Asif N. Daud

Molecular characterization of a neuronal low-voltage-activated T-type calcium channel

The molecular diversity of voltage-activated calcium channels was established by studies showing that channels could be distinguished by their voltage-dependence, deactivation and single-channel conductance. Low-voltage-activated channels are called ‘T’ type because their currents are both transient (owing to fast inactivation) and tiny (owing to small conductance). T-type channels are thought to be involved in pacemaker activity, low-threshold calcium spikes, neuronal oscillations and resonance, and rebound burst firing. Here we report the identification of a neuronal T-type channel. Our cloning strategy began with an analysis of Genbank sequences defined as sharing homology with calcium channels. We sequenced an expressed sequence tag (EST), then used it to clone a full-length complementary DNA from rat brain. Northern blot analysis indicated that this gene is expressed predominantly in brain, in particular the amygdala, cerebellum and thalamus. We mapped the human gene to chromosome 17q22, and the mouse gene to chromosome 11. Functional expression of the channel was measured in Xenopus oocytes. Based on the channels distinctive voltage dependence, slow deactivation kinetics, and 7.5-pS single-channel conductance, we conclude that this channel is a low-voltage-activated T-type calcium channel.

Cloning and Characterization of α1H From Human Heart, a Member of the T-Type Ca2+ Channel Gene Family

Voltage-activated Ca2+ channels exist as multigene families that share common structural features. Different Ca2+ channels are distinguished by their electrophysiology and pharmacology and can be classified as either low or high voltage-activated channels. Six alpha1 subunit genes cloned previously code for high voltage-activated Ca2+ channels; therefore, we have used a database search strategy to identify new Ca2+ channel genes, possibly including low voltage-activated (T-type) channels. A novel expressed sequence-tagged cDNA clone of alpha1G was used to screen a cDNA library, and in the present study, we report the cloning of alpha1H (or CavT.2), a low voltage-activated Ca2+ channel from human heart. Northern blots of human mRNA detected more alpha1H expression in peripheral tissues, such as kidney and heart, than in brain. We mapped the gene, CACNA1H, to human chromosome 16p13.3 and mouse chromosome 17. Expression of alpha1H in HEK-293 cells resulted in Ca2+ channel currents displaying voltage dependence, kinetics, and unitary conductance characteristic of native T-type Ca2+ channels. The alpha1H channel is sensitive to mibefradil, a nondihydropyridine Ca2+ channel blocker, with an IC50 of 1.4 micromol/L, consistent with the reported potency of mibefradil for T-type Ca2+ channels. Together with alpha1G, a rat brain T-type Ca2+ channel also cloned in our laboratory, these genes define a unique family of Ca2+ channels.

Cloning and Expression of a Novel Member of the Low Voltage-Activated T-Type Calcium Channel Family

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, α1I or CavT.3, from rat brain. Northern analysis indicated that it is predominantly expressed in brain. Expression of the cloned channel in either Xenopusoocytes 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 α1G and α1H and to the high voltage-activated channels formed by α1Eβ3. The α1I channels opened after small depolarizations of the membrane similar to α1G and α1H 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 α1I 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 α1I plays important roles in determining the electroresponsiveness of neurons, and hence, may be a novel drug target.

Comparison of the Ca2 + currents induced by expression of three cloned α1 subunits, α1G, α1H and α1I, of low-voltage-activated T-type Ca2 + channels

Expression of rat α1G, human α1H and rat α1I 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 α1G show the fastest activation and inactivation kinetics. The α1H and α1I 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 α1G and α1H deactivate with time constants of up to 6 ms at a test potential of − 80 mV, but currents induced by α1I deactivate about three‐fold faster. Recovery from short‐term inactivation is more than three‐fold slower for currents induced by α1H and α1I in comparison to α1G. In contrast to these characteristics, reactivation after long‐term inactivation was fastest for currents arising from expression of α1I and slowest in cells expressing α1H calcium channels. The calcium inward current induced by expression of α1I is increased by positive prepulses while currents induced by α1H and α1G show little ( <  5%) or no facilitation. The data thus provide a characteristic fingerprint of each channels activity, which may allow correlation of the α1G, α1H and α1I induced currents with their in vivo counterparts.

Molecular cloning and functional expression of Cav3.1c, a T-type calcium channel from human brain

Low voltage‐activated T‐type calcium channels are encoded by a family of at least three genes, with additional diversity created by alternative splicing. This study describes the cloning of the human brain α1G, which is a novel isoform, Cav3.1c. Comparison of this sequence to genomic sequences deposited in the GenBank allowed us to identify the intron/exon boundaries of the human CACNA1G gene. A full‐length cDNA was constructed, then used to generate a stably‐transfected mammalian cell line. The resulting currents were analyzed for their voltage‐ and time‐dependent properties. These properties identify this gene as encoding a T‐type Ca2+ channel.

Synthetic Heparin Pentasaccharide Depolymerization by Heparinase 1: Molecular and Biological Implications

A synthetic pentasaccharide (SR90107/ ORG31540) representing the antithrombin III (ATIII) binding sequence in heparin is under clinical development for the prophylaxis and management of venous thromboembolism. This pentasaccharide exhibits potent anti-factor Xa (AXa) effects (>750 lU/mg> and does not exhibit any anti-factor IIa (AIIa) activity. Previous reports have suggested that synthetic heparin pentasaccharides are resistant to the digestive effects of heparinase 1. To investigate the effect of heparinase I on the AXa activity of pentasaccharide SR90107/ORG31540. graded concentrations (1.25-100 μg/ml) were incubated with a fixed amount of heparinase I (0.1 U/ml). Heparinase I produced a strong neutralizing effect on this pentasaccharide, as measured by AXa activity. This observation led to further studies where high performance liquid chromatography (HPLC) analysis was eniployed to determine the potential breakdown products of the pentasaccharide. The experiment with the pentasaccharide included incubation (37°C) at mg/ml and exposure to graded concentrations of heparinase I (0.125-1 U/ml). After 30 min of incubation, the enzymatic activity was stopped by heat treatment and the mixture was analyzed using high performance size exclusion chromatography (HPSEC). Heparinase I concentration-dependent cleavage of the pentasaccharide was evident. The breakdown products exhibited a mass of 1,034 d and 743 d, respectively, suggesting the generation of a trisaccharide and a disaccharide moiety. The extinction of a disaccharide moiety in the UV region was high, indicating the presence of a double bond in this molecule. These data clearly, suggest that pentasaccharide SR90107/ORG31540 is digestible by heparinase I into its two components. Furthermore, these data support the hypothesis that heparinase I can be used as a neutralizing agent for pentasaccharide overdose. Additionally, a highly methylated analog of the previously mentioned synthetic pentasaccharide, SanOrg34006, which has also been subjected to similar experiments, has shown complete resistance to the depolymerizing function of heparinase I; therefore, its use may be appropriate in chronic situations as a long-acting form of the pentasaccharide.

Prostaglandin E2 mediates growth arrest in NFS-60 cells by down-regulating interleukin-6 receptor expression.

Interleukin-6 (IL-6), a potent myeloid mitogen, and the immunosuppressive prostanoid prostaglandin E2 (PGE2) are elevated following thermal injury and sepsis. We have previously demonstrated that bone marrow myeloid commitment shifts toward monocytopoiesis and away from granulocytopoiesis during thermal injury and sepsis and that PGE2 plays a central role in this alteration. Here we investigated whether PGE2 can modulate IL-6-stimulated growth in the promyelocytic cell line, NFS-60, by down-regulating IL-6 receptor (IL-6r) expression. Exposure of NFS-60 cells to PGE2 suppressed IL-6-stimulated proliferation as well as IL-6r expression. Receptor down-regulation is functionally significant since IL-6-induced signal transduction through activators of transcription (STAT)-3 is also decreased. Down-regulation of IL-6r correlated with the ability of PGE2 to arrest cells in the G0/G1 phase of the cell cycle. PGE2 appears to signal through EP2 receptors. Butaprost (EP2 agonist) but not sulprostone (EP3 agonist) inhibited IL-6-stimulated proliferation. In addition, an EP2 antagonist (AH6809) alleviated the anti-proliferative effects of PGE2. NFS-60 cells express predominantly EP2 and EP4 receptors. While PGE2 down-regulated both the IL-6r protein and mRNA expression, it had no influence on EP2 or EP4 mRNA expression. The present study demonstrates that PGE2 is a potent down-regulator of IL-6r expression and thus may provide a mechanistic explanation for the granulocytopenia seen in thermal injury and sepsis.

Anticoagulant and Antiprotease Effects of a Novel Heparinlike Compound From Shrimp (Penaeus brasiliensis) and Its Neutralization by Heparinase I

Heparin is usually obtained from mammalian organs, such as beef lung, beef mucosa, porcine mucosa, and sheep intestinal mucosa. Because of the increased use of heparin in the production of low-molecular-weight heparin (LMWH), there is a growing shortage of the raw material needed to produce LMWHs. A previous report described the structural features of a novel LMWH from the shrimp (Penaeus brasiliensis). In order to compare anticoagulant and antiprotease effects of this heparin, global anticoagulant tests, such as the prothrombin time, activated partial thromboplastin time, thrombin time, and Heptest®, were used. Amidolytic anti-Xa and anti-IIa activities were also measured. The relative susceptibility of this heparin to flavobacterial heparinase was also evaluated. The United States Pharmacopeia (USP) potency of shrimp heparin (SH) was found to be 28 U/mg. SH produced a concentration-dependent prolongation of all of the clotting tests and exhibited marked inhibition of FXa and FIIa. Heparinase treatment resulted in a marked decrease of the anticoagulant effects and neutralized the in vitro anti-IIa actions. However, the anti-Xa activities were only partially neutralized. Protamine sulfate was only partially effective in neutralizing the anticoagulant and antithrombin effects of SH. SH also produced marked prolongation of activated clotting time, which was neutralized by heparinase but not by protamine sulfate. These results suggest that SH is a strong anticoagulant with comparable properties to mammalian heparins and can be used in the development of clinically useful antithrombotic-anticoagulant drugs.

Corrigendum to: Molecular cloning and functional expression of Cav3.1c, a T-type calcium channel from human brain: [FEBS Letters 466 (2000) 54–58]1

The original publication of this paper reported the full-length sequence of an apparent splice variation of the Cav3.1 clone, with an additional exon in the domain III^IV linker. The authors have discovered that the human cDNA they expressed does not include the additional exon, and therefore represents the human Cav3.1a cDNA. The correct sequence is identical to that in Fig. 1, excluding the erroneous 18 amino acid exon, and can be found in the GenBank database, accession number AF190860.