Cloning and expression of the human T-type channel Ca(v)3.3: insights into prepulse facilitation

The full-length human Ca(v)3.3 (alpha(1I)) T-type channel was cloned, and found to be longer than previously reported. Comparison of the cDNA sequence to the human genomic sequence indicates the presence of an additional 4-kb exon that adds 214 amino acids to the carboxyl terminus and encodes the 3' untranslated region. The electrophysiological properties of the full-length channel were studied after transient transfection into 293 human embryonic kidney cells using 5 mM Ca(2+) as charge carrier. From a holding potential of -100 mV, step depolarizations elicited inward currents with an apparent threshold of -70 mV, a peak of -30 mV, and reversed at +40 mV. The kinetics of channel activation, inactivation, deactivation, and recovery from inactivation were very similar to those reported previously for rat Ca(v)3.3. Similar voltage-dependent gating and kinetics were found for truncated versions of human Ca(v)3.3, which lack either 118 or 288 of the 490 amino acids that compose the carboxyl terminus. A major difference between these constructs was that the full-length isoform generated twofold more current. These results suggest that sequences in the distal portion of Ca(v)3.3 play a role in channel expression. Studies on the voltage-dependence of activation revealed that a fraction of channels did not gate as low voltage-activated channels, requiring stronger depolarizations to open. A strong depolarizing prepulse (+100 mV, 200 ms) increased the fraction of channels that gated at low voltages. In contrast, human Ca(v)3.3 isoforms with shorter carboxyl termini were less affected by a prepulse. Therefore, Ca(v)3.3 is similar to high voltage-activated Ca(2+) channels in that depolarizing prepulses can regulate their activity, and their carboxy termini play a role in modulating channel activity.     

CDKN3 mRNA as a Biomarker for Survival and Therapeutic Target in Cervical Cancer

The cyclin-dependent kinase inhibitor 3 (CDKN3) gene, involved in mitosis, is upregulated in cervical cancer (CC). We investigated CDKN3 mRNA as a survival biomarker and potential therapeutic target for CC. CDKN3 mRNA was measured in 134 CC and 25 controls by quantitative PCR. A 5-year survival study was conducted in 121 of these CC patients. Furthermore, CDKN3-specific siRNAs were used to investigate whether CDKN3 is involved in proliferation, migration, and invasion in CC-derived cell lines (SiHa, CaSki, HeLa). CDKN3 mRNA was on average 6.4-fold higher in tumors than in controls (p = 8 x 10⁻⁶, Mann-Whitney). A total of 68.2% of CC patients over expressing CDKN3 gene (fold change ≥ 17) died within two years of diagnosis, independent of the clinical stage and HPV type (Hazard Ratio = 5.0, 95% CI: 2.5–10, p = 3.3 x 10⁻⁶, Cox proportional-hazards regression). In contrast, only 19.2% of the patients with lower CDKN3 expression died in the same period. In vitro inactivation of CDKN3 decreased cell proliferation on average 67%, although it had no effect on cell migration and invasion. CDKN3 mRNA may be a good survival biomarker and potential therapeutic target in CC.     

Adenosine Triphosphate Activates a Noninactivating K+ Current in Adrenal Cortical Cells through Nonhydrolytic Binding

Bovine adrenal zona fasciculata (AZF) cells express a noninactivating K⁺ current (I_AC) that is inhibited by adrenocorticotropic hormone and angiotensin II at subnanomolar concentrations. Since I_AC appears to set the membrane potential of AZF cells, these channels may function critically in coupling peptide receptors to membrane depolarization, Ca²⁺ entry, and cortisol secretion. I_AC channel activity may be tightly linked to the metabolic state of the cell. In whole cell patch clamp recordings, MgATP applied intracellularly through the patch electrode at concentrations above 1 mM dramatically enhanced the expression of I_AC K⁺ current. The maximum I_AC current density varied from a low of 8.45 ± 2.74 pA/pF (n = 17) to a high of 109.2 ± 26.3 pA/pF (n = 6) at pipette MgATP concentrations of 0.1 and 10 mM, respectively. In the presence of 5 mM MgATP, I_AC K⁺ channels were tonically active over a wide range of membrane potentials, and voltage-dependent open probability increased by only ∼30% between −40 and +40 mV. ATP (5 mM) in the absence of Mg²⁺ and the nonhydrolyzable ATP analog AMP-PNP (5 mM) were also effective at enhancing the expression of I_AC, from a control value of 3.7 ± 0.1 pA/pF (n = 3) to maximum values of 48.5 ± 9.8 pA/pF (n = 11) and 67.3 ± 23.2 pA/pF (n = 6), respectively. At the single channel level, the unitary I_AC current amplitude did not vary with the ATP concentration or substitution with AMP-PNP. In addition to ATP and AMP-PNP, a number of other nucleotides including GTP, UTP, GDP, and UDP all increased the outwardly rectifying I_AC current with an apparent order of effectiveness: MgATP > ATP = AMP-PNP > GTP = UTP > ADP >> GDP > AMP and ATP-γ-S. Although ATP, GTP, and UTP all enhanced I_AC amplitude with similar effectiveness, inhibition of I_AC by ACTH (200 pM) occurred only in the presence of ATP. As little as 50 μM MgATP restored complete inhibition of I_AC, which had been activated by 5 mM UTP. Although the opening of I_AC channels may require only ATP binding, its inhibition by ACTH appears to involve a mechanism other than hydrolysis of this nucleotide. These findings describe a novel form of K⁺ channel modulation by which I_AC channels are activated through the nonhydrolytic binding of ATP. Because they are activated rather than inhibited by ATP binding, I_AC K⁺ channels may represent a distinctive new variety of K⁺ channel. The combined features of I_AC channels that allow it to sense and respond to changing ATP levels and to set the resting potential of AZF cells, suggest a mechanism where membrane potential, Ca²⁺ entry, and cortisol secretion could be tightly coupled to the metabolic state of the cell through the activity of I_AC K⁺ channels.     

Alternative splicing of the rat Ca(v)3.3 T-type calcium channel gene produces variants with distinct functional properties(1)

Molecular diversity in T-type Ca(2+) channels is produced by expression of three genes, and alternative splicing of those genes. Prompted by differences noted between rat and human Ca(v)3.3 sequences, we searched for splice variants. We cloned six variants, which are produced by splicing at exon 33 and exon 34. Expression of the variants differed between brain regions. The electrophysiological properties of the variants displayed similar voltage-dependent gating, but differed in their kinetic properties. The functional impact of splicing was inter-related, suggesting an interaction. We conclude that alternative splicing of the Ca(v)3.3 gene produces channels with distinct properties.     

Pharmacological evidence that LH-RH action on lordosis behavior is mediated through a rise in cAMP

The effect of two phosphodiesterase inhibitors, theophylline (THP) and 1-methyl-3-isobutyl-xanthine (MIX), on the lordosis response induced by three dose levels (0.5, 1, and 5 μg) of LH-RH was studied in ovariectomized estrogen-primed rats (estradiol benzoate, 4 μg). Neither THP (10 mg) nor MIX (2mg) facilitated lordosis behavior in estrogen-primed rats. Combined administration of 10 mg of THP with LH-RH exerted only a slight facilitatory effect on lordosis behavior. Administration of 2 mg of MIX significantly synergized with LH-RH for the facilitation of lordosis. The results suggest that LH-RH elicits sexual behavior by increasing cAMP levels in neurons related to the expression of lordosis behavior.     

Identification of a disulfide bridge essential for structure and function of the voltage-gated Ca(2+) channel α(2)δ-1 auxiliary subunit

Voltage-gated calcium (Ca(V)) channels are transmembrane proteins that form Ca(2+)-selective pores gated by depolarization and are essential regulators of the intracellular Ca(2+) concentration. By providing a pathway for rapid Ca(2+) influx, Ca(V) channels couple membrane depolarization to a wide array of cellular responses including neurotransmission, muscle contraction and gene expression. Ca(V) channels fall into two major classes, low voltage-activated (LVA) and high voltage-activated (HVA). The ion-conducting pathway of HVA channels is the α(1) subunit, which typically contains associated β and α(2)δ ancillary subunits that regulate the properties of the channel. Although it is widely acknowledged that α(2)δ-1 is post-translationally cleaved into an extracellular α(2) polypeptide and a membrane-anchored δ protein that remain covalently linked by disulfide bonds, to date the contribution of different cysteine (Cys) residues to the formation of disulfide bridges between these proteins has not been investigated. In the present report, by predicting disulfide connectivity with bioinformatics, molecular modeling and protein biochemistry experiments we have identified two Cys residues involved in the formation of an intermolecular disulfide bond of critical importance for the structure and function of the α(2)δ-1 subunit. Site directed-mutagenesis of Cys404 (located in the von Willebrand factor-A region of α(2)) and Cys1047 (in the extracellular domain of δ) prevented the association of the α(2) and δ peptides upon proteolysis, suggesting that the mature protein is linked by a single intermolecular disulfide bridge. Furthermore, co-expression of mutant forms of α(2)δ-1 Cys404Ser and Cys1047Ser with recombinant neuronal N-type (Ca(V)2.2α(1)/β(3)) channels, showed decreased whole-cell patch-clamp currents indicating that the disulfide bond between these residues is required for α(2)δ-1 function.     

Modulation of I A Potassium Current in Adrenal Cortical Cells by a Series of Ten Lanthanide Elements

The modulation of I _A K⁺ current by ten trivalent lanthanide (Ln³⁺) cations spanning the series with ionic radii ranging from 0.99 ? to 1.14 ? was characterized by the whole-cell patch clamp technique in bovine adrenal zona fasciculata (AZF) cells. Each of the ten Ln³⁺s reduced I _A amplitude measured at +20 mV in a concentration-dependent manner. Smaller Ln³⁺s were the most potent and half-maximally effective concentrations (EC₅₀s) varied inversely with ionic radius for the larger elements. Estimation of EC₅₀s yielded the following potency sequence: Lu³⁺ (EC₅₀= 3.0 μm) ≈ Yb³⁺ (EC₅₀= 2.7 μm) > Er³⁺ (EC₅₀= 3.7 μm) ≥ Dy³⁺ (EC₅₀= 4.7 μm) > Gd³⁺ (EC₅₀= 6.7 μm) ≈ Sm³⁺ (EC₅₀= 6.9 μm) > Nd³⁺ (EC₅₀= 11.2 μm) > Pr³⁺ (EC₅₀= 22.3 μm) > Ce³⁺ (EC₅₀= 28.0 μm) > La³⁺ (EC₅₀= 33.7 μm). Ln³⁺s altered selected voltage-dependent gating and kinetic parameters of I _A with a potency and order of effectiveness that paralleled the reduction of I _A amplitude. Ln³⁺s markedly slowed activation kinetics and shifted the voltage-dependence of I _A gating such that activation and steady-state inactivation occurred at more depolarized potentials. In contrast, Ln³⁺s did not measurably alter inactivation or deactivation kinetics and only slightly slowed kinetics of inactivated channels returning to the closed state. Replacement of external Ca²⁺ with Mg²⁺ had no effect on the concentration-dependent inhibition of I _A by Ln³⁺s. In contrast to their action on I _A K⁺ current, Ln³⁺s inhibited T-type Ca²⁺ currents in AZF cells without slowing activation kinetics. These results indicate that Ln³⁺ modulate I _A K⁺ channels through binding to a site on I _A channels located within the electric field but which is not specific for Ca²⁺. They are consistent with a model where Ln³⁺ binding to negative charges on the gating apparatus alters the voltage-dependence and kinetics of channel opening. Ln³⁺s modulate transient K⁺ and Ca²⁺ currents by two fundamentally different mechanisms. Received: 21 January 1997/Revised: 3 April 1998     

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.