Chemical synthesis and characterization of ShK toxin: a potent potassium channel inhibitor from a sea anemone.

ShK-toxin, a 35 residue peptide isolated from the sea anemone Stichodactyla helianthus, was synthesized using an Fmoc strategy and successfully folded to the biologically active form containing three intramolecular disulfide bonds. The ability of synthetic ShK toxin to inhibit specific [125I]-dendrotoxin I binding to rat brain membranes slightly exceeded (was more potent than) that of the natural ShK toxin sample, but was comparable with previously reported data for ShK toxin. The peptide toxin inhibited [125I]-charybdotoxin binding to Jurkat T lymphocytes with an IC50 value of 32 pM. In addition, Jurkat T lymphocytes Kv1.3 potassium channels were inhibited with an IC50 value of 133 pM. Owing to their unique structure and high affinity for at least some potassium channels, ShK toxin and related sea anemone potassium channel toxins may become useful molecular probes for investigating potassium channels.

Synthesis and structure-activity relationships of 6-heterocyclic-substituted purines as inactivation modifiers of cardiac sodium channels.

Purine-based analogs of SDZ 211-500 (5) were prepared and evaluated as inactivation modifiers of guinea pig or human cardiac sodium (Na) channels expressed in Xenopus oocytes. Substances which remove or slow the Na channel inactivation process in cardiac tissue are anticipated to prolong the effective refractory period and increase inotropy and thus have potential utility as antiarrhythmic agents. Heterocyclic substitution at the 6-position of the purine ring resulted in compounds with increased Na activity and potency, with 5-membered heterocycles being optimal. Only minor modifications to the benzhydrylpiperazine side chain were tolerated. Selected compounds which delayed the inactivation of Na channels were found to increase refractoriness and contractility in a rabbit Langendorff heart model, consistent with the cellular mechanism. Activity in both the oocyte and rabbit heart assays was specific to the S enantiomers. Preliminary in vivo activity has been demonstrated following intravenous infusion. The most promising compound on the basis of in vitro data is the formylpyrrole (S)-74, which is 25-fold more potent than DPI 201-106 (1) in the human heart Na channel assay.

Novel inhibitors of potassium ion channels on human T lymphocytes.

The in vitro biological characterization of a series of 4-(alkylamino)-1,4-dihydroquinolines is reported. These compounds are novel inhibitors of voltage-activated n-type potassium ion (K+) channels in human T lymphocytes. This series, identified from random screening, was found to inhibit [125I]charybdotoxin binding to n-type K+ channels with IC50 values ranging from 10(-6) to 10(-8) M. These analogs also inhibit whole cell n-type K+ currents with IC50 values from 10(-5) to 10(-7) M. The preparation of a series of new 4-(alkylamino)-1,4-dihydroquinolines is described. Structure-activity relationships are discussed. Naphthyl analog 7c, the best compound prepared, exhibited > 100-fold selectivity for inhibition of [125I]charybdotoxin binding to n-type K+ channels compared with inhibition of [3H]dofetilide binding to cardiac K+ channels. These compounds represent a potent and selective series of n-type K+ channel inhibitors that have the potential for further development as anti-inflammatory agents.

Stable expression and functional characterization of a human cardiac Na+ channel gene in mammalian cells.

In order to develop mammalian cell lines expressing a functional human heart Na+ channel gene (hH1), Chinese hamster ovary (CHO-K1) cells and HeLa cells were transfected with the hH1 gene and the bacterial neomycin (neo) resistance gene. In CHO-K1 cells, direct screening for hH1-positive, G418-resistant colonies by functional patch clamp analysis was complicated due to low-level endogenous expression of a brain-type Na+ channel. Therefore, we developed a stepwise strategy for isolation of cell lines expressing functional hH1 Na+ channels: G418-resistant colonies were sequentially analysed for (1) chromosomal integration of hH1 DNA by PCR, (2) specific hH1 mRNA expression by RT-PCR, (3) hH1 protein production by immunoprecipitation with hH1-specific antisera, and (4) hH1 Na+ channel function by patch-clamp analysis. Using this strategy we obtained two CHO-K1 cell lines which express functional human heart Na+ channels. However, using the same strategy, we were unsuccessful in obtaining functional, hH1-positive HeLa cell lines, even though hH1 mRNA and protein was produced in these cells. The two CHO-K1 cell lines stably express human cardiac Na+ channels which retain normal electrophysiological characteristics with respect to activation and inactivation. In addition, the Na+ channels expressed in these cells are blocked by tetrodotoxin with an IC50 value of 2.5 microM; consistent with known cardiac Na+ channel pharmacology. The density of channels is high enough to permit recording of pseudomacroscopic currents in excised outside-out patches of membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Voltage dependence of cardiac delayed rectifier block by methanesulfonamide class III antiarrhythmic agents.

Voltage-clamp experiments were performed on isolated guinea pig ventricular myocytes to examine the voltage dependence of delayed rectifier block by methanesulfonamide channel blockers. Voltage-dependent channel block, in which block decreases as membrane potential is made more positive, could contribute to the phenomenon of reverse use dependence, in which the magnitude of the drug-induced prolongation in action potential duration (APD) is inversely proportional to stimulation rate. To determine whether such a voltage dependence exists, concentration-response curves were generated for dofetilide, E-4031, sematilide, and D,L-sotalol at test potentials ranging from 0 to 60 mV. All agents blocked current in a manner consistent with selective blockade of the rapidly activating component of delayed rectifier current, IKr. The rank order of potency was E-4031 approximately dofetilide > sematilide > sotalol. Block of tail currents by this class of compounds was more potent after test potentials to +60 mV than after those < or = 0-10 mV. These data are inconsistent with voltage-dependent channel block being a contributing factor to reverse use-dependence and suggest that other mechanisms must be responsible for this phenomenon.

Characterization of endogenous sodium channel gene expressed in Chinese hamster ovary cells.

Chinese hamster ovary (CHO-K1) cells were observed to display transient inward Na+ currents of average amplitude (-92 +/- 20 pA), which activated at voltages more than -40 mV, and peak inward currents were observed at potentials equal to or more than +10 mV. Inward Na+ currents in these cells were eliminated after treatment with 500 or 50 nM tetrodotoxin (TTX), whereas 5 nM TTX resulted in 64 +/- 10% inhibition of Na+ current. Using DNA primers designed to bind to the rat brain IIA Na+ channel subtype, we amplified specific polymerase chain reaction (PCR) fragments from CHO-K1 poly-(A)+RNA. The cloning and sequencing of two of these fragments confirmed the presence of an endogenously expressed Na+ channel gene in these cells, which we have termed cho 1. Comparison of the DNA sequence of cho 1 PCR fragments with other known Na+ channel genes indicated a high degree of homology with rat brain Na+ channel subtypes. Northern blots using riboprobes generated from the cho 1 PCR fragments revealed the presence of a specific 9-kb mRNA in these cells. The molecular and electrophysiological data suggest that the cho 1 Na+ channel gene from CHO-K1 cells is closely related to brain-type Na+ channels.

Identification of a specific radioligand for the cardiac rapidly activating delayed rectifier K+ channel.

Class III antiarrhythmic drugs show promise as effective treatments for the suppression of potentially lethal cardiac arrhythmias. Dofetilide (UK-68,798), is a potent class III antiarrhythmic agent that is presently under clinical investigation. The objective of this study was to determine whether [3H]dofetilide could be used as a specific radioligand for the rapidly activating delayed rectifier K+ channel of the heart. We find that [3H]dofetilide binds to high-affinity sites on guinea pig cardiac myocytes. Competition studies using unlabeled dofetilide indicate that binding is characterized by an IC50 of 100 +/- 30 nM (mean +/- SD, n = 13). Scatchard analyses of binding indicate a Kd of 70 +/- 6 nM and a maximal binding capacity of 0.30 +/- 0.02 pmol/mg protein. [3H]Dofetilide is displaced from guinea pig myocytes by dofetilide, clofilium, quinidine, sotalol, and sematilide with a rank order of potency that correlates with functional blockade of the rapidly activating delayed rectifier K+ current (correlation coefficient, 0.951; slope, 0.99 +/- 0.19; p = 0.014). High-affinity [3H]dofetilide binding is not detected in rat myocytes, which are devoid of delayed rectifier K+ current. We conclude that [3H]dofetilide specifically binds to sites associated with the rapidly activating delayed rectifier K+ channel of guinea pig myocardium.

Properties of endogenous voltage-dependent sodium currents in Xenopus laevis oocytes.

Endogenous voltage-dependent sodium currents were recorded using standard 2-microelectrode techniques in Xenopus laevis oocytes. Maximal inward current occurred at -10 mV with an average amplitude of -279 +/- 17 nA and steady-state inactivation was half-maximal at a voltage of -38 +/- 0.5 mV. Currents were blocked by low concentrations of tetrodotoxin (TTX) with an IC50 value of 6 nM. These properties make the endogenous sodium current in Xenopus oocytes similar to sodium currents expressed following injection of mammalian brain RNA. While endogenous sodium channels have the potential to complicate analysis when using the oocyte expression system, they are only present at significant levels in rare batches of oocytes (less than 5%). Our results do stress the need, however, to reproduce results from exogenous expression studies across several batches of oocytes from different donors.

Expression of cardiac Na channels with appropriate physiological and pharmacological properties in Xenopus oocytes.

The objective of this study was to determine whether the Xenopus laevis oocyte can express an exogenous cardiac Na channel that retains its normal physiological and pharmacological properties. Cardiac Na channels were expressed in oocytes following injection of RNA from guinea pig, rat, and human heart and detailed analysis was performed for guinea pig cardiac Na channels. Average current amplitudes were -351 +/- 37 nA with peak current observed at -8 +/- 1 mV. Steady-state inactivation was half-maximal at -49 +/- 0.6 mV for the expressed channels. All heart Na currents were resistant to block by tetrodotoxin compared to Na currents expressed from brain RNA with IC50 values for guinea pig, rat, and human heart of 651 nM, 931 nM, and 1.3 microM, respectively. In contrast, rat brain Na channels were blocked by tetrodotoxin with an IC50 value of 9.1 nM. In addition, the effects of the cardiac-selective agents lidocaine and DPI 201-106 were examined on Na currents expressed from brain and heart RNA. Lidocaine (10 microM) blocked cardiac Na current in a use-dependent manner but had no effect on brain Na currents. DPI 201-106 (10 microM) slowed the rate of cardiac Na channel inactivation but had no effect on inactivation of brain Na channels. These results indicate the Xenopus oocyte system is capable of synthesizing and expressing cardiac Na channels that retain normal physiological and pharmacological properties.

Inhibition of calmodulin and protein kinase C by amiodarone and other class III antiarrhythmic agents.

Class III antiarrhythmic agents may prolong refractoriness via modulation of ion channels, which may be sensitive to Ca2+ regulatory proteins or enzymes. Accordingly, the purpose of this study was to quantitate the effects of several structurally diverse class III antiarrhythmic agents on calmodulin-regulated enzymes and protein kinase C activity, and to evaluate the ability of these agents and known calmodulin antagonists to prolong cardiac refractoriness in vivo. The rank order of potency (IC50;microM) of selected class III antiarrhythmic agents and reference calmodulin antagonists as inhibitors of calmodulin-regulated phosphodiesterase activity were: calmidazolium (0.12 microM) greater than amiodarone (0.62 microM) greater than desethylamiodarone (1.5 microM) greater than trifluoperazine (4.3 microM), bepridil (5 microM) greater than W-7 (7.5 microM), clofilium (13 microM). Similar concentration-related inhibition was evident in a second calmodulin-regulated system, inhibition of myosin light-chain phosphorylation and superprecipitation of arterial actomyosin. Sotalol and tetraethylammonium were inactive at 100 microM. Protein kinase C activity was also inhibited by some of these agents; desethylamiodarone (IC50 = 11 microM) was more potent than the reference agent, H-7 (IC50 = 79 microM), or amiodarone (38% inhibition at 100 microM) and clofilium (32% inhibition at 100 microM). In vivo, the minimally effective doses required to increase ventricular effective refractory periods in paced guinea pigs were (in mg/kg) bepridil, sotalol [1] greater than clofilium [3] greater than amiodarone [10] greater than W-7, desethylamiodarone [20]. No changes in refractory period were noted with maximum testable doses of calmidazolium or trifluoperazine. These studies show that some, but not all, class III antiarrhythmic agents are effective and potent calmodulin antagonists or protein kinase C inhibitors. Moreover, some calmodulin antagonists are effective at prolonging refractoriness in vivo. However, a lack of correlation between these agents suggests that these mechanisms are not solely responsible for the prolongation of refractoriness of all class III agents.