Changes in sodium channel gating produced by point mutations in a cytoplasmic linker.

Voltage-gated sodium channels are transmembrane proteins of approximately 2000 amino acids and consist of four homologous domains (I through IV). In current topographical models, domains III and IV are linked by a highly conserved cytoplasmic sequence of amino acids. Disruptions of the III-IV linker by cleavage or antibody binding slow inactivation, the depolarization-induced closed state characteristic of sodium channels. This linker might be the positively charged "ball" that is thought to cause inactivation by occluding the open channel. Therefore, groups of two or three contiguous lysines were neutralized or a glutamate was substituted for an arginine in the III-IV linker of type III rat brain sodium channels. In all cases, inactivation occurred more rapidly rather than more slowly, contrary to predictions. Furthermore, activation was delayed in the arginine to glutamate mutation. Hence, the III-IV linker does not simply act as a charged blocker of the channel but instead influences all aspects of sodium channel gating.

Beta-adrenergic inhibition of cardiac sodium channels by dual G-protein pathways.

The signaling pathways by which beta-adrenergic agonists modulate voltage-dependent cardiac sodium currents are unknown, although it is likely that adenosine 3'5'-monophosphate (cAMP) is involved. Single-channel and whole-cell sodium currents were measured in cardiac myocytes and the signal transducing G protein Gs was found to couple beta-adrenergic receptors to sodium channels by both cytoplasmic (indirect) and membrane-delimited (direct) pathways. Hence, Gs can act on at least three effectors in the heart: sodium channels, calcium channels, and adenylyl cyclase. The effect on sodium currents was inhibitory and was enhanced by membrane depolarization. During myocardial ischemia the sodium currents of depolarized cells may be further inhibited by the accompanying increase in catecholamine levels.

Exchange of conduction pathways between two related K+ channels.

The structure of the ion conduction pathway or pore of voltage-gated ion channels is unknown, although the linker between the membrane spanning segments S5 and S6 has been suggested to form part of the pore in potassium channels. To test whether this region controls potassium channel conduction, a 21-amino acid segment of the S5-S6 linker was transplanted from the voltage-activated potassium channel NGK2 to another potassium channel DRK1, which has very different pore properties. In the resulting chimeric channel, the single channel conductance and blockade by external and internal tetraethylammonium (TEA) ion were characteristic of the donor NGK2 channel. Thus, this 21-amino acid segment controls the essential biophysical properties of the pore and may form the conduction pathway of these potassium channels.

K+ pore structure revealed by reporter cysteines at inner and outer surfaces.

The structure of the carboxyl half of the pore-forming region of Kv2.1 was studied by replacing each of 15 consecutive residues between positions 383 and 369 with a reporter cysteine residue. Extracellular application of charged, membrane-impermeant methanethiosulfonates irreversibly modified currents at four cysteine-substituted positions, K382, Y380, I379, and D378. Intracellular exposure to methanethiosulfonate ethyltrimethylammonium revealed another set of reactive mutants (V374, T373, T372, and T370). Our results indicate that positions 378 and 374 are exposed at outer and inner mouths of the channel, respectively, and immersed in the aqueous phase. In contrast to present topological models, the 383-369 region appears to span the pore mainly as a nonperiodic structure.

Segmental exchanges define 4-aminopyridine binding and the inner mouth of K+ pores.

4-Aminopyridine (4AP) blocks the intracellular mouth of voltage-gated K+ channels. We identified critical regions for 4AP binding with chimeric channels in which segments of a low affinity clone (Kv2.1, IC50 = 18 mM) were replaced with those of a high affinity clone (Kv3.1, IC50 = 0.1 mM). 4AP sensitivity was not transferred with the S5-S6 linker (pore or P region). Instead, a chimera of the cytoplasmic half of S6 increased block 20-fold, without affecting gating. A double chimera of the cytoplasmic halves of S5 and S6 fully transferred 4AP sensitivity. Because 4AP block was inhibited by tetrapentylammonium, we conclude that determinants of 4AP binding lie in the S6 segment that forms the cytoplasmic vestibule of the pore and that this site may overlap a quaternary ammonium site.

Fast and slow gating of sodium channels encoded by a single mRNA.

We investigated the kinetics of rat brain type III Na+ currents expressed in Xenopus oocytes. We found distinct patterns of fast and slow gating. Fast gating was characterized by bursts of longer openings. Traces with slow gating occurred in runs with lifetimes of 5 and 30 s and were separated by periods with lifetimes of 5 and 80 s. Cycling of fast and slow gating was present in excised outside-out patches at 10 degrees C, suggesting that metabolic factors are not essential for both forms of gating. It is unlikely that more than one population of channels was expressed, as patches with purely fast or purely slow gating were not observed. We suggest that structural mechanisms for fast and slow gating are encoded in the primary amino acid sequence of the channel protein.

Differences between the deep pores of K+ channels determined by an interacting pair of nonpolar amino acids.

The pore of a chimeric K+ channel, CHM, differed from its parental host channel, Kv2.1, by 9 amino acids. Four were located in a putative deep region and 5 in a nearby outer mouth. Point reversions were without restorative effects, and reversions V369I or L374V in the deep pore produced novel phenotypes. Among double mutations, only V369I and L374V were effective in restoring the Kv2.1 pore phenotype. Adding a change in charge at Q382K in the outer pore fully restored the parental phenotype. Thus, the pore appears to have an inner, deep region where ions such as K+ and TEA+ may be regulated by nonpolar residues and an outer region where ions may be regulated by charged residues.

Synthesis and characterization of dicyanovinyl-substituted thienylpyrroles as new nonlinear optical chromophores.

[structure: see text] New dicyanovinyl-substituted 1-(alkyl)aryl-2-(2'-thienyl)pyrroles 2 were synthesized and characterized. The solvatochromic behavior of the synthesized compounds was investigated. All the derivatives showed reversible oxidation and reduction on the CV time scale. The hyperpolarizabilities (beta) of compounds 2 were measured using hyper-Rayleigh scattering. The results are among the highest beta values reported for donor-acceptor-substituted thienylpyrroles.

Sodium channel blockers identify risk for sudden death in patients with ST-segment elevation and right bundle branch block but structurally normal hearts.

BACKGROUND: A mutation in the cardiac sodium channel gene (SCN5A) has been described in patients with the syndrome of right bundle branch block, ST-segment elevation in leads V1 to V3, and sudden death (Brugada syndrome). These electrocardiographic manifestations are transient in many patients with the syndrome. The present study examined arrhythmic risk in patients with overt and concealed forms of the disease and the effectiveness of sodium channel blockers to unmask the syndrome and, thus, identify patients at risk. METHODS AND RESULTS: The effect of intravenous ajmaline (1 mg/kg), procainamide (10 mg/kg), or flecainide (2 mg/kg) on the ECG was studied in 34 patients with the syndrome and transient normalization of the ECG (group A), 11 members of 3 families in whom a SCN5A mutation was associated with the syndrome and 8 members in whom it was not (group B), and 53 control subjects (group C). Ajmaline, procainamide, or flecainide administration resulted in ST-segment elevation and right bundle branch block in all patients in group A and in all 11 patients with the mutation in group B. A similar pattern could not be elicited in the 8 patients in group B who lacked the mutation or in any person in group C. The follow-up period (37+/-33 months) revealed no differences in the incidence of arrhythmia between the 34 patients in whom the phenotypic manifestation of the syndrome was transient and the 24 patients in whom it was persistent (log-rank, 0.639). CONCLUSIONS: The data demonstrated a similar incidence of potentially lethal arrhythmias in patients displaying transient versus persistent ST-segment elevation and right bundle branch block, as well as the effectiveness of sodium channel blockers to unmask the syndrome and, thus, identify patients at risk.

Gating-dependent mechanism of 4-aminopyridine block in two related potassium channels.

4-aminopyridine (4AP) is widely used as a selective blocker of voltage-activated K+ currents in excitable membranes, but its mechanism and site of action at the molecular level are not well understood. To address this problem we have analyzed 4AP block in Kv2.1 and Kv3.1, mammalian representatives of the Drosophila Shab and Shaw subfamilies of voltage-gated K+ channels, respectively. The two channels were expressed in Xenopus oocytes and analyzed at both the macroscopic and single channel levels. Whole cell analysis showed that 4AP sensitivity of Kv3.1 was approximately 150 times greater than that of Kv2.1. Patch clamp analysis revealed that the mechanism of 4AP block in both channels was qualitatively similar. 4AP reached its blocking site via the cytoplasmic side of the channels, the ON rate for block was strongly accelerated when channels opened and the drug was trapped in closed channels. Single channel analysis showed that 4AP decreased burst duration and increased the latency-to-first-opening. These effects were found to be related, respectively to drug ON and OFF rates in the activated channel. Kv3.1's high 4AP sensitivity relative to Kv2.1 was associated with both a slower OFF rate and therefore increased stability of the blocked state, as well as a faster ON rate and therefore increased access to the binding site. Our results indicate that in both channels 4AP enters the intracellular mouth to bind to a site that is guarded by the gating mechanism. Differences in channel gating as well as differences in the structure of the intracellular mouth may be important for specifying the 4AP sensitivity in related voltage-gated K+ channels.

Temperature dependence of Na currents in rabbit and frog muscle membranes.

The effect of temperature (0-22 degrees C) on the kinetics of Na channel conductance was determined in voltage-clamped rabbit and frog skeletal muscle fibers using the triple-Vaseline-gap technique. The Hodgkin-Huxley model was used to extract kinetic parameters; the time course of the conductance change during step depolarization followed m3h kinetics. Arrhenius plots of activation time constants (tau m), determined at both moderate (-10 to -20 mV) and high (+100 mV) depolarizations, were linear in both types of muscle. In rabbit muscle, Arrhenius plots of the inactivation time constant (tau h) were markedly nonlinear at +100 mV, but much less so at -20 mV. The reverse situation was found in frog muscle. The contrast between the highly nonlinear Arrhenius plot of tau h at +100 mV in rabbit muscle, compared with that of frog muscle, was interpreted as revealing an intrinsic nonlinearity in the temperature dependence of mammalian muscle Na inactivation. These results are consistent with the notion that mammalian cell membranes undergo thermotropic membrane phase transitions that alter lipid-channel interactions in the 0-22 degrees C range. Furthermore, the observation that Na channel activation appears to be resistant to this effect suggests that the gating mechanisms that govern activation and inactivation reside in physically distinct regions of the channel.

Kinetic properties of single sodium channels in rat heart and rat brain.

Single Na channel currents were compared in ventricular myocytes and cortical neurons of neonatal rats using the gigaseal patch-clamp method to determine whether tissue-specific differences in gating can be detected at the single-channel level. Single-channel currents were recorded in cell-attached and excised membrane patches at test potentials of -70 to -20 mV and at 9-11 degrees C. In both cell-attached and excised patches brain Na channel mean open time progressively increased from less than 1 ms at -70 mV to approximately 2 ms at -20 mV. Near threshold, single openings with dispersed latencies were observed. By contrast, in cell-attached patches, heart Na channel mean open time peaked near -50 mV, was three times brain Na channel mean open time, and declined continuously to approximately 2 ms at -20 mV. Near threshold, openings occurred frequently usually as brief bursts lasting several milliseconds and rarely as prolonged bursts lasting tens of milliseconds. Unlike what occurs in brain tissue where excision did not change gating, in excised heart patches both the frequency of prolonged bursting and the mean open time of single units increased markedly. Brain and cardiac Na channels can therefore be distinguished on the basis of their mean open times and bursting characteristics.

Delayed rectification in the transverse tubules: origin of the late after-potential in frog skeletal muscle.

Tetanic stimulation of skeletal muscle fibers elicits a train of spikes followed by a long-lasting depolarization called the late after-potential (LAP). We have conducted experiments to determine the origin of the LAP. Isolated single muscle fibers were treated with a high potassium solution (5 mM or 10 mM K) followed by a sudden reduction of potassium concentration to 2.5 mM. This procedure produced a slow repolarization (K repolarization), which reflects a diffusional outflow of potassium from inside the lumen of the transverse tubular system (T system). Tetanic stimulation was then applied to the same fiber and the LAP was recorded. The time courses of K repolarization and LAP decay were compared and found to be roughly the same. This approximate equality held under various conditions that changed the time courses of both events over a wide range. Both K repolarization and the LAP became slower as fiber radius increased. These results suggest that LAP decay and K repolarization represent the same process. Thus, we conclude that the LAP is caused by potassium accumulation in the T system. A consequence of this conclusion is that delayed rectification channels exist in the T system. A rough estimation suggests that the density of delayed rectification channels is less in the T system than in the surface membrane.

Interaction of n-alkylguanidines with the sodium channels of squid axon membrane.

The effects of n-alkylguanidine derivatives on sodium channel conductance were measured in voltage clamped, internally perfused squid giant axons. After destruction of the sodium inactivation mechanism by internal pronase treatment, internal application of n-amylguanidine (0.5 mM) or n-octylguanidine (0.03 mM) caused a time-dependent block of sodium channels. No time-dependent block was observed with shorter chain derivatives. No change in the rising phase of sodium current was seen and the block of steady-state sodium current was independent of the membrane potential. In axons with intact sodium inactivation, an apparent facilitation of inactivation was observed after application of either n-amylguanidine or n-octylguanidine. These results can be explained by a model in which alkylguanidines enter and occlude open sodium channels from inside the membrane with voltage-independent rate constants. Alkylguanidine block bears a close resemblance to natural sodium inactivation. This might be explained by the fact that alkylguanidines are related to arginine, which has a guanidino group and is thought to be an essential amino acid in the molecular mechanism of sodium inactivation. A strong correlation between alkyl chain length and blocking potency was found, suggesting that a hydrophobic binding site exists near the inner mouth of the sodium channel.

Modification of Na channel gating by an alpha scorpion toxin from Tityus serrulatus.

The effects of TsIV-5, a toxin isolated from the Brazilian scorpion Tityus serrulatus, on whole-cell and single-channel Na currents were determined in N18 neuroblastoma cells. In whole-cell records at a test potential of -10 mV, external application of 500 nM TsIV-5 slowed inactivation 20-fold and increased peak current by about one-third without changing time-to-peak. Both the steady-state activation and inactivation curves were shifted to more negative potentials. Other alpha scorpion toxins produce similar effects but the single-channel mechanism is not known. TsIV-5 caused a voltage-dependent prolongation of mean single-channel open time such that at a test potential of -60 mV no change was observed, whereas at -20 mV mean open time increased about threefold and prolonged bursting was observed. Macroscopic current reconstructed from summed single-channel records showed a characteristic toxin-induced potentiation of peak current and a 20-fold slowing of the decay phase. TsIV-5 does not discriminate between tissue-specific Na channel subtypes. Prolonged open times and bursting were also observed in toxin-treated Na channels from rat ventricular myocytes, rat cortical neurons, and mouse skeletal muscle. The toxin effects are shown to be consistent with a kinetic model in which TsIV-5 selectively interferes with the ability of the channel to reach the inactivated state.

Role of transmembrane segment S5 on gating of voltage-dependent K+ channels.

The cytoplasmic half of S5 (5'S5) has been identified as part of the inner mouth of the pore based on evidence that mutations in this region greatly alter single channel conductance, 4-aminopyridine (4-AP) block and the rate of channel closing upon repolarization (deactivation). The latter effect, suggestive of a role for 5'S5 in channel gating was investigated in the present study. The biophysical properties of chimeric channels, in which the 5'S5 regions were exchanged between two host channels (Kv2.1 and Kv3.1) that differ in 4-AP sensitivity and deactivation rate, were examined in a Xenopus oocyte expression system. Exchange of 5'S5 between Kv2.1 and Kv3.1 confers steady-state voltage dependence of activation and rates of channel deactivation similar to those of the donor channel. The involvement of voltage-dependent gating was confirmed by the observation that exchanging the 5'S5 segment of Kv2.1 with that of Kv3.1 confers a change from slow to fast deactivation kinetics by accelerating the decay of off-gating charge movement. We suggest that a conformational change that extends from the voltage-sensor in S4 to the region of the pore lined by S5 regulates the stability of the open state. Therefore, the cytoplasmic end of S5, in addition to forming part of the conduction pathway near the inner mouth of the pore, also participates in the conformational rearrangements associated with late steps in channel activation and early steps in deactivation.

Kv2.1 and electrically silent Kv6.1 potassium channel subunits combine and express a novel current.

Heteromultimer formation between Kv potassium channel subfamilies with the production of a novel current is reported for the first time. Protein-protein interactions between Kv2.1 and electrically silent Kv6.1 alpha-subunits were detected using two microelectrode voltage clamp and yeast two-hybrid measurements. Amino terminal portions of Kv6.1 were unable to form homomultimers but interacted specifically with amino termini of Kv2.1. Xenopus oocytes co-injected with Kv6.1 and Kv2.1 cRNAs exhibited a novel current with decreased rates of deactivation, decreased sensitivity to TEA block, and a hyperpolarizing shift of the half maximal activation potential when compared to Kv2.1. Our results indicate that Kv channel subfamilies can form heteromultimeric channels and, for the first time, suggest a possible functional role for the Kv6 subfamily.

Electrophysiological characterization of a new member of the RCK family of rat brain K+ channels.

A novel member of the RCK family of rat brain K+ channels, called RCK2, has been sequenced and expressed in Xenopus oocytes. The K+ currents were voltage-dependent, activated within 20 ms (at 0 mV), did not inactivate in 5 s, and had a single channel conductance in frog Ringers of 8.2 pS. Compared to other members of the RCK family the pharmacological profile of RCK2 was unique in that the channel was resistant to block (IC50 = 3.3 microM) by charybdotoxin [(1988) Proc. Natl. Acad. Sci. USA 85, 3329-3333] but relatively sensitive to 4-aminopyridine (0.3 mM), tetraethylammonium (1.7 mM), alpha-dendrotoxin (25 nM), noxiustoxin (200 nM), and mast cell degranulating peptide (200 nM). Thus, RCK2 is a non-inactivating delayed rectifier K+ channel with interesting pharmacological properties.

New synthetic vitamin D analogs with antiproliferative activities.

The introduction of oxygen atoms into different positions of the vitamin D side chain is described. By combining the arising 23-oxa and 25-oxa elements with other structural modifications (19-nor, iso-19-nor, 20-methyl, 20-ene, 20,21-cyclo) calcitriol analogs with remarkable levels of dissociation between beneficial acitivities on cell growth regulation and undesired hypercalcemia were identified. Structure-activity relations are elaborated in a very systematic outline of the Schering drug finding program in this particular class of vitamin D compounds.

Synthesis and evaluation of radioiodinated terminal p-iodophenyl-substituted alpha- and beta-methyl-branched fatty acids.

Methods have been developed for the preparation of terminal p-idophenyl-substituted alpha- and beta-methyl-branched long-chain fatty acids. The syntheses and physical properties of 14-(p-iodophenyl)-2(RS)-methyltetradecanoic acid and 15-p-iodophenyl)-3(RS)-methylpentadecanoic acid are described. The radioiodinated agents are of interest as a result of the expected pronounced uptake and prolonged myocardial retention that may result from the inhibition of fatty acid metabolism. Tissue distribution studies in rats with 14-(p-[125I]iodophenyl)-2(RS)-methyltetradecanoic acid and 15-(p-[125I]iodophenyl)-3(RS)-methylpentadecanoic acid show significant heart uptake and prolonged retention accompanied by low in vivo deiodination and high blood levels. A comparison of the heart uptake of the radioiodinated methyl-branched fatty acids and their unbranched analogues has demonstrated a greater myocardial retention of the methyl-branched fatty acids than the unbranched analogues. These results suggest that the mechanism of myocardial retention results from steric or chemical inhibition of the metabolism of these fatty acids by the presence of the methyl group.