Testosterone-mediated modulation of HERG blockade by proarrhythmic agents.

Diverse drugs from many therapeutic classes exert cardiotoxic side effects by inducing torsades de pointes (TdP), a life threatening cardiac arrhythmia, which often results from drug interaction with HERG (human ether-a-go-go related gene) encoded K(+) channels, that generate an I(Kr) component of the delayed rectifier cardiac K(+) current. Men are known to be at a lower risk for drug-induced TdP than women suggesting a role of sex steroid hormones, androgens and estrogens, in modulation of drug sensitivity of cardiac K(+) channels, particularly those encoded by HERG. Here by using neuroleptic agents haloperidol, pimozide, and fluspirilene, all of which can induce TdP, and a steroid hormone-sensitive system Xenopus oocytes for HERG channels expression we show that testosterone is able to reduce HERG-blocking potency of neuroleptics. Haloperidol, pimozide, and fluspirilene inhibited HERG current with IC(50) of 1.36, 1.74, and 2.34 microM, and maximal block of 73%, 76% and 65%, respectively. The action of these neuroleptics was voltage-dependent, most consistent with an open-channel blocking mechanism. Pretreatment of HERG-expressing oocytes with 1 microM testosterone increased the IC(50) values to 2.73, 2.08, and 5.04 microM, reduced the maximal block to 65%, 59%, and 64%, and strongly diminished voltage-dependence of the blockade. Testosterone treatment per se produced about a 35% reduction of HERG current compared with untreated oocytes. Our data suggest that androgens may protect against the arrhythmogenic actions of some cardiotoxic drugs.

Syntaxin modulation of slow inactivation of N-type calcium channels.

Syntaxin, a membrane protein vital in triggering vesicle fusion, interacts with voltage-gated N- and P/Q-type Ca(2+) channels. This biochemical association is proposed to colocalize Ca(2+) channels and presynaptic release sites, thus supporting rapid and efficient initiation of neurotransmitter release. The syntaxin channel interaction may also support a novel signaling function, to modulate Ca(2+) channels according to the state of the associated release machinery (Bezprozvanny et al., 1995; Wiser et al., 1996; see also Mastrogiacomo et al., 1994). Here we report that syntaxin 1A (syn1A) coexpressed with N-type channels in Xenopus oocytes greatly promoted slow inactivation gating, but had little or no effect on the onset of and recovery from fast inactivation. Accordingly, the effectiveness of syntaxin depended strongly on voltage protocol. Slow inactivation was found for N-type channels even in the absence of syntaxin and could be distinguished from fast inactivation on the basis of its slow kinetics, distinct voltage dependence (voltage-independent at potentials higher than the level of half-inactivation), and temperature independence (Q(10), approximately 0.8). Trains of action potential-like stimuli were more effective than steady depolarizations in stabilizing the slowly inactivated condition. Agents that stimulate protein kinase C decreased the inhibitory effect of syntaxin on N-type channels. Application of BoNtC1 to cleave syntaxin sharply attenuated the modulatory effects on Ca(2+) channel gating, consistent with structural analysis of syntaxin modulation, supporting use of this toxin to test for the impact of syntaxin on Ca(2+) influx in nerve terminals.

Calcium channel block by (-)devapamil is affected by the sequence environment and composition of the phenylalkylamine receptor site.

The pore-forming alpha 1 subunit of L-type calcium (Ca2+) channels is the molecular target of Ca2+ channel blockers such as phenylalkylamines (PAAs). Association and dissociation rates of (-)devapamil were compared for a highly PAA-sensitive L-type Ca2+ channel chimera (Lh) and various class A Ca2+ channel mutants. These mutants carry the high-affinity determinants of the PAA receptor site in a class A sequence environment. Apparent drug association and dissociation rate constants were significantly affected by the sequence environment (class A or L-type) of the PAA receptor site. Single point mutations affecting the high-affinity determinants in segments IVS6 of the PAA receptor site, introduced into a class A environment, reduced the apparent drug association rates. Mutation I1811M in transmembrane segment IVS6 (mutant AL25/-I) had the highest impact and decreased the apparent association rate for (-)devapamil by approximately 30-fold, suggesting that this pore-lining isoleucine in transmembrane segment IVS6 plays a key role in the formation of the PAA receptor site. In contrast, apparent drug dissociation rates of Ca2+ channels in the resting state were almost unaffected by point mutations of the PAA receptor site.

Receptors couple to L-type calcium channels via distinct Go proteins in rat neuroendocrine cell lines.

1. The present study examines the hypothesis of G protein subtype selectivity in receptor-induced inhibition of calcium channel currents (ICa) in the insulin-secreting RINm5F and pituitary GH3 rat cell lines. Specificity of receptor coupling to G proteins was studied by infusion of purified G alpha isoforms into cells via a patch pipette. 2. In RINm5F cells, the neuropeptide galanin inhibited dihydropyridine (DHP)- and omega-conotoxin-sensitive components of ICa and slowed down their activation kinetics. In GH3 cells, DHP-sensitive ICa was inhibited by galanin, as well as by somatostatin and carbachol. Agonist-induced ICa inhibition was suppressed by pertussis toxin (PTX) pretreatment of the cells. In PTX-pretreated cells of either cell line, the response to galanin was restored only by the G alpha o1 subunit. Following PTX treatment of GH3 cells, only the G alpha o1 subunit restored carbachol-induced inhibition of ICa, whereas only the G alpha o2 subunit restored somatostatin-induced inhibition of ICa. G(i) subtypes had no effect on ICa inhibition. 3. Both cell lines expressed two distinct immunoreactive Go proteins. Whereas in RINm5F cell membranes Go1 was found to be the predominant isoform, we detected more Go2 than Go1 in GH3 cell membranes. Nevertheless, all agonists stimulated incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into both G(o) isoforms. 4. The results indicate that the same Go subtype, i.e. Go1, mediates galanin-induced inhibition of ICa in both cell lines and that the Go subtype specificity of receptor-G protein coupling is confined to intact cells.

Transfer of high sensitivity for benzothiazepines from L-type to class A (BI) calcium channels.

To investigate the molecular basis of the calcium channel block by diltiazem, we transferred amino acids of the highly sensitive and stereoselective L-type (alpha1S or alpha1C) to a weakly sensitive, nonstereoselective class A (alpha1A) calcium channel. Transfer of three amino acids of transmembrane segment IVS6 of L-type alpha1 into the alpha1A subunit (I1804Y, S1808A, and M1811I) was sufficient to support a use-dependent block by diltiazem and by the phenylalkylamine (-)-gallopamil after expression in Xenopus oocytes. An additional mutation F1805M increased the sensitivity for (-)-gallopamil but not for diltiazem. Our data suggest that the receptor domains for diltiazem and gallopamil have common but not identical molecular determinants in transmembrane segment IVS6. These mutations also identified single amino acid residues in segment IVS6 that are important for class A channel inactivation.

Transfer of L-type calcium channel IVS6 segment increases phenylalkylamine sensitivity of alpha1A.

Conditioned ("use-dependent") inhibition by phenylalkylamines (PAAs) is a characteristic property of L- type calcium (Ca2+) channels. To determine the structural elements of the PAA binding domain we transferred sequence stretches of the pore-forming regions of repeat III and/or IV from the skeletal muscle alpha1 subunit (alpha1S) to the class A alpha1 subunit (alpha1A and expressed these chimeras together with beta1a and alpha2/delta subunits in Xenopus oocytes. The corresponding barium currents (IBa) were tested for PAA sensitivity during trains of depolarizing test pulses (conditioned block). IBa of oocytes expressing the alpha1A subunit were only weakly inhibited by PAAs (less than 10% conditioned block of IBa during a 100-ms pulse train of 0.1 Hz). Transfer of the transmembrane segment IVS6 from alpha1S to alpha1A produced an enhancement of PAA sensitivity of the resulting alpha1A/alpha1S chimera comparable to L-type alpha1 subunits (about 35% conditioned block Of IBa during a 100-ms pulse train of 0.1 Hz). Our results demonstrate that substitution of 11 amino acids within the segment RVS6 of alpha1A with the corresponding residues of alpha1S is sufficient to transfer L-type PAA sensitivity into the low sensitive class A Ca2+ channel.

A specific G(o) heterotrimer couples somatostatin receptors to voltage-gated calcium channels in RINm5F cells.

The peptide hormone somatostatin inhibits glucose-induced insulin secretion in the rat insulinoma RINm5F cells by inhibition of voltage-gated calcium channels. Here we used micro-injection of antisense oligonucleotides directed against subtypes of G-protein subunits to determine the subunit composition involved in somatostatin-induced inhibition of voltage-gated calcium channels in RINmF5 cells. Injection of antisense oligonucleotides annealing to the respective mRNA of G alpha o2, G beta 1 and G gamma 3 reduced the somatostatin-induced inhibition of calcium channels in these cells. Injection of antisense oligonucleotides directed against other G-protein subunits did not, suggesting that in RINm5F cells the somatostatin receptor couples to a G protein of G alpha o2 beta 1 gamma 3 composition. By using a selective agonist of type 2 somatostatin receptors (SSTR 2) NC 8-12, we identified this receptor as the subtype coupling to calcium channels in RINm5F cells.

Ca(2+)-permeable large-conductance nonselective cation channels in rat basophilic leukemia cells.

Spreading of Ca2+ signals in rat basophilic leukemia (RBL) cells occurs by release of ATP. Therefore we studied the effect of ATP on membrane currents. ATP (1-10 microM) activated large-conductance channels. Single channel events were resolved in the whole cell mode. Similar channel activity was observed in RBL cells transfected with the muscarinic M1 receptor after stimulation with carbachol as well as after intracellular infusion of aluminum fluoride. Activation was independent of internal Ca2+ (0-10 microM). The channels had a conductance of 250 pS in 135 mM Na+ and 70 pS in 100 mM Ca2+. The permeability (P) ratio was PCa/PNa/PCs/PMg = 16:1:0.6:0.6. These channels may contribute to secretory responses by allowing Ca2+ entry, leading to high Ca2+ concentrations in the vicinity of the channel pore.

Subunit composition of G(o) proteins functionally coupling galanin receptors to voltage-gated calcium channels.

The neuropeptide galanin is widely expressed in the central nervous system and other tissues and induces different cellular reactions, e.g. hormone release from pituitary and inhibition of insulin release from pancreatic B cells. By microinjection of antisense oligonucleotides we studied the question as to which G proteins mediate the galanin-induced inhibition of voltage-gated Ca2+ channels in the rat pancreatic B-cell line RINm5F and in the rat pituitary cell line GH3. Injection of antisense oligonucleotides directed against alpha 01, beta 2, beta 3, gamma 2 and gamma 4 G protein subunits reduced the inhibition of Ca2+ channel current which was induced by galanin, whereas no change was seen after injection of cells with antisense oligonucleotides directed against alpha i, alpha q, alpha 11, alpha 14, alpha 15, beta 1, beta 4, gamma 1, gamma 3, gamma 5, or gamma 7 G protein subunits or with sense control oligonucleotides. In view of these data and of previous results, we conclude that the galanin receptors in GH3 and in RINm5F cells couple mainly to the G(0) protein consisting of alpha 01 beta 2 gamma 2 to inhibit Ca2+ channels and use alpha 01beta 3 gamma 4 less efficiently. The latter G protein composition was previously shown to be used by muscarinic M4 receptors to inhibit Ca2+ channels.

The isoquinoline derivative LOE 908 selectively blocks vasopressin-activated nonselective cation currents in A7r5 aortic smooth muscle cells.

The effect of (R,S)-(3,4-dihydro 6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl- N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide (LOE 908), a cation channel blocker in HL-60 promyeloblasts, was studied in the A7r5 smooth muscle cell line from rat thoracic aorta, using the whole-cell patch-clamp technique. At a holding potential of -60 mV, application of vasopressin induced a nonselective cation conductance in voltage-clamped A7r5 cells. The current-voltage relation was linear, and currents reversed close to 0 mV regardless of the chloride gradient. The activation of the nonselective cation conductance by vasopressin was not affected by dialysing cells with Ca(2+)-free internal solution. LOE 908 blocked this current in a concentration-dependent manner with an IC50 of 560 nM, whereas dihydropyridine-sensitive Ba2+ current through voltage-dependent Ca2+ channels was blocked with an IC50 of 28 microM. Another organic blocker of receptor-mediated Ca2+ entry, 1-beta-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl-1H-imidazole hydrochloride (SK&F 96365), blocked both, the vasopressin-induced nonselective conductance and the voltage-activated Ba2+ current with similar IC50 values of 13 microM and 8 microM, respectively. The rank order of potency of inorganic blockers on the vasopressin-induced inward current was Gd3+ > La3+ > Cd2+. Vasopressin-induced non-selective cation current was also observed in pertussis toxin-pretreated A7r5 cells but was completely abolished after infusion of the GDP analogue, guanosine 5'-O-[3-thio]diphosphate, from the patch pipette. Furthermore, vasopressin induced a transient outward current, suggesting a Ca(2+)-activated K(+)-current, which overlapped with the nonselective cation conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effect of ammonium perchlorate on calcium currents in neurons and leukocyte mobility]. / Vliianie perkhlorata ammoniia na kal'tsievye toki v neironakh podvizhnost' leikotsitov.

NH4ClO4 has been studied for its effect on the voltage-activated calcium current (ICa) in voltage-clamp experiments on intracellularly perfused snail neurons and on the chemokinesis of rat leukocytes. It has been found that NH4ClO4 enhances ICa amplitude 1.5-5 times (EC50 = 3 mmol/l), decreases the rate of ICa decay and stimulates mobility of leukocytes 3 times (EC50 = 2.5 mmol/l). It is suggested that these changes may be intermediated by similar mechanisms, e.g. by an increase in intracellular pH.

[Features of the effect of leukotriene LTC4 and the blocker of its biosynthesis BW755C on the contractile capability of smooth muscles and the functioning of potential-dependent calcium channels of the nerve cell]. / Osobennosti vliianiia leikotriena LTC4 i blokatora ego biosinteza BW755C na sokratitel'nuiu sposobnost' gladkikh myshts i funktsionirovanie potentsialozavisimykh kal'tsievykh kanalov nervnoi kletki.

The effect of LTC4, a synthetic leukotriene, and of BW 755C, a blocker of its biosynthesis on the functioning of Ca-channels in the membrane of snail neurons and on the contractile activity of rat uterus muscles in late terms of pregnancy was studied by means of intracellular dialysis and spontaneous contractility recording techniques. It was found that the 10(-7) mol/l of LTC4 stimulated the contractile function of the rat uterus and activated effectively the Ca-conductance in the somatic membrane of the nervous cell. The action of BW 755C was similar to that of leukotriene on Ca-conductance; in addition BW 755C produced transient activation of contractions of the rat uterus followed by a complete suppression of smooth muscle contractions. Possible mechanisms of the effects described were discussed.

[Action of the leukotriene LTC4 on the calcium channels of the somatic membrane of the nerve cell and tone of smooth muscle organs]. / Deistvie leikotriena LTC4 na kal'tsievye kanaly somaticheskoi membrany nervnoi kletki i tonus gladkomyshechnykh organov.

Effects of synthetic leukotriene LTC4 on the internally perfused voltage-clamped snail neurons as well as on the electrical and mechanical activities of the stomach smooth muscle cells were studied. It was found that LTC4 increased calcium inward current in the soma membrane of a cell by activation of both voltage and receptor operated Ca-channels. LTC4 was effective only from outside but not from inside of the membrane which indicated that there was a receptor for leukotrienes in the neuronal membrane.

[Effect of the lipoxygenase blockader BW755C on calcium currents in the nerve cell membrane of the snail and on the contractile responses of the ureteral smooth-muscle fibers in the guinea pig]. / Vliianie blokatora lipooksigenazy BW755C na kal'tsievye toki v membrane nervnoi kletki ulitki i sokratitel'nye otvety gladkomyshechnykh volokon mochetochnika morskoi svinki.

Lipoxygenase blocker BW755C has been studied for its effect on calcium inward current in intracellularly perfused voltage clamped and contraction of guinea-pig ureter smooth muscle. BW755C increased the Ca inward current in somatic membrane and inhibited contraction of the smooth muscle evoked by high-potassium solution. It is suggested that the effects observed are related to the changes in the level of cyclic nucleotides in cytoplasm due to the action of the final products of lypoxygenase breakdown of fatty acids from the cell membrane.