 μg/mL) reduced the beating-rate of CM by 20.1% and 24.6%, respectively. Coincubation of CM with both, LPS and ivabradine, did not further reduce the beating-rate, indicating interaction of both compounds with HCN-channels, while coincubation with Ca(2+) -channel blockers and LPS caused additive beating-rate reduction. In CD1-mice (containing an active autonomic-nervous-system), injection of LPS (0.4 mg/kg) expectedly resulted in increased heart-rate. However, if the autonomic nervous system was blocked by propranolol and atropine, in line with the in vitro data, LPS induced a significant reduction of heart-rate, which was not additive to ivabradine. The in vivo and in vitro results indicate that LPS interacts with HCN-channels of cardiomyocytes. Thus, LPS indirectly sensitizes HCN-channels for sympathetic activation (tachycardic-effect), and in parallel directly inhibits channel activity (bradycardic-effect). Both effects may contribute to the detrimental effects of septic cardiomyopathy and septic autonomic dysfunction., (© 2015 Wiley Publishing Asia Pty Ltd.)

Depressed heart rate variability in severe inflammatory diseases can be partially explained by the lipopolysaccharide (LPS)-dependent modulation of cardiac pacemaker channels. Recently, we showed that LPS inhibits pacemaker current in sinoatrial node cells and in HEK293 cells expressing cloned pacemaker channels, respectively. The present study was designed to verify whether this inhibition involves LPS-dependent intracellular signalling and to identify structures of LPS responsible for pacemaker current modulation. We examined the effect of LPS on the activity of human hyperpolarization-activated cyclic nucleotide-gated channel 2 (hHCN2) stably expressed in HEK293 cells. In whole-cell recordings, bath application of LPS decreased pacemaker current (IhHCN2) amplitude. The same protocol had no effect on channel activity in cell-attached patch recordings, in which channels are protected from the LPS-containing bath solution. This demonstrates that LPS must interact directly with or close to the channel protein. After cleavage of LPS into lipid A and the polysaccharide chain, neither of them alone impaired IhHCN2, which suggests that modulation of channel activity critically depends on the integrity of the entire LPS molecule. We furthermore showed that β-cyclodextrin interfered with LPS-dependent channel modulation predominantly via scavenging of lipid A, thereby abrogating the capability of LPS to intercalate into target cell membranes. We conclude that LPS impairs IhHCN2 by a local mechanism that is restricted to the vicinity of the channels. Furthermore, intercalation of lipid A into target cell membranes is a prerequisite for the inhibition that is suggested to depend on the direct interaction of the LPS polysaccharide chain with cardiac pacemaker channels.

Recently it was shown that lipopolysaccharide (LPS) impairs the pacemaker current in human atrial myocytes. It was speculated that reduced heart rate variability (HRV), typical of patients with severe sepsis, may partially be explained by this impairment. We evaluated the effect of various types of LPS on the activity of human hyperpolarization-activated cyclic nucleotide-gated channel 2 (hHCN2) expressed in HEK293 cells, and on pacemaker channels in native murine sino-atrial node (SAN) cells, in order to determine the structure of LPS necessary to modulate pacemaker channel function. Application of LPS caused a robust inhibition of hHCN2-mediated current (I(hHCN2)) owing to a negative shift of the voltage dependence of current activation and to a reduced maximal conductance. In addition, kinetics of channel gating were modulated by LPS. Pro-inflammatory LPS-types lacking the O-chain did not reduce I(hHCN2), whereas pro-inflammatory LPS-types containing the O-chain reduced I(hHCN2). On the other hand, a detoxified LPS without inflammatory activity, but containing the O-chain reduced I(hHCN2). Similar observations were made in HEK293 cells expressing hHCN4 and in murine SAN cells. This mechanistic analysis showed the novel finding that the O-chain of LPS is required for reduction of HCN channel activity. In the clinical situation the observed modulation of HCN channels may slow down diastolic depolarization of pacemaker cells and, hence, influence heart rate variability and heart rate., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Since aging increases oxidative stress, we analyzed the contribution of reactive oxygen species (ROS) to the contractile dysfunction of aged ventricular myocytes and investigated whether short-term interference with ROS formation could normalize contractile performance. Isolated ventricular myocytes from young (2-4 months) and aged (24-26 months) male mice (C57BL/6) were used. We analyzed sarcomere shortening and calcium transients (Indo-1 fluorescence) of voltage clamped ventricular myocytes and myofilament ATPase activity (malachite green assay). Expression of calcium handling proteins (Western blots) and NADPH oxidase subunits (real-time PCR) was quantified, as well as NADPH oxidase activity (lucigenin chemiluminescence). We found that aged myocytes showed decelerated shortening/relengthening without changes in fractional shortening. Calcium transient decay was similarly decelerated, but the amplitude of calcium transients was increased with aging. Calcium sensitivity of myofilaments of aged myocytes was reduced. These age-dependent changes occurred without altered calcium handling protein expression but were reversed by the superoxide scavenger tiron. Aged myocytes showed increased NADPH oxidase expression and activity. Pharmacological inhibition of NADPH oxidase (diphenylene iodonium; apocynin) normalized age-dependent deceleration of shortening/relengthening. In summary, we show that increased superoxide formation by upregulated NADPH oxidase contributes significantly to age-dependent alterations in calcium handling and contractility of murine ventricular myocytes., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Serotonin (5-HT) exerts pleiotropic effects in the human cardiovascular system. Some of the effects are thought to be mediated via 5-HT(4) receptors, which are expressed in the human atrium and in ventricular tissue. However, a true animal model to study these receptors in more detail has been hitherto lacking. Therefore, we generated, for the first time, a transgenic (TG) mouse with cardiac myocyte-specific expression of the human 5-HT(4) receptor. RT-PCR and immunohistochemistry revealed expression of the receptor at the mRNA and protein levels. Stimulation of isolated cardiac preparations by isoproterenol increased phospholamban phosphorylation at Ser(16) and Thr(17) sites. 5-HT increased phosphorylation only in TG mice but not in wild-type (WT) mice. Furthermore, 5-HT increased contractility in isolated perfused hearts from TG mice but not WT mice. These effects of 5-HT could be blocked by the 5-HT(4) receptor-selective antagonist GR-125487. An intravenous infusion of 5-HT increased left ventricular contractility in TG mice but not in WT mice. Similarly, the increase in contractility by 5-HT in isolated cardiomyocytes from TG mice was accompanied by and probably mediated through an increase in L-type Ca(2+) channel current and in Ca(2+) transients. In intact animals, echocardiography revealed an inotropic and chronotropic effect of subcutaneously injected 5-HT in TG mice but not in WT mice. In isolated hearts from TG mice, spontaneous polymorphic atrial arrhythmias were noted. These findings demonstrate the functional expression of 5-HT(4) receptors in the heart of TG mice, and a potential proarrhythmic effect in the atrium. Therefore, 5-HT(4) receptor-expressing mice might be a useful model to mimic the human heart, where 5-HT(4) receptors are present and functional in the atrium and ventricle of the healthy and failing heart, and to investigate the influence of 5-HT in the development of cardiac arrhythmias and heart failure.

We studied the effects of L-lysine in cardiac preparations of mice and men. Of note, L-lysine increased force of contraction in a concentration- and time-dependent manner in isolated electrically paced left atrium of mouse and in human right atrium. It further increased heart rate and left ventricular pressure in the isolated perfused mouse heart. In isolated adult mouse cardiomyocytes, the contractility as assessed by edge detection was increased as well as the Ca(2+) transients after electrically pacing by field stimulation. However, using the patch clamp technique, no effect of L-lysine on action potential duration from a constant holding potential or on current through L-type calcium channels could be observed. However, L-lysine led to a depolarization of unclamped cells. Furthermore, effects of L-lysine were stereospecific, as they were not elicited by D-lysine. The inotropic effects of L-lysine were not abrogated by additionally applied L-ornithine or L-arginine (known inhibitors of lysine transport). However, L-lysine (5 mM) shifted the concentration-response curve for a positive inotropic effect of 5-hydroxytryptamine (5-HT; serotonin) in atrium of transgenic mice (with cardiac specific overexpression of 5-HT(4) receptors) to higher concentrations. In summary, we describe a novel positive inotropic effect of an essential amino acid, L-lysine, in the mammalian heart. One might speculate that L-lysine treatment under certain conditions could sustain cardiac performance. Moreover, L-lysine is able to block, at least in part, cardiac 5-HT(4) receptors.

Multiple types of voltage-activated Ca2+ channels (T, L, N, P, Q, R type) coexist in excitable cells and participate in synaptic differentiation, secretion, transmitter release, and neuronal plasticity. Ca2+ ions entering cells trigger these events through their interaction with the ion channel itself or through Ca2+ binding to target proteins initiating signalling cascades at cytosolic loops of the ion conducting subunit (Cava1). These loops interact with target proteins in a Ca2+-dependent or independent manner. In Cav2.3-containing channels the cytosolic linker between domains II and III confers a novel Ca2+ sensitivity to E-type Ca2+ channels including phorbol ester sensitive signalling via protein kinase C (PKC) in Cav2.3 transfected HEK-293 cells. To understand Ca2+ and phorbol ester mediated activation of Cav2.3 Ca2+ channels, protein interaction partners of the II-III loop were identified. FLAG-tagged II-III - loop of human Cav2.3 was over-expressed in HEK 293 cells, and the molecular chaperone hsp70, which is known to interact with PKC, was identified as a novel functional interaction partner. Immunopurified II-III loop-protein of neuronal and endocrine Cav2.3 splice variants stimulate autophosphorylation of PKCa, leading to the suggestion that hsp70--binding to the II-III loop--may act as an adaptor for Ca2+ dependent targeting of PKC to E-type Ca2+ channels.

There is growing evidence that E-type voltage dependent Ca(2+) channels (Ca(v)2.3) are involved in triggering and controlling pivotal cellular processes like neurosecretion and long-term potentiation. The mechanism underlying a novel Ca(2+) dependent stimulation of E-type Ca(2+) channels was investigated in the context of the recent finding that influx of Ca(2+) through other voltage dependent Ca(2+) channels is necessary and sufficient to directly activate protein kinase C (PKC). With Ba(2+) as charge carrier through Ca(v)2.3 channel alpha(1) subunits expressed in HEK-293 cells, activation of PKC by low concentrations of phorbol ester augmented peak I(Ba) by approximately 60%. In addition, the non-inactivating fraction of I(Ba) was increased by more than three-fold and recovery from short-term inactivation was accelerated. The effect of phorbol ester on I(Ba) was inhibited by application of the specific PKC inhibitor bisindolylmaleimide I. With Ca(2+) as charge carrier, application of phorbol ester did not change the activity of Ca(v)2.3 currents but they were modified by the PKC inhibitor bisindolylmaleimide I. These results suggest that with Ca(2+) as charge carrier the incoming Ca(2+) can activate PKC, thereby augmenting Ca(2+) influx into the cytosol. No modulation of Ca(v)2.3 channels by PKC was observed when an arginine rich region in the II-III loop of Ca(v)2.3 was eliminated. Receptor independent stimulation of PKC and its interaction with Ca(v)2.3 channels therefore represents an important positive feedback mechanism to decode electrical signals into a variety of cellular functions.

Ca2+-dependent regulation of L-type and P/Q-type Ca2+ channel activity is an important mechanism to control Ca2+ entry into excitable cells. Here we addressed the question whether the activity of E-type Ca2+ channels can also be controlled by Ca2+. Switching from Ba2+ to Ca2+ as charge carrier increased within 50 s, the density of currents observed in HEK-293 cells expressing a human Cav2.3d subunit and slowed down the inactivation kinetics. Furthermore, with Ca2+ as permeant ion, recovery from inactivation was accelerated, compared to the recovery process recorded under conditions where the accumulation of [Ca2+]i was prevented. In a Ba2+ containing bath solution the Ca2+-dependent changes of E-type channel activity could be induced by dialysing the cells with 1 micro m free [Ca2+]i suggesting that an elevation of [Ca2+]i is responsible for these effects. Deleting 19 amino acids in the intracellular II-III linker (exon 19) as part of an arginine-rich region, severely impairs the Ca2+ responsiveness of the expressed channels. Interestingly, deletion of an adjacent homologue arginine-rich region activates channel activity but now independently from [Ca2+]i. As a positive feedback-regulation of channel activity this novel activation mechanism might determine specific biological functions of E-type Ca2+ channels.

There is growing evidence that Ca(v)2.3 (alpha1E, E-type) transcripts may encode the ion-conducting subunit of a subclass of R-type Ca(2+) channels, a heterogeneous group of channels by definition resistant to blockers of L-, N-, and P/Q-type Ca(2+) channels. To understand whether splice variation of Ca(v)2.3 contributes to the divergence of R-type channels, individual variants of Ca(v)2.3 were constructed and expressed in HEK-293 cells. With Ba(2+) as charge carrier, the tested biophysical properties were similar. In Ca(2+), the inactivation time course was slower and the recovery from short-term inactivation was faster; however, this occurred only in variants containing a 19-amino-acid-long insertion, which is typical for neuronal Ca(v)2.3 Ca(2+) channel subunits. This different Ca(2+) sensitivity is not responsible for the major differences between various R-type channels, and future studies might clarify its importance for in vivo synaptic or dendritic integration and the reasons for its loss in endocrine Ca(v)2.3 splice variants.

Objective: Multiple types of voltage-activated Ca(2+) channels (T, L, N, P, Q and R type) coordinate a variety of Ca(2+)-dependent processes in neurons and neuroendocrine cells. In insulinoma cell lines as well as in endocrine tissues, the non-L-type alpha1E (Ca(v)2.3) subunit is expressed as the tissue-specific splice variant alpha1Ee., Design and Methods: To understand the functional role of alpha1E-containing Ca(2+) channels, antisense alpha1E mRNA was overexpressed in INS-1 cells by stable transfection of an antisense alpha1E cassette cDNA. As controls, either a sense alpha1E cassette or a control vector containing enhanced green fluorescent protein as an unrelated gene was stably transfected. The overexpression of each transfected cassette cDNA was recorded by RT-PCR., Results: In three independent antisense alpha1E INS-1 clones, the glucose-induced insulin release was significantly reduced as compared with wild-type INS-1 cells and with a sense alpha1E INS-1 clone. However, in the antisense INS-1 clones, the KCl-induced insulin release was less impaired by overexpressing the antisense alpha1E cassette than the glucose-induced insulin release, leading to the assumption that glucose (15 mmol/l) and KCl (25 mmol/l) finally depolarize the membrane potential to a different extent., Conclusion: alpha1E is involved in glucose-induced insulin secretion probably by influencing the excitability of INS-1 cells.

Among voltage-gated Ca2+ channels the non-dihydropyridine-sensitive alpha1E subunit is functionally less well characterized than the structurally related alpha1A (omega-agatoxin-IVA sensitive, P- /Q-type) and alpha1B (omega-conotoxin-GVIA sensitive, N-type) subunits. In the rat insulinoma cell line, INS-1, a tissue-specific splice variant of alpha1E (alpha1Ee) has been characterized at the mRNA and protein levels, suggesting that INS-1 cells are a suitable model for investigating the function of alpha1Ee. In alpha1E-transfected human embryonic kidney (HEK-293) cells the alpha1E-selective peptide antagonist SNX-482 (100 nM) reduces alpha1Ed- and alpha1Ee-induced Ba2+ inward currents in the absence and presence of the auxiliary subunits beta3 and alpha2delta-2 by more than 80%. The inhibition is fast and only partially reversible. No effect of SNX-482 was detected on the recombinant T-type Ca2+ channel subunits alpha1G, alpha1H, and alpha1I showing that the toxin from the venom of Hysterocrates gigas is useful as an alpha1E-selective antagonist. After blocking known components of Ca2+ channel inward current in INS-1 cells by 2 microM (+/-)-isradipine plus 0.5 microM omega-conotoxin-MVIIC, the remaining current is reduced by 100 nM SNX-482 from -12.4 +/- 1.2 pA/pF to -7.6 +/- 0.5 pA/pF (n = 9). Furthermore, in INS-1 cells, glucose- and KCl-induced insulin release are reduced by SNX-482 in a dose-dependent manner leading to the conclusion that alpha1E, in addition to L-type and non-L-type (alpha1A-mediated) Ca2+ currents, is involved in Ca2+ dependent insulin secretion of INS-1 cells.

Expression of rat alpha1G, human alpha1H and rat alpha1I subunits of voltage-activated Ca2 + channels in HEK-293 cells yields robust Ca2 + inward currents with 1.25 mM Ca2 + as the charge carrier. Both similarities and marked differences are found between their biophysical properties. Currents induced by expression of alpha1G show the fastest activation and inactivation kinetics. The alpha1H and alpha1I currents activate and inactivate up to 1.5- and 5-fold slower, respectively. No differences in the voltage dependence of steady state inactivation are detected. Currents induced by expression of alpha1G and alpha1H deactivate with time constants of up to 6 ms at a test potential of - 80 mV, but currents induced by alpha1I deactivate about three-fold faster. Recovery from short-term inactivation is more than three-fold slower for currents induced by alpha1H and alpha1I in comparison to alpha1G. In contrast to these characteristics, reactivation after long-term inactivation was fastest for currents arising from expression of alpha1I and slowest in cells expressing alpha1H calcium channels. The calcium inward current induced by expression of alpha1I is increased by positive prepulses while currents induced by alpha1H and alpha1G show little ( < 5%) or no facilitation. The data thus provide a characteristic fingerprint of each channel's activity, which may allow correlation of the alpha1G, alpha1H and alpha1I induced currents with their in vivo counterparts.

Polyclonal antibodies were raised against a common and a specific epitope present only in longer alpha1E isoforms of voltage-gated Ca(2+) channels, yielding an "anti-E-com" and an "anti-E-spec" serum, respectively. The specificity of both sera was established by immunocytochemistry and immunoblotting using stably transfected HEK-293 cells or membrane proteins derived from them. Cells from the insulinoma cell line INS-1, tissue sections from cerebellum, and representative regions of gastrointestinal tract were stained immunocytochemically. INS-1 cells expressed an alpha1E splice variant with a longer carboxy terminus, the so-called alpha1Ee isoform. Similarily, in rat cerebellum, which was used as a reference system, the anti-E-spec serum stained somata and dendrites of Purkinje cells. Only faint staining was seen throughout the cerebellar granule cell layer. After prolonged incubation times, neurons of the molecular layer were stained by anti-E-com, suggesting that a shorter alpha1E isoform is expressed at a lower protein density. In human gastrointestinal tract, endocrine cells of the antral mucosa (stomach), small and large intestine, and islets of Langerhans were stained by the anti-E-spec serum. In addition, staining by the anti-E-spec serum was observed in Paneth cells and in the smooth muscle cell layer of the lamina muscularis mucosae. We conclude that the longer alpha1Ee isoform is expressed in neuroendocrine cells of the digestive system and that, in pancreas, alpha1Ee expression is restricted to the neuroendocrine part, the islets of Langerhans. alpha1E therefore appears to be a common voltage-gated Ca(2+) channel linked to neuroendocrine and related systems of the body.

Low voltage-activated Ca2+ channels play important roles in pacing neuronal firing and producing network oscillations, such as those that occur during sleep and epilepsy. Here we describe the cloning and expression of the third member of the T-type family, alpha1I or CavT.3, from rat brain. Northern analysis indicated that it is predominantly expressed in brain. Expression of the cloned channel in either Xenopus oocytes or stably transfected human embryonic kidney-293 cells revealed novel gating properties. We compared these electrophysiological properties to those of the cloned T-type channels alpha1G and alpha1H and to the high voltage-activated channels formed by alpha1Ebeta3. The alpha1I channels opened after small depolarizations of the membrane similar to alpha1G and alpha1H but at more depolarized potentials. The kinetics of activation and inactivation were dramatically slower, which allows the channel to act as a Ca2+ injector. In oocytes, the kinetics were even slower, suggesting that components of the expression system modulate its gating properties. Steady-state inactivation occurred at higher potentials than any of the other T channels, endowing the channel with a substantial window current. The alpha1I channel could still be classified as T-type by virtue of its criss-crossing kinetics, its slow deactivation (tail current), and its small (11 pS) conductance in 110 mM Ba2+ solutions. Based on its brain distribution and novel gating properties, we suggest that alpha1I plays important roles in determining the electroresponsiveness of neurons, and hence, may be a novel drug target.

The expression of Ca2+ channel alpha1E isoforms has been analyzed in different cell lines, embryoid bodies and tissues. The comparison of the different cloned alpha1E cDNA sequences led to the prediction of alpha1E splice variants. Transcripts of two cloned alpha1E isoforms, which are discriminated by a carboxy terminal 129-bp sequence, have been detected in different cell lines and tissues. Transcripts of the shorter alpha1E isoform have been assigned to the rat cerebrum and to neuron-like cells from in vitro, differentiated embryonic stem cells. The shorter isoform is the major transcript amplified from total RNA by reverse transcription (RT)-PCR and visualized on the protein level by Western blotting with common and isoform-specific antibodies. Transcripts of the longer alpha1E isoform have been identified in mouse, rat and human cerebellum, in in vitro, differentiated embryoid bodies, in the insulinoma cell lines INS-1 (rat) and betaTC-3 (mouse), in the pituitary cell line AtT-20 (mouse) when grown in 5 mM glucose, and in islets of Langerhans (rat) and kidney (rat and human). The detection of different isoforms of alpha1E in cell lines and tissues shows that the wide expression of alpha1E has to be specified by identifying the corresponding isoforms in each tissue. In islets of Langerhans and in kidney, a distinct isoform called alpha1Ee has been determined by RT-PCR, while in cerebellum a set of different alpha1E structures has been detected, which might reflect the functional heterogeneity of cerebellar neurons. The tissue-specific expression of different isoforms might be related to specific functions, which are not yet known, but the expression of the new isoform alpha1Ee in islets of Langerhans and kidney leads to the suggestion that alpha1E might be involved in the modulation of the Ca2+-mediated hormone secretion.

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 alpha1 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 alpha1 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 alpha1 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.

There is growing evidence for diversity of cardiac-type (class C) voltage-dependent calcium-channel alpha1-subunits arising from the alternative splicing of a primary transcript. In this study, we show the existence of carboxy-terminal variability in the human cardiac alpha1-gene by genomic cloning. We found that the genomic DNA segment encoding the COOH-terminal tail of the protein is composed of nine invariable and two alternative exons. The alternative utilization of these latter two exons gives rise to the formation of three message variants for this region. Reverse transcription followed by polymerase chain reaction and radioanalytic quantitation of the reverse transcription-polymerase chain reaction products showed significant variations in the distribution of these isoforms (hHt alpha1, rHt alpha1, fHt alpha1) in distinct parts of the heart, the aorta, and fibroblasts. Expression of the three alpha1-isoforms in Xenopus oocytes or in HEK-293 cells and analysis of the kinetics and voltage dependence of the induced calcium-channel currents revealed only insignificant differences in the behavior of these isoforms. When the alpha1-isoforms were coexpressed with a human beta-subunit, no alpha1-specific divergences were observed, but the effects of beta-subunit coexpression on alpha1-isoform biophysical properties were confirmed. The differential abundance of the three isoforms and the influence of an accessory subunit are of potential physiological significance.

The oxime derivative 2,3-butanedione monoxime (BDM) is used as an inorganic phosphatase to probe the phosphorylation state of many cellular proteins including the L-type calcium channel in various tissues. We used BDM further to shed light on the controversy surrounding direct phosphorylation of the L-type Ca2+ channel. We employed a recombinant system that utilizes HEK 293 cells expressing wild type and mutant human heart calcium channels. BDM reversibly reduced the calcium channel current induced by expression of the wild type channel in a concentration-dependent manner with an apparent IC50 value of 15.3 mM. Deletion of part of the carboxyl terminus of the alpha 1 subunit, which contains one putative protein kinase A site, or mutating all of the protein kinase A consensus sites of the pore forming subunit, did not significantly change the apparent IC50 value or alter in any other way the blocking effect of BDM on the expressed currents. Our data suggest that BDM produces reversible modifications of the cardiac calcium channel protein leading to an expected reduction in the amplitude of the expressed currents, but the site of action must be different from that of the consensus sites for protein kinase A dependent phosphorylation.