Functional analysis of a rice putative voltage-dependent Ca2+ channel, OsTPC1, expressed in yeast cells lacking its homologous gene CCH1

We isolated a cDNA (OsTPC1) from rice that was homologous to AtTPC1, a putative voltage-dependent Ca(2+) channel (VDCC) gene of Arabidopsis thaliana. The hydropathy profile of its deduced amino acid sequence showed significant structural features of the alpha 1-subunit of animal VDCCs. Functional analysis using a heterologous yeast expression system showed that OsTPC1 facilitates Ca(2+) permeation. The K(m) value for Ca(2+) of OsTPC1, 47.5 micro M, was comparable to that of intrinsic CCH1, a candidate VDCC in yeast. Ca(2+) permeation by OsTPC1 was inhibited by verapamil, a VDCC blocker. These findings indicate for the first time that OsTPC1 is a putative VDCC in rice.     

The biochemical activation of T-type Ca2+ channels in HEK293 cells stably expressing alpha1G and Kir2.1 subunits

In order to investigate the currently unknown cellular signaling pathways of T-type Ca(2+) channels, we decided to construct a new cell line which would stably express alpha(1G) and Kir2.1 subunits in HEK293 cells (HEK293/alpha(1G)/Kir2.1). Compared to cells which only expressed alpha(1G) (HEK293/alpha(1G)), HEK293/alpha(1G)/Kir2.1 cells produced an enormous inward rectifying current which was blocked by external Ba(2+) and Cs(+) in a concentration-dependent manner. The expression of Kir2.1 channels contributed significantly to the shift of membrane potential from -12.2+/-2.8 to -57.3+/-3.7mV. However, biophysical and pharmacological properties of alpha(1G)-mediated Ca(2+) channels remained unaffected by the expression of Kir2.1 subunits, except for the enlarging of the window current region. Biochemical activation of alpha(1G) channels using 150mM KCl brought about an increase in [Ca(2+)](i), which was blocked by mibefradil, the T-type Ca(2+) channel blocker. These data suggest that the HEK293/alpha(1G)/Kir2.1 cell line would have potential uses in the study of T-type Ca(2)(+) channel-mediated signaling pathways and possibly useful in the development of new therapeutic drugs associated with T-type Ca(2)(+) channels.     

T-type Ca2+ channel expression in human esophageal carcinomas: a functional role in proliferation

In the present study the role of T-type Ca(2+) channels in cancer cell proliferation was examined. Seventeen human esophageal cancer cell lines were screened for T-type channels using RT-PCR and voltage-clamp recordings. mRNAs for all three T-type channel alpha(1)-subunits (alpha(1G), alpha(1H), and alpha(1I)) were detected in all 17 cell lines: either alpha(1H) alone, alpha(1H) and alpha(1G), or all three T-type alpha(1)-subunits. Eleven cell lines were further subjected to voltage-clamp recordings: one, i.e. the TE8 cell line, was found to exhibit a typical T-type current while others exhibited a minimal or no T-type current. Cell proliferation assays were performed in the presence or absence of T-type channel blocker mibefradil in KYSE150, KYSE180 and TE1 cells expressing mRNA for T-type channel alpha(1)-subunits but lacking T-type current, and TE8 cells exhibiting T-type current. Only TE8 cell proliferation was reduced by mibefradil. Silencing the alpha(1G)-gene that encodes functional T-type Ca(2+) channels in TE8 cells with type-specific shRNA transduction also significantly decreased TE8 cell proliferation. The reduction of cell proliferation in TE8 cells was found to be associated with an up-regulation of p21(CIP1). Moreover, p53 silencing nearly abolished the up-regulation of p21(CIP1) resulting from mibefradil T-type channel blockade. Together, these findings suggest a functional role of T-type channels in certain esophageal carcinomas, and that inhibition of T-type channels reduces cell proliferation via a p53-dependent p21(CIP1) pathway.     

Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha1H

Nickel has been proposed to be a selective blocker of low-voltage-activated, T-type calcium channels. However, studies on cloned high-voltage-activated Ca(2+) channels indicated that some subtypes, such as alpha1E, are also blocked by low micromolar concentrations of NiCl(2). There are considerable differences in the sensitivity to Ni(2+) among native T-type currents, leading to the hypothesis that there may be more than one T-type channel. We confirmed part of this hypothesis by cloning three novel Ca(2+) channels, alpha1G, H, and I, whose currents are nearly identical to the biophysical properties of native T-type channels. In this study we examined the nickel block of these cloned T-type channels expressed in both Xenopus oocytes and HEK-293 cells (10 mM Ba(2+)). Only alpha1H currents were sensitive to low micromolar concentrations (IC(50) = 13 microM). Much higher concentrations were required to half-block alpha1I (216 microM) and alpha1G currents (250 microM). Nickel block varied with the test potential, with less block at potentials above -30 mV. Outward currents through the T channels were blocked even less. We show that depolarizations can unblock the channel and that this can occur in the absence of permeating ions. We conclude that Ni(2+) is only a selective blocker of alpha1H currents and that the concentrations required to block alpha1G and alpha1I will also affect high-voltage-activated calcium currents.     

Conducted vasoconstriction in rat mesenteric arterioles: role for dihydropyridine-insensitive Ca(2+) channels

The aim of this study was to evaluate the role of voltage-operated Ca(2+) channels in the initiation and conduction of vasoconstrictor responses to local micropipette electrical stimulation of rat mesenteric arterioles (28 +/- 1 microm, n = 79) in vivo. Local and conducted (600 microm upstream from the pipette) vasoconstriction was not blocked by TTX (1 micromol/l, n = 5), nifedipine, or nimodipine (10 micromol/l, n = 9). Increasing the K(+) concentration of the superfusate to 75 mmol/l did not evoke vasoconstriction, but this depolarizing stimulus reversibly abolished vasoconstrictor responses to current stimulation (n = 7). Addition of the T-type Ca(2+) antagonist mibefradil (10 micromol/l, n = 6) to the superfusate reversibly blocked local and conducted vasoconstriction to current stimulation. With the use of RT-PCR techniques, it was demonstrated that rat mesenteric arterioles <40 microm do not express mRNA for L-type Ca(2+) channels (alpha(1C)-subunit), whereas mRNA coding for T-type subunits was found (alpha(1G)- and alpha(1H)-subunits). The data indicate that L-type Ca(2+) channels are absent from rat mesenteric arterioles (<40 microm). Rather, the vasoconstrictor responses appear to rely on other types of voltage-gated, dihydropyridine-insensitive Ca(2+) channels, possibly of the T-type.     

The role of Ca2+ channel modulation in the neuroprotective actions of estrogen in beta-amyloid protein and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) cytotoxic models

Physiologically relevant concentrations of 17beta-estradiol (E2) are neuroprotective in both beta-amyloid protein 25-35 (Abeta) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced cytotoxicity in SK-N-SH cells. MPTP, but not Abeta, induces apoptosis in this cell line. The L-type calcium channel blocker nifedipine or decreased extracellular Ca(2+) concentration blocked Abeta-induced cell death, but not MPTP-induced cell death. Other blockers selective for different Ca(2+) channel subtypes had no effects on either Abeta or MPTP induced death. Western blot analysis for L-type Ca(2+) channel alpha(1)-subunits demonstrated that Abeta increases the expression of the neuronal alpha(1C) and alpha(1D) subunits of L-type channels. Both E2 and nifedipine inhibit the increase in expression of these by Abeta. MPTP also increases expression of alpha(1C) and alpha(1D), but the increases were not influenced by E2 or nifedipine. These observations suggested that Abeta cytotoxicity in SK-N-SH cells may involve increased availability of calcium to cells, whereas MPTP induced cytotoxicity does not require extracellular Ca(2+). Both cytotoxic models were associated with increased expression of Ca(2+) channel alpha(1) subunits, and neuroprotection associated with inhibition of that increase. These studies reveal that nifedipine, in addition to its direct action of nifedipine on Ca(2+) channels, may also protect neurons from Abeta toxicity through the suppression of the channel protein overexpression. A new action of dihydropyridines (DHPs) may be considered in the regulation of calcium homeostasis.     

Interaction of bestrophin-1 and Ca2+ channel β-subunits: identification of new binding domains on the bestrophin-1 C-terminus

Bestrophin-1 modulates currents through voltage-dependent L-type Ca(2+) channels by physically interacting with the β-subunits of Ca(2+) channels. The main function of β-subunits is to regulate the number of pore-forming Ca(V)-subunits in the cell membrane and modulate Ca(2+) channel currents. To understand the influence of full-length bestrophin-1 on β-subunit function, we studied binding and localization of bestrophin-1 and Ca(2+) channel subunits, together with modulation of Ca(V)1.3 Ca(2+) channels currents. In heterologeous expression, bestrophin-1 showed co-immunoprecipitation with either, β3-, or β4-subunits. We identified a new highly conserved cluster of proline-rich motifs on the bestrophin-1 C-terminus between amino acid position 468 and 486, which enables possible binding to SH3-domains of β-subunits. A bestrophin-1 that lacks these proline-rich motifs (ΔCT-PxxP bestrophin-1) showed reduced efficiency to co-immunoprecipitate with β3 and β4-subunits. In the presence of ΔCT-PxxP bestrophin-1, β4-subunits and Ca(V)1.3 subunits partly lost membrane localization. Currents from Ca(V)1.3 subunits were modified in the presence of β4-subunit and wild-type bestrophin-1: accelerated time-dependent activation and reduced current density. With ΔCTPxxP bestrophin-1, currents showed the same time-dependent activation as with wild-type bestrophin-1, but the current density was further reduced due to decreased number of Ca(2+) channels proteins in the cell membrane. In summary, we described new proline-rich motifs on bestrophin-1 C-terminus, which help to maintain the ability of β-subunits to regulate surface expression of pore-forming Ca(V) Ca(2+)-channel subunits.     

Functional and molecular analysis of L-type calcium channels in human esophagus and lower esophageal sphincter smooth muscle

Excitation of human esophageal smooth muscle involves the release of Ca(2+) from intracellular stores and influx. The lower esophageal sphincter (LES) shows the distinctive property of tonic contraction; however, the mechanisms by which this is maintained are incompletely understood. We examined Ca(2+) channels in human esophageal muscle and investigated their contribution to LES tone. Functional effects were examined with tension recordings, currents were recorded with patch-clamp electrophysiology, channel expression was explored by RT-PCR, and intracellular Ca(2+) concentration was monitored by fura-2 fluorescence. LES muscle strips developed tone that was abolished by the removal of extracellular Ca(2+) and reduced by the application of the L-type Ca(2+) channel blocker nifedipine (to 13 +/- 6% of control) but was unaffected by the inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA). Carbachol increased tension above basal tone, and this effect was attenuated by treatment with CPA and nifedipine. Voltage-dependent inward currents were studied using patch-clamp techniques and dissociated cells. Similar inward currents were observed in esophageal body (EB) and LES smooth muscle cells. The inward currents in both tissues were blocked by nifedipine, enhanced by Bay K8644, and transiently suppressed by acetylcholine. The molecular form of the Ca(2+) channel was explored using RT-PCR, and similar splice variant combinations of the pore-forming alpha(1C)-subunit were identified in EB and LES. This is the first characterization of Ca(2+) channels in human esophageal smooth muscle, and we establish that L-type Ca(2+) channels play a critical role in maintaining LES tone.     

Structure and function of neuronal Ca2+ channels and their role in neurotransmitter release

Electrophysiological studies of neurons reveal different Ca2+ currents designated L-, N-, P-, Q-, R-, and T-type. High-voltage-activated neuronal Ca2+ channels are complexes of a pore-forming alpha 1 subunit of about 190-250 kDa, a transmembrane, disulfide-linked complex of alpha 2 and delta subunits, and an intracellular beta subunit, similar to the alpha 1, alpha 2 delta, and beta subunits previously described for skeletal muscle Ca2+ channels. The primary structures of these subunits have all been determined by homology cDNA cloning using the corresponding subunits of skeletal muscle Ca2+ channels as probes. In most neurons, L-type channels contain alpha 1C or alpha 1D subunits, N-type contain alpha 1B subunits, P- and Q-types contain alternatively spliced forms of alpha 1A subunits, R-type contain alpha 1E subunits, and T-type contain alpha 1G or alpha 1H subunits. Association with different beta subunits also influences Ca2+ channel gating substantially, yielding a remarkable diversity of functionally distinct molecular species of Ca2+ channels in neurons.     

Aspartate residues of the Glu-Glu-Asp-Asp (EEDD) pore locus control selectivity and permeation of the T-type Ca(2+) channel alpha(1G)

The structural determinant of the permeation and selectivity properties of high voltage-activated (HVA) Ca(2+) channels is a locus formed by four glutamate residues (EEEE), one in each P-region of the domains I-IV of the alpha(1) subunit. We tested whether the divergent aspartate residues of the EEDD locus of low voltage-activated (LVA or T-type) Ca(2+) channels account for the distinctive permeation and selectivity features of these channels. Using the whole-cell patch-clamp technique in the HEK293 expression system, we studied the properties of the alpha(1G) T-type, the alpha(1C) L-type Ca(2+) channel subunits, and alpha(1G) pore mutants, containing aspartate-to-glutamate conversions in domain III, domain IV, or both. Three characteristic features of HVA Ca(2+) channel permeation, i.e. (a) Ba(2+) over Ca(2+) permeability, (b) Ca(2+)/Ba(2+) anomalous mole fraction effect (AMFE), and (c) high Cd(2+) sensitivity, were conferred on the domain III mutant (EEED) of alpha(1G). In contrast, the relative Ca(2+)/Ba(2+) permeability and the lack of AMFE of the alpha(1G) wild type channel were retained in the domain IV mutant (EEDE). The double mutant (EEEE) displayed AMFE and a Cd(2+) sensitivity similar to that of alpha(1C), but currents were larger in Ca(2+)- than in Ba(2+)-containing solutions. The mutation in domain III, but not that in domain IV, consistently displayed outward fluxes of monovalent cations. H(+) blocked Ca(2+) currents in all mutants more efficiently than in alpha(1G). In addition, activation curves of all mutants were displaced to more positive voltages and had a larger slope factor than in alpha(1G) wild type. We conclude that the aspartate residues of the EEDD locus of the alpha(1G) Ca(2+) channel subunit not only control its permeation properties, but also affect its activation curve. The mutation of both divergent aspartates only partially confers HVA channel permeation properties to the alpha(1G) Ca(2+) channel subunit.     

Ca2+ channel currents and contraction in CaVbeta3-deficient ileum smooth muscle from mouse

Voltage activated L-type Ca(2+) channels are the principal Ca(2+) channels in intestinal smooth muscle cells. They comprise the ion conducting Ca(V)1 pore and the ancillary subunits alpha(2)delta and beta. Of the four Ca(V)beta subunits Ca(V)beta(3) is assumed to be the relevant Ca(V)beta protein in smooth muscle. In protein lysates isolated from mouse ileum longitudinal smooth muscle we could identify the Ca(V)1.2, Ca(V)alpha(2), Ca(V)beta(2) and Ca(V)beta(3) proteins, but not the Ca(V)beta(1) and Ca(V)beta(4) proteins. Protein levels of Ca(V)1.2, Ca(V)alpha(2) and Ca(V)beta(2) are not altered in ileum smooth muscle obtained from Ca(V)beta(3)-deficient mice indicating that there is no compensatory increase of the expression of these channel proteins. Neither the Ca(V)beta(2) nor the other Ca(V)beta proteins appear to substitute for the lacking Ca(V)beta(3). L-type Ca(2+) channel properties including current density, inactivation kinetics as well as Cd(2+)- and dihydropyridine sensitivity were identical in cells of both genotypes suggesting that they do not require the presence of a Ca(V)beta(3) protein. However, a key hallmark of the Ca(V)beta modulation of Ca(2+) current, the hyperpolarisation of channel activation is slightly but significantly reduced by 4 mV. In addition to L-type Ca(2+) currents T-type Ca(2+) currents could be recorded in the murine ileum smooth muscle cells, but T-type currents were not affected by the lack of Ca(V)beta(3). Both proteins, Ca(V)beta(2) and Ca(V)beta(3) are localized near the plasma membrane and the localization of Ca(V)beta(2) is not altered in Ca(V)beta(3) deficient cells. Spontaneous contractions and potassium and carbachol induced contractions are not significantly different between ileum longitudinal smooth muscle strips from mice of both genotypes. In summary the data show that in ileum smooth muscle cells, Ca(V)beta(3) has only subtle effects on L-type Ca(2+) currents, appears not to be required for spontaneous and potassium induced contraction but might have a function beyond being a Ca(2+) channel subunit.     

Remodeling excitation-contraction coupling of hypertrophied ventricular myocytes is dependent on T-type calcium channels expression

We utilized Wistar rats with monocrotaline (MCT)-induced right ventricular hypertrophy (RVH) in order to evaluate the T-type Ca2+ channel current (ICaT) for myocardial contraction. RT-PCR provides that mRNA for T-type Ca2+ channel alpha1-subunits in hypertrophied myocytes was significantly higher than those in control rats (alpha1G; 264+/-36%, alpha1H; 191+/-34%; P<0.05). By whole-cell patch-clamp study, ICaT was recorded only in hypertrophied myocytes but not in control myocytes. The application of 50 nmol/L nifedipine reduced the twitch tension of the right ventricles equally in the control and RVH rats. On the other hand, 0.5 micromol/L mibefradil, a T-type Ca2+ channel blocker, strongly inhibited the twitch tension of the RVH muscle (control 6.4+/-0.8% vs. RVH 20.0+/-2.3% at 5 Hz; P<0.01). In conclusion, our results indicate the functional expression of T-type Ca2+ channels in the hypertrophied heart and their contribution to the remodeling of excitation-contraction coupling in the cardiac myocyte.     

Double-pulse facilitation of smooth muscle alpha 1-subunit Ca2+ channels expressed in CHO cells.

Frequent strong depolarizations facilitate Ca2+ channels in various cell types by shifting their gating behavior towards mode 2, which is characterized by long openings and high probability of being open. In cardiac cells, the same type of gating behavior is potentiated by beta-adrenoceptors presumably acting via phosphorylation of a protein identical to or associated with the channel. Voltage-dependent phosphorylation has also been reported to underlie Ca2+ channel facilitation in chromaffin adrenal medulla and in skeletal muscle cells. We studied a possible voltage-dependent facilitation of the principal channel forming alpha 1-subunit of the dihydropyridine-sensitive smooth muscle Ca2+ channel. Single channel and whole-cell Ca2+ currents were recorded in Chinese hamster ovary cells stably expressing the class Cb Ca2+ channel alpha 1-subunit. Strong depolarizing voltage-clamp steps preceding the test pulse resulted in a 2- to 3-fold increase of the single Ca2+ channel activity and induction of mode 2-like gating behavior. Accordingly we observed a significant potentiation of the whole-cell current by approximately 50%. In contrast to the previous suggestions we found no experimental evidence for involvement of channel phosphorylation by protein kinases (cAMP-dependent protein kinase, protein kinase C and other protein kinases utilizing ATP gamma S) in the control and facilitated current. The data demonstrate that the L-type Ca2+ channel alpha 1-subunit solely expressed in Chinese hamster ovary cells is subject to a voltage-dependent facilitation but not to phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific properties of T-type calcium channels generated by the human alpha 1I subunit

We have cloned and expressed a human alpha(1I) subunit that encodes a subtype of T-type calcium channels. The predicted protein is 95% homologous to its rat counterpart but has a distinct COOH-terminal region. Its mRNA is detected almost exclusively in the human brain, as well as in adrenal and thyroid glands. Calcium currents generated by the functional expression of human alpha(1I) and alpha(1G) subunits in HEK-293 cells were compared. The alpha(1I) current activated and inactivated approximately 10 mV more positively. Activation and inactivation kinetics were up to six times slower, while deactivation kinetics was faster and showed little voltage dependence. A slower recovery from inactivation, a lower sensitivity to Ni(2+) ions (IC(50) approximately 180 micrometer), and a larger channel conductance (approximately 11 picosiemens) were the other discriminative features of the alpha(1I) current. These data demonstrate that the alpha(1I) subunit encodes T-type Ca(2+) channels functionally distinct from those generated by the human alpha(1G) or alpha(1H) subunits and point out that human and rat alpha(1I) subunits have species-specific properties not only in their primary sequence, but also in their expression profile and electrophysiological behavior.