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The selectivity filter (SF) of bacterial voltage-gated sodium channels consists of four glutamate residues arranged in a C 4 symmetry. The protonation state population of this tetrad is unclear. To address this question, we simulate the pore domain of bacterial voltage-gated sodium channel of Magnetococcus sp. (Na v Ms) through constant pH methodology in explicit solvent and free energy perturbation calculations. We find that at physiological pH the fully deprotonated as well as singly and doubly protonated states of the SF appear feasible, and that the calculated pKa decreases with each additional bound ion, suggesting that a decrease in the number of ions in the pore can lead to protonation of the SF. Previous molecular dynamics simulations have suggested that protonation can lead to a decrease in the conductance, but no pKa calculations were performed. We confirm a decreased ionic population of the pore with protonation, and also observe structural symmetry breaking triggered by protonation; the SF of the deprotonated channel is closest to the C 4 symmetry observed in crystal structures of the open state, while the SF of protonated states display greater levels of asymmetry which could lead to transition to the inactivated state which possesses a C 2 symmetry in the crystal structure. We speculate that the decrease in the number of ions near the mouth of the channel, due to either random fluctuations or ion depletion due to conduction, could be a self-regulatory mechanism resulting in a nonconducting state that functionally resembles inactivated states., (© 2019 Wiley Periodicals, Inc.)

ERIN, Educational Resources in Neuroscience, is the Society for Neuroscience's web portal to selected, high-quality materials for higher education. A Board of Editors approves resources after describing them and classifying them by topic, subtopic, media type, author, and appropriate educational level. Some resources are also accompanied by reviews and ratings from faculty who have used the resource. These features make a search of ERIN far more useful than a typical Google search. ERIN's development was funded by the National Science Foundation with a three-year grant to SfN. Along the way, various unexpected problems arose and solutions were found, many of which are described in this overview of ERIN's history and the various decisions that were made in its design and development.

Introduction: At night pedestrians tend to overestimate their conspicuity to oncoming drivers, but little is known about factors affecting pedestrians' conspicuity estimates. This study examines how headlamp intensity and pedestrians' clothing influence judgments of their own conspicuity., Method: Forty-eight undergraduate students estimated their own conspicuity on an unilluminated closed road by walking in front of a stationary vehicle to the point at which they judged that they were just recognizable to the driver. Unknown to the participants, high beam intensity was manipulated between subjects by placing neutral density filters on the headlamps., Results: Estimated conspicuity distances did not significantly vary with changes in headlamp intensity even when only 3% of the illumination from the headlamps was present., Practical Applications: These findings underscore the need to educate pedestrians about the visual challenges that drivers face at night and the need to minimize pedestrians' exposure to traffic flow at night., (© 2013.)

Nicotinic acetylcholine receptors are implicated in several neuropsychiatric disorders, including nicotine addiction, Alzheimer's, schizophrenia, and depression. Therefore, they represent a critical molecular target for drug development and targeted therapeutic intervention. Understanding the molecular mechanisms by which allosteric modulators enhance activation of these receptors is crucial to the development of new drugs. We used the substituted cysteine accessibility method to study conformational changes induced by the positive allosteric modulator N-(5-chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)-urea (PNU-120596) in the extracellular ligand binding domain of alpha7 nicotinic receptors carrying the L247T mutation. PNU-120596 caused changes in cysteine accessibility at the inner beta sheet, transition zone, and agonist binding site. These changes in accessibility are similar to but not identical to those caused by ACh alone. In particular, PNU-120596 induced changes in MTSEA accessibility at N170C (in the transition zone) that were substantially different from those evoked by acetylcholine (ACh). We found that PNU-120596 induced changes at position E172C in the absence of allosteric modulation. We identified a cysteine mutation of the agonist binding site (W148C) that exhibited an unexpected phenotype in which PNU-120596 acts as a full agonist. In this mutant, ACh-evoked currents were more sensitive to thiol modification than PNU-evoked currents, suggesting that PNU-120596 does not bind at unoccupied agonist-binding sites. Our results provide evidence that binding sites for PNU-120596 are not in the agonist-binding sites and demonstrate that positive allosteric modulators such as PNU-120596 enhance agonist-evoked gating of nicotinic receptors by eliciting conformational effects that are similar but nonidentical to the gating conformations promoted by ACh.

Allosteric modulation of membrane receptors is a widespread mechanism by which endogenous and exogenous agents regulate receptor function. For example, several members of the nicotinic receptor family are modulated by physiological concentrations of extracellular calcium ions. In this paper, we examined conformational changes underlying this modulation and compare these with changes evoked by ACh. Two sets of residues in the alpha 7 acetylcholine receptor extracellular domain were mutated to cysteine and analyzed by measuring the rates of modification by the thiol-specific reagent 2-aminoethylmethane thiosulfonate. Using Ba2+ as a surrogate for Ca2+, we found a divalent-dependent decrease the modification rates of cysteine substitutions at M37 and M40, residues at which rates were also slowed by ACh. In contrast, Ba2+ had no significant effect at N52C, a residue where ACh increased the rate of modification. Thus divalent modulators cause some but not all of the conformational effects elicited by agonist. Cysteine substitution of either of two glutamates (E44 or E172), thought to participate in the divalent cation binding site, caused a loss of allosteric modulation, yet Ba2+ still had a significant effect on modification rates of these residues. In addition, the effect of Ba2+ at these residues did not appear to be due to direct occlusion. Our data demonstrate that modulation by divalent cations involves substantial conformational changes in the receptor extracellular domain. Our evidence also suggests the modulation occurs via a binding site distinct from one which includes either (or both) of the conserved glutamates at E44 or E172.

Cys-loop ligand-gated ion channels assemble as pentameric proteins, and each monomer contributes two structural elements: an extracellular ligand-binding domain (LBD) and a transmembrane ion channel domain. Models of receptor activation include rotational movements of subunits leading to opening of the ion channel. We tested this idea using substituted cysteine accessibility to track conformational changes in the inner beta sheet of the LBD. Using a nondesensitizing chick alpha7 background (L(247)T), we constructed 18 consecutive cysteine replacement mutants (Leu(36) to Ile(53)) and tested each for expression of acetylcholine (ACh)-evoked currents and functional sensitivity to thiol modification. We measured rates of modification in the presence and absence of ACh to identify conformational changes associated with receptor activation. Resting modification rates of eight substituted cysteines in the beta1 and beta2 strands and the sequence between them (loop 2) varied over several orders of magnitude, suggesting substantial differences in the accessibility or electrostatic environment of individual side chains. These differences were in general agreement with structural models of the LBD. Eight of 18 cysteine replacements displayed ACh-dependent changes in modification rates, indicating a change in the accessibility or electrostatic environment of the introduced cysteine during activation. We were surprised that the effects of agonist exposure were difficult to reconcile with rotational models of activation. Acetylcholine reduced the modification rate of M(40)C but increased it at N(52)C despite the close physical proximity of these residues. Our results suggest that models that depend strictly on rigid-body rotation of the LBD may provide an incomplete description of receptor activation.

alpha-7 Nicotinic acetylcholine receptors (AChRs) exhibit a positive modulation by divalent cations similar to that observed in other AChRs. In the chick alpha7 AChR, this modulation involves a conserved glutamate in loop 9 (Glu172) that undergoes agonist-dependent movements during activation. From these observations, we hypothesized that movements of the nearby beta-sheet formed by the beta7, beta9, and beta10 strands may be involved in agonist activation and/or divalent modulation. To test this hypothesis, we examined functional properties of cysteine mutations of the beta7 and beta10 strands, alone or in pairs. We postulated that reduced flexibility or mobility of the beta7/beta9/beta10-sheet as a result of introduction of a disulfide bond between the beta strands would alter activation by agonists. Using a nondesensitizing alpha7 mutant background (L247T), we identified one mutant pair, K144C + T198C, that exhibited a unique characteristic: it was fully activated by divalent cations (Ca2+, Ba2+, or Sr2+) in the absence of acetylcholine (ACh). Divalent-evoked currents were blocked by the alpha7 antagonist methyllycaconitine and were abolished when Glu172 was mutated to glutamine. When the K144C + T198C pair was expressed in wild-type alpha7 receptors, activation required both ACh and divalent cations. We conclude that the introduction of a disulfide bond into beta7/beta9/beta10 lowers the energetic barrier between open and closed conformations, probably by reducing the torsional flexibility of the beta-sheet. In this setting, divalent cations, acting at the conserved glutamate in loop 9, act as full agonists or requisite coagonists.

Neisseria gonorrhoeae has two porins, PIA and PIB, whose genes (porA and porB, respectively) are alleles of a single por locus. We recently demonstrated that penB mutations at positions 120 and 121 in PIB, which are presumed to reside in loop 3 that forms the pore constriction zone, confer intermediate-level resistance to penicillin and tetracycline (M. Olesky, M. Hobbs, and R. A. Nicholas, Antimicrob. Agents Chemother. 46:2811-2820, 2002). In the present study, we investigated the electrophysiological properties as well as solute and antibiotic permeation rates of recombinant PIB proteins containing penB mutations (G120K, G120D/A121D, G120P/A121P, and G120R/A121H). In planar lipid bilayers, the predominant conducting state of each porin variant was 30 to 40% of the wild type, even though the anion selectivity and maximum channel conductance of each PIB variant was similar to that of the wild type. Liposome-swelling experiments revealed no significant differences in the permeation of sugars or beta-lactam antibiotics through the wild type or PIB variants. Although these results are seemingly contradictory with the ability of these variants to increase antibiotic resistance, they are consistent with MIC data showing that these porin mutations confer resistance only in strains containing an mtrR mutation, which increases expression of the MtrC-MtrD-MtrE efflux pump. Moreover, both the mtrR and penB mutations were required to decrease in vivo permeation rates below those observed in the parental strain containing either mtrR or porin mutations alone. Thus, these data demonstrate a novel mechanism of porin-mediated resistance in which mutations in PIB have no affect on antibiotic permeation alone but instead act synergistically with the MtrC-MtrD-MtrE efflux pump in the development of antibiotic resistance in gonococci.

The molecular mechanisms that couple agonist binding to the gating of Cys-loop ionotropic receptors are not well understood. The crystal structure of the acetylcholine (ACh) binding protein has provided insights into the structure of the extracellular domain of nicotinic receptors and a framework for testing mechanisms of activation. Key ligand binding residues are located at the C-terminal end of the beta9 strand. At the N-terminal end of this strand (loop 9) is a conserved glutamate [E172 in chick alpha7 nicotinic acetylcholine receptors (nAChRs)] that is important for modulating activation. We hypothesize that agonist binding induces the movement of loop 9. To test this, we used the substituted-cysteine accessibility method to examine agonist-dependent changes in the modification of cysteines introduced in loop 9 of L247T alpha7 nAChRs. In the absence of agonist, ACh-evoked responses of E172C/L247T alpha7 nAChRs were inhibited by 2-trimethylammonioethylmethane thiosulfonate (MTSET). Agonist coapplication with MTSET reduced the extent and rate of modification. The dose-dependence of ACh activation was nearly identical with that of ACh-dependent protection from modification. ACh increased the inhibition by methanethiosulfonate reagents of N170C and did not change inhibition of G171C receptors. The antagonist dihydro-beta-erythroidine did not mimic the effects of ACh. Combined with a structural model, the data suggest that receptor activation includes subunit rotation and/or intrasubunit conformational changes that move N170 to a more accessible position and E172 to a more protected position away from the vestibule. Thus, loop 9, located near the junction between the extracellular and transmembrane domains, participates in conformational changes triggered by ligand binding.

Neuronal alpha7 nicotinic ACh receptors (nAChRs) are permeable to and modulated by Ca2+, Ba2+, and Sr2+. These permeant divalent cations interact with slowly desensitizing L247T alpha7 nAChRs to increase the potency and maximal efficacy of ACh, increase the efficacy of dihydro-beta-erythroidine (DHbetaE), and increase agonist-independent activity. Mutation of glutamate 172 (E172) to glutamine or cysteine eliminated these effects of permeant divalent cations. 2-(Trimethylammonium)ethyl methanethiosulfonate (MTSET), a cysteine-modifying reagent directed at water-accessible thiols, inhibited ACh-evoked currents of E172C/L247T alpha7 nAChRs by >90%, demonstrating that E172 was accessible to permeant ions. The data are consistent with a model of alpha7 receptors, derived from the crystal structure of the ACh binding protein (AChBP) from Lymnaea stagnalis, in which E172 projects toward the lumen of the extracellular vestibule. The observations that E172 was essential for divalent cation modulation of L247T alpha7 nAChRs and was accessible to permeating ions suggest that this residue participates in coupling ion permeation with modulation of receptor activity.

Neuronal alpha(7) nicotinic acetylcholine receptors (nAChRs) are permeable to Ca(2+) and other divalent cations. We characterized the modulation of the pharmacological properties of nondesensitizing mutant (L(247)T and S(240)T/L(247)T) alpha(7) nAChRs by permeant (Ca(2+), Ba(2+), and Sr(2+)) and impermeant (Cd(2+) and Zn(2+)) divalent cations. alpha(7) receptors were expressed in Xenopus oocytes and studied with two-electrode voltage clamp. Extracellular permeant divalent cations increased the potency and maximal efficacy of ACh, whereas impermeant divalent cations decreased potency and maximal efficacy. The antagonist dihydro-beta-erythroidine (DHbetaE) was a strong partial agonist of L(247)T and S(240)T/L(247)T alpha(7) receptors in the presence of divalent cations but was a weak partial agonist in the presence of impermeant divalent cations. Mutation of the "intermediate ring" glutamates (E(237)A) in L(247)T alpha(7) nAChRs eliminated Ca(2+) conductance but did not alter the Ca(2+)-dependent increase in ACh potency, suggesting that site(s) required for modulation are on the extracellular side of the intermediate ring. The difference between permeant and impermeant divalent cations suggests that sites within the pore are important for modulation by divalent cations.

alpha7 nicotinic receptors are highly permeable to Ca(2+) as well as monovalent cations. We extended the characterization of the Ca(2+) permeation of non-desensitizing chick alpha7 receptors (S240T/L247T alpha7 nAChRs) expressed in Xenopus oocytes by (1) measuring the concentration dependence of conductance under conditions in which Ca(2+) or Ba(2+) were the only permeant cations in the extracellular solution, and (2) measuring the concentration dependence of Ca(2+) block of K(+) currents through the receptors. The first set of experiments yielded an apparent affinity of 0.96 mM Ca(2+) activity (2.4 mM concentration) for Ca(2+) permeation and an apparent affinity of 0.65 mM Ba(2+) activity (1.7 mM concentration) for Ba(2+) permeation. The apparent affinity of Ca(2+) inhibition of K(+) currents was 0.49 mM activity (1.5 mM concentration). The similarity of these apparent affinities in the millimolar range suggests that the pore of alpha7 receptors has one or more low-affinity Ca(2+) binding sites and no high-affinity sites.

A cDNA encoding a new cytochrome P450 was isolated from a mouse brain library. Sequence analysis reveals that this 1,958-base pair cDNA encodes a 57-58-kDa 502-amino acid polypeptide that is 70-91% identical to CYP2J subfamily P450s and is designated CYP2J9. Recombinant CYP2J9 was co-expressed with NADPH-cytochrome P450 oxidoreductase (CYPOR) in Sf9 cells using a baculovirus system. Microsomes of CYP2J9/CYPOR-transfected cells metabolize arachidonic acid to 19-hydroxyeicosatetraenoic acid (HETE) thus CYP2J9 is enzymologically distinct from other P450s. Northern analysis reveals that CYP2J9 transcripts are present at high levels in mouse brain. Mouse brain microsomes biosynthesize 19-HETE. RNA polymerase chain reaction analysis demonstrates that CYP2J9 mRNAs are widely distributed in brain and most abundant in the cerebellum. Immunoblotting using an antibody raised against human CYP2J2 that cross-reacts with CYP2J9 detects a 56-kDa protein band that is expressed in cerebellum and other brain segments and is regulated during postnatal development. In situ hybridization of mouse brain sections with a CYP2J9-specific riboprobe and immunohistochemical staining with the anti-human CYP2J2 IgG reveals abundant CYP2J9 mRNA and protein in cerebellar Purkinje cells. Importantly, 19-HETE inhibits the activity of recombinant P/Q-type Ca(2+) channels that are known to be expressed preferentially in cerebellar Purkinje cells and are involved in triggering neurotransmitter release. Based on these data, we conclude that CYP2J9 is a developmentally regulated P450 that is abundant in brain, localized to cerebellar Purkinje cells, and active in the biosynthesis of 19-HETE, an eicosanoid that inhibits activity of P/Q-type Ca(2+) channels. We postulate that CYP2J9 arachidonic acid products play important functional roles in the brain.

The co-release of ATP with norepinephrine from sympathetic nerve terminals in the heart may augment adrenergic stimulation of cardiac Ca(2+) channel activity. To test for a possible direct effect of extracellular ATP on L-type Ca(2+) channels, single channels were reconstituted from porcine sarcolemma into planar lipid bilayers so that intracellular signaling pathways could be controlled. Extracellular ATP (2-100 microM) increased the open probability of the reconstituted channels, with a maximal increase of approximately 2.6-fold and an EC(50) of 3.9 microM. The increase in open probability was due to an increase in channel availability and a decrease in channel inactivation rate. Other nucleotides displayed a rank order of effectiveness of ATP > alpha,beta-methylene-ATP > 2-methylthio-ATP > UTP > adenosine 5'-O-(3-thiotriphosphate) >> ADP; adenosine had no effect. Several antagonists of P2 receptors had no impact on the ATP-dependent increase in open probability, indicating that receptor activation was not required. These results suggest that extracellular ATP and other nucleotides can stimulate the activity of cardiac L-type Ca(2+) channels via a direct interaction with the channels.

The alpha7 nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that modulates neurotransmitter release in the central nervous system. We show here that functional, homo-oligomeric alpha7 nAChRs can be synthesized in vitro with a rabbit reticulocyte lysate translation system supplemented with endoplasmic reticulum microsomes, reconstituted into planar lipid bilayers, and evaluated using single-channel recording techniques. Because wild-type alpha7 nAChRs desensitize rapidly, we used a nondesensitizing form of the alpha7 receptor with mutations in the second transmembrane domain (S2'T and L9'T) to record channel activity in the continuous presence of agonist. Endoglycosidase H treatment of microsomes containing nascent alpha7 S2'T/L9'T nAChRs indicated that the receptors were glycosylated. A proteinase K protection assay revealed a 36-kDa fragment in the ER lumen, consistent with a large extracellular domain predicted by most topological models, indicating that the protein was folded integrally through the ER membrane. alpha7 S2'T/L9'T receptors reconstituted into planar lipid bilayers had a unitary conductance of approximately 50 pS, were highly selective for monovalent cations over Cl(-), were nonselective between K(+) and Na(+), and were blocked by alpha-bungarotoxin. This is the first demonstration that a functional ligand-gated ion channel can be synthesized using an in vitro expression system.

Epoxyeicosatrienoic acids (EETs), products of the cytochrome P-450 monooxygenase metabolism of arachidonic acid, can regulate the activity of ion channels. We examined the effects of EETs on cardiac L-type Ca2+ channels that play important roles in regulating cardiac contractility, controlling heart rate, and mediating slow conduction in normal nodal cells and ischemic myocardium. Our experimental approach was to reconstitute porcine L-type Ca2+ channels into planar lipid bilayers where we could control the aqueous and lipid environments of the channels and the regulatory pathways that change channel properties. We found that 20 to 125 nM EETs inhibited the open probability of reconstituted L-type Ca2+ channels, accelerated the inactivation of the channels, and reduced the unitary current amplitude of open channels. There was no selectivity among different EET regioisomers or stereoisomers. When 11,12-EET was esterified to the sn-2 position of phosphatidylcholine, restricting it to the hydrophobic phase of the planar lipid bilayer, the reconstituted channels were similarly inhibited, suggesting that the EET interacts directly with Ca2+ channels through the lipid phase. The inhibitory effects of EET persisted in the presence of microcystin, an inhibitor of protein phosphatases 1 and 2A, suggesting that dephosphorylation was not the mechanism through which these eicosanoids down-regulate channel activity. This inhibition may be an important protective mechanism in the setting of cardiac ischemia where arachidonic acid levels are dramatically increased and EETs have been shown to manifest preconditioning-like effects.

Capsaicin (Cap) is a pungent extract of the Capsicum pepper family, which activates nociceptive primary sensory neurons. Inward current and membrane potential responses of cultured neonatal rat dorsal root ganglion neurons to capsaicin were examined using whole-cell and perforated patch recording methods. The responses exhibited strong desensitization operationally classified as acute (diminished response during constant Cap exposure) and tachyphylaxis (diminished response to successive applications of Cap). Both acute desensitization and tachyphylaxis were greatly diminished by reductions in external Ca2+ concentration. Furthermore, chelation of intracellular Ca2+ by addition of either EGTA or bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid to the patch pipette attenuated both forms of desensitization even in normal Ca2+. Release of intracellular Ca2+ by caffeine triggered acute desensitization in the absence of extracellular Ca2+, and barium was found to effectively substitute for calcium in supporting desensitization. Cap activated inward current at an ED50 of 728 nM, exhibiting cooperativity (Hill coefficient, 2.2); however, both forms of desensitization were only weakly dependent on [Cap], suggesting a dissociation between activation of Cap-sensitive channels and desensitization. Removal of ATP and GTP from the intracellular solutions resulted in nearly complete tachyphylaxis even with intracellular Ca2+ buffered to low levels, whereas changes in nucleotide levels did not significantly alter the acute form of desensitization. These data suggest a key role for intracellular Ca2+ in desensitization of Cap responses, perhaps through Ca2+-dependent dephosphorylation at a locus that normally sustains Cap responsiveness via ATP-dependent phosphorylation. It also seems that the signaling mechanisms underlying the two forms of desensitization are not identical in detail.

The malaria parasite, Plasmodium falciparum, requires large amounts of nutrients to sustain its rapid growth within the human red blood cell. A recently identified ion channel on the surface of the intraerythrocytic parasite may provide direct access to these nutrients in the red blood cell cytosol. Evidence supporting this role was obtained by incorporating this channel into planar lipid bilayers. In bilayers, this channel has conductance and gating properties identical to the in situ channel, passes soluble macromolecules of up to 1400 Da, and functions as a high capacity, low affinity molecular sieve. These properties, remarkably similar to those of a pore on Toxoplasma gondii (another protozoan parasite causing human disease), suggest a novel class of channels used by these intracellular parasites to acquire nutrients from host cytosol.

We have studied the effect of a palmitoyl-L-carnitine (L-PC) on single cardiac L-type Ca channels incorporated from porcine ventricular sarcolemma into planar lipid bilayers where we could control the concentration, intracellular and/or extracellular location, and duration of L-PC treatment. We found that 1.0 microM L-PC in either the intracellular or extracellular chamber caused an approximately 8 fold increase in channel open probability when measured within the first minute after L-PC addition. Higher concentrations of L-PC did not increase open probability to the same extent as 1.0 microM. In addition, we found that L-PC had biphasic effects on the open probability of L-type Ca channels, causing an increase in activity immediately after the addition of L-PC, but leading to a decrease in open probability after a few minutes. Higher concentrations of L-PC (10 microM) also caused a decrease in single-channel conductance from 26 to 21 pS (measured in 100 mM external Ba2+). The effects of L-PC were similar on both sides of the channels, suggesting that alterations in the physical properties of the membrane surrounding the channels may be responsible for the effects of L-PC. These changes in Ca channel activity may participate in the generation of abnormal electrical activity and arrhythmogenesis during ischemia.

We have characterized a voltage-sensitive chloride channel from cardiac sarcoplasmic reticulum (SR) following reconstitution of porcine heart SR into planar lipid bilayers. In 250 mM KCl, the channel had a main conductance level of 130 pS and exhibited two substrates of 61 and 154 pS. The channel was very selective for Cl- over K+ or Na+ (PK+/PCl- = 0.012 and PNa+/PCl- approximately 0.040). It was permeable to several anions and displayed the following sequence of anion permeability: SCN- > I- > NO3- approximately Br- > Cl- > F- > HCOO-. Single-channel conductance saturated with increasing Cl- concentrations (Km = 900 mM and gamma max = 488 pS). Channel activity was voltage dependent, with an open probability ranging from approximately 1.0 around 0 mV to approximately 0.5 at +80 mV. From -20 to +80 mV, channel gating was time-independent. However, at voltages below -40 mV the channel entered a long-lasting closed state. Mean open times varied with voltage, from approximately 340 msec at -20 mV to approximately 6 msec at +80 mV, whereas closed times were unaffected. The channel was not Ca(2+)-dependent. Channel activity was blocked by disulfonic stilbenes, arylaminobenzoates, zinc, and cadmium. Single-channel conductance was sensitive to trans pH, ranging from approximately 190 pS at pH 5.5 to approximately 60 pS at pH 9.0. These characteristics are different from those previously described for Cl- channels from skeletal or cardiac muscle SR.

Intracellular Ca2+ can inhibit the activity of voltage-gated Ca channels by modulating the rate of channel inactivation. Ca(2+)-dependent inactivation of these channels may be a common negative feedback process important for regulating Ca2+ entry under physiological and pathological conditions. This article demonstrates that the inactivation of cardiac L-type Ca channels, reconstituted into planar lipid bilayers and studied in the presence of a dihydropyridine agonist, is sensitive to Ca2+. The rates and extents of inactivation, determined from ensemble averages of unitary Ba2+ currents, decreased when the calcium concentration facing the intracellular surface of the channel ([Ca2+]i) was lowered from approximately 10 microM to 20 nM by the addition of Ca2+ chelators. The rates and extents of Ba2+ current inactivation could also be increased by subsequent addition of Ca2+ raising the [Ca2+]i to 15 microM, thus demonstrating that the Ca2+ dependence of inactivation could be reversibly regulated by changes in [Ca2+]i. In addition, reconstituted Ca channels inactivated more quickly when the inward current was carried by Ca2+ than when it was carried by Ba2+, suggesting that local increases in [Ca2+]i could activate Ca(2+)-dependent inactivation. These data support models in which Ca2+ binds to the channel itself or to closely associated regulatory proteins to control the rate of channel inactivation, and are inconsistent with purely enzymatic models for channel inactivation.

We have studied the effects of activated G proteins (Gs alpha and Gi1 alpha), adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA), and okadaic acid on L-type Ca channels incorporated from porcine ventricular sarcolemma into planar lipid bilayers. Channel activity evoked by membrane depolarizations diminished to extremely low levels within 2 min of incorporation (channel "rundown"). When Gs alpha [activated with guanosine 5'-O-(3-thiotriphosphate)] was present in the intracellular chamber, the initial level of channel activity was increased and rundown was delayed, so that channel activity was sustained for longer times after incorporation. The effect was specific for activated Gs alpha; activated Gi1 alpha, heat-denatured, activated Gs alpha, and unactivated Gs alpha did not augment channel activity. Activated Gi1 alpha inhibited the stimulation of Ca channel activity by Gs alpha. Treatment of the sarcolemmal membranes with PKA and Mg-ATP also increased the initial channel open probability and delayed their rundown. Addition of intracellular Gs alpha to PKA-treated channels increased the initial level of activity above that seen with PKA or Gs alpha alone, suggesting different nonocclusive pathways for the channel stimulation. This was also supported by the observation that activated Gi1 alpha had no effect on PKA-treated channels. Okadaic acid (100 nM) increased the level of Ca channel activity, suggesting that dephosphorylation by endogenous phosphatases participated in the downregulation of the channels in cell-free membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

The functional activity of ion channels and other membrane proteins requires that the proteins be correctly assembled in a transmembrane configuration. Thus, the functional expression of ion channels, neurotransmitter receptors and complex membrane-limited signalling mechanisms from complementary DNA has required the injection of messenger RNA or transfection of DNA into Xenopus oocytes or other target cells that are capable of processing newly translated protein into the surface membrane. These approaches, combined with voltage-clamp analysis of ion channel currents, have been especially powerful in the identification of structure-function relationships in ion channels. But oocytes express endogenous ion channels, neurotransmitter receptors and receptor-channel subunits, complicating the interpretation of results in mRNA-injected eggs. Furthermore, it is difficult to control experimentally the membrane lipids and post-translational modifications that underlie the regulation and modulation of ion channels in intact cells. A cell-free system for ion channel expression is ideal for good experimental control of protein expression and modulatory processes. Here we combine cell-free protein translation, microsomal membrane processing of nascent channel proteins, and reconstitution of newly synthesized ion channels into planar lipid bilayers to synthesize, glycosylate, process into membranes, and record in vitro the activity of functional Shaker potassium channels.

Kupffer cells, the resident hepatic macrophages, are activated by calcium, but conclusive evidence that they contain voltage-dependent calcium channels has not been presented previously. In this study, the cytosolic free calcium concentration ([Ca2+]i) of cultured Kupffer cells was measured with the fluorescent Ca2+ indicator fura-2. Partial replacement of extracellular Na+ by K+ caused an increase in [Ca2+]i in a concentration-dependent manner (half-maximal effect at 81 mM K+), presumably due to membrane depolarization. At 65 mM K+, where there were minimal changes in [Ca2+]i, addition of the dihydropyridine-type calcium channel agonist BAY K 8644 (1 microM) caused a large increase in [Ca2+]i. Overall, the effect of BAY K 8644 (1 microM) was to shift the concentration-response curve for K+ to the left (half-maximal effect at 61 mM K+). Under depolarizing conditions (65 mM K+), BAY K 8644 increased [Ca2+]i in a concentration-dependent manner (half-maximal effect at approximately 400 nM BAY K 8644). Moreover, the dihydropyridine-type calcium channel blocker nitrendipine inhibited the BAY K 8644-induced increase in [Ca2+]i in a concentration-dependent manner (half-maximal inhibition with about 25 nM nitrendipine). When extracellular Ca2+ was omitted from the incubation medium, the increases in [Ca2+]i due to BAY K 8644 were prevented completely. In addition, an intracellular Ca2+ antagonist, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (200 microM), did not inhibit the BAY K 8644-sensitive, voltage-dependent increase in [Ca2+]i. Thus, these data collectively indicate that BAY K 8644 causes a transmembrane Ca2+ influx in Kupffer cells in a voltage-dependent manner, providing the first direct evidence that Kupffer cells contain L-type voltage-dependent Ca2+ channels.

Ethaverine is a derivative of papaverine used in the treatment of peripheral vascular disease and is thought to cause vasodilation by reducing intracellular Ca2+ concentrations in vascular smooth muscle cells. We tested its effects on single, dihydropyridine-sensitive, L-type calcium channels from porcine cardiac muscle, incorporated into planar lipid bilayers. L-type calcium channels were activated by step depolarizations from a holding potential of -60 mV to a test potential of 0 mV, and unitary currents carried by 100 mM BaCl2 were recorded. Channel activity was enhanced by the presence of the dihydropyridine agonist (+)-202-791 (0.5 microM) and the activated alpha subunit of the stimulatory GTP-binding protein, Gs. We found that 0.3-30 microM ethaverine on either side of the channel caused a reduction in the channel open probability (EC50 approximately 1 microM), with the higher concentrations inhibiting channel activity almost completely. In addition, the ethaverine caused a small reduction in the unitary current amplitude of single open channels (approximately 20%). To test whether the effect of ethaverine on open probability was due to a displacement of the dihydropyridine agonist, we studied the effect of ethaverine on the binding of [3H]nitrendipine to cardiac sarcolemma and found that ethaverine inhibited [3H]nitrendipine binding with a Ki of approximately 8.5 microM. Ethaverine also inhibited the binding of [3H]diltiazem and [3H]verapamil, with Ki values of 1-2 microM. Because ethaverine is structurally related to verapamil, it is likely that ethaverine acts by binding to the verapamil binding sites on the L-type calcium channels to inhibit channel activation and dihydropyridine binding.

We have previously shown that monoclonal antibody E12 (MAb E12), one of several such antibodies raised against theophylline-treated Necturus gallbladder (NGB) epithelial cells, inhibits the chloride conductance in the apical membrane of that tissue. Since chloride channels are critical to the secretory function of epithelia in many different animals, we have used this antibody to determine whether the channels are conserved, and in an immunoaffinity column to isolate the channel protein. We now demonstrate that MAb E12 cross-reacts with detergent-solubilized extracts of different tissues from various species by enzyme-linked immunosorbent assay (ELISA). Western blot analysis shows that this monoclonal antibody recognizes proteins of Mr 219,000 in NGB, toad gallbladder, urinary bladder, and small intestine, A6 cells, rat colon, rabbit gastric mucosa, human lymphocytes, and human nasal epithelial cells, and inhibits the chloride conductance in toad gallbladder, rat colon, and human nasal epithelium. Detergent-solubilized protein eluted from an immunoaffinity column and then further purified via FPLC yields a fraction (Mr 200,000-220,000) which has been reconstituted into a planar lipid bilayer. There it behaves as a chloride-selective channel (PCl/PNa = 20.2 in a 150/50 mM trans-bilayer NaCl gradient) whose unit conductance is 62.4 +/- 4.6 pS, and which is blocked in the bilayer by the antibody. The gating characteristics of this channel indicate that it can exist as aggregates or as independent single channels, and that the antibody interferes with gating of the aggregates, leaving the unit channels unchanged. From these data we conclude that the protein of Mr 219,000 recognized by this monoclonal antibody is an important component of an epithelial chloride channel, and that this channel is conserved across a wide range of animal species.

L-type Ca channels from porcine cardiac sarcolemma were incorporated into planar lipid bilayers. We characterized interactions of permeant and blocking ions with the channel's pore by (a) studying the current-voltage relationships for Ca2+ and Na+ when equal concentrations of the ions were present in both internal and external solutions, (b) testing the dose-dependent block of Ba2+ currents through the channels by internally applied cadmium, and (c) examining the dose and voltage dependence of the block of Na+ currents through the channels by internally and externally applied Ca2+. We found that the I-V relationship for Na+ appears symmetrical through the origin when equal concentrations of Na+ are present on both sides of the channel (gamma = 90 pS in 200 mM NaCl). The conductance for outward Ca2+ currents with 100 mM Ca2+ on both sides of the channel is approximately 8 pS, a value identical to that observed for inward currents when 100 mM Ca2+ was present outside only. This provides evidence that ions pass through the channel equally well regardless of the direction of net flux. In addition, we find that internal Cd2+ is as effective as external Cd2+ in blocking Ba2+ currents through the channels, again suggesting identical interactions of ions with each end of the pore. Finally, we find that micromolar Ca2+, either in the internal or in the external solution, blocks Na+ currents through the channels. The affinity for internally applied Ca2+ appears the same as that for externally applied Ca2+. The voltage dependence of the Ca(2+)-block suggests that the sites to which Ca2+ binds are located approximately 15% and approximately 85% of the electric field into the pore. Taken together, these data provide direct experimental evidence for the existence of at least two ion binding sites with high affinity for Ca2+, and support the idea that the sites are symmetrically located within the electric field across L-type Ca channels.

The tetrodotoxin-binding protein purified from electroplax of Electrophorus electricus has been reincorporated into multilamellar vesicles that were used for patch recording. When excised patches of these reconstituted membranes were voltage clamped in the absence of neurotoxins, voltage-dependent single-channel currents were recorded. These displayed properties qualitatively and quantitatively similar to those reported for Na channels from nerve and muscle cells, including uniform single-channel conductances of the appropriate magnitude (approximately equal to 11 pS in 95 mM Na+), mean open times of approximately equal to 1.9 msec, and 7-fold selectively for Na+ over K+. Currents averaged from many depolarizations showed initial voltage-dependent activation and subsequent inactivation. In the presence of batrachotoxin, channels were observed with markedly different properties, including conductances of 20-25 pS (95 mM Na+), mean open times of approximately equal to 28 msec, and no indication of inactivation. Collectively, these findings indicate that the tetrodotoxin-binding protein of electroplax is a voltage-regulated sodium channel.

Social insecurity is a psychological orientation that has been found to be associated significantly with both coronary atherosclerosis and psychological distress. Although sympathetic arousal has been assumed to be a cause of coronary atherosclerosis and an effect of psychological distress, neither of the prior studies included a measure of sympathetic arousal in its design. The present study fills this gap by examining social insecurity in relation to blood pressure, as an index of sympathetic arousal. The results, derived from 50 male, medical/surgical patients, are consistent with the pattern of findings in previous studies in that social insecurity was independent of the Type A pattern and additive to it in its association with blood pressure. Differences between social insecurity and the Type A pattern are discussed in terms of their differential associations with systolic versus diastolic pressure and their differential trait-like and state-like patterns of association with blood pressure.

A fluorescence assay for measuring Na channel activation in liposomes containing voltage-sensitive Na channels isolated from Electrophorus electricus is described. The assay is based on transport of a heavy-metal cation, T1+, through the activated channel to quench fluorescence of an internalized, water-soluble chromophore. The channel is "locked" in a chronically opened configuration with alkaloid neurotoxins such as veratridine or batrachotoxin. Diffusion potentials are used to amplify the signal, and enlarged liposomes (greater than 8000 A) result in time courses extended to the range of seconds. Analysis of the kinetics of quenching yields parameters that behave as linear functions of channel activation and reflect vesicle size and channel abundance. The k1/2's for activation by veratridine and batrachotoxin were 5 microM and 169 nM, respectively, and that for tetrodotoxin blockade was 4 nM. Externally applied QX-222 and tetrodotoxin each acted to partially block the stimulated signal, as expected for compounds that act on oppositely oriented channels in the membrane. Single-channel conductances estimated with either veratridine or batrachotoxin ranged between 0.6 and 40.7 pS, corresponding to transport numbers of (1.2 X 10(5)) to (8.1 X 10(6)) ions s-1 channel-1 under the conditions of assay. The assay is approximately 100-fold more sensitive than radiotracer influx assays, requiring 1 fmol of protein per time course.

Cynical Mistrust was examined among 64 medical and surgical patients, 23 of whom were selected for a history of CHD and 41 for an absence of such a history. Cynical Mistrust was found to differentiate subjects with a positive history from those without such a history. As hypothesized, persons scoring high in Cynical Mistrust also scored high in Self-Worth by Social Comparison, Playing Hardball with Others and Self-Criticism. Surprisingly, high scorers in Cynical Mistrust among subjects with a coronary history also scored high in Dependency on others for validation that they were deserving of being loved. We suggest that the pathogenicity of Cynical Mistrust for CHD may be potentiated by the presence of certain other dispositions. Further investigation of dependency on others in particular appears to be worthwhile in order to determine whether consideration of this disposition can improve the identification of risk for CHD.

In time of food shortage, accurate assessment of nutrition status is important so that effective aid may be directed to those in greatest need and, in the future, the impact of this aid on the nutritional situation may be assessed. Using a population proportional sampling method and the index weight for height (which is relatively independent of sex, race, and age) as a criterion of nutritional status, CDC investigators determined that about 10% of the sampled population (aged about 6 months through 6 years) in Mali, Mauritania, Niger, and Upper Volta and over 20% in the sampled population in Chad were severely and acutely undernourished. Apparently younger children were affected first, but as the food shortage became more severe the prevalence of undernourished older children increased. This method of nutrition surveillance has proven itself during a stressful period and should be considered a fundamental tool when rapid assessment of acute undernutrition in children in developing nations is required.

Neurons and neuroendocrine cells contain a unique isoenzyme of the glycolytic enzyme enolase which is not found in other cells. This acidic enolase isoenzyme has been designated neuron specific enolase or NSE and is easily identified by its elution on DEAE sephadex. The present study shows that brain tissue from species such as yeast, fish and frog do not contain appreciable amounts of acidic "NSE-like" enolase suggesting that lower species do not have this neuronal isoenzyme.

Immunodeficient patients who were presumed to be susceptible received zoster immune globulin prophylaxis after exposure to varicella. The highest clinical attack rate (35.9%) was seen in household contacts; the lowest attack rate (0%) was observed in children exposed at school. Among household contacts, 48 of 100 patients who received high titer ZIG (reciprocal complement fixation titer greater than or equal to 2,560) developed fourfold rises in serum CF antibody between pre- and 48-hour post-treatment specimens, compared to only one of 34 patients treated with lower titer ZIG lots (P less than 0.001). Patients who developed fourfold antibody rises were significantly less likely to contract clinical varicella (P less than 0.01). Patients who received high titer ZIG also had significantly lower risks of death (P = 0.025) and complications (P = 0.006). Among ZIG-treated patients who contracted clinical varicella, 80% developed mild disease (less than 100 pox), and the median incubation period was prolonged. Immunodeficient children exposed to varicella benefit from ZIG prophylaxis and higher titer ZIG is of greatest benefit.

Several purines have been shown to be competitive inhibitors of [3H] diazepam binding. Inosine has also been shown to have benzodiazepine-like neurophysiologic, pharmacologic and behavioral effects, and to partially inhibit caffeine-induced seizures in mice. Using presumptive therapeutic doses of inosine, levels were determined in mouse brain at various times following injection. Inosine and hypoxanthine concentrations in brain increased several fold following inosine administration, indicating that inosine permeated the blood-brain barrier. The levels of inosine and hypoxanthine attained in brain were sufficient to inhibit by more than 50% the GABA-stimulated [3H] diazepam binding. These data suggest that the anticonvulsant properties of inosine are related to its interaction with the benzodiazepine receptor.

Planar lipid bilayer recordings were used to study Ca channels from bovine cardiac sarcolemmal membranes. Ca channel activity was recorded in the absence of nucleotides or soluble enzymes, over a range of membrane potentials and ionic conditions that cannot be achieved in intact cells. The dihydropyridine-sensitive L-type Ca channel, studied in the presence of Bay K 8644, was identified by a detailed comparison of its properties in artificial membranes and in intact cells. L-type Ca channels in bilayers showed voltage dependence of channel activation and inactivation, open and closed times, and single-channel conductances in Ba2+ and Ca2+ very similar to those found in cell-attached patch recordings. Open channels were blocked by micromolar concentrations of external Cd2+. In this cell-free system, channel activity tended to decrease during the course of an experiment, reminiscent of Ca2+ channel "rundown" in whole-cell and excised-patch recordings. A purely voltage-dependent component of inactivation was observed in the absence of Ca2+ stores or changes in intracellular Ca2+. Millimolar internal Ca2+ reduced unitary Ba2+ influx but did not greatly increase the rate or extent of inactivation or the rate of channel rundown. In symmetrical Ba2+ solutions, unitary conductance saturated as the Ba2+ concentration was increased up to 500 mM. The bilayer recordings also revealed activity of a novel Ca2+-permeable channel, termed "B-type" because it may contribute a steady background current at negative membrane potentials, which is distinct from L-type or T-type Ca channels previously reported. Unlike L-type channels, B-type channels have a small unitary Ba2+ conductance (7 pS), but do not discriminate between Ba2+ and Ca2+, show no obvious sensitivity to Bay K 8644, and do not run down. Unlike either L- or T-type channels, B-type channels did not require a depolarization for activation and displayed mean open times of greater than 100 ms.

The constructs of the Type A behavior pattern and the Inhibited Power Motive Syndrome (IPMS) have many features in common. The empirical relation between the two constructs was investigated in this study with 45 employed, male medical and surgical patients. Four different measures of the Type A pattern were examined. Results showed that, of the four measures, the Structured Interview and the Hostility Scale were related significantly to the IPMS. Systolic blood pressure reactivity was also related significantly to the IPMS. These relations could be ascribed largely to activity inhibition alone. The contribution of activity inhibition to an understanding of the biological and psychological substrates of the Type A behavior pattern is discussed.

These experiments provide a starting point for biochemical characterization of Ca channels from neuronal membranes, using omega-CgTX as a specific marker. The purification of the omega-CgTX receptors is far from complete. Each of the purification steps described results in only a two- to fivefold enrichment of the receptor proteins, and is accompanied by a loss of receptor concentration and stability, so the maximal specific activity achieved by a combination of these steps falls several orders of magnitude short of that of a large, homogeneous, active protein. Nevertheless, these studies have yielded important information about the omega-CgTX receptor. The Stokes' radius, determined from gel exclusion chromatography, is approximately 87 A, and the sedimentation coefficient, determined from sucrose gradient sedimentation, is approximately 19 S. These values are similar to those found for the DHP receptors solubilized in digitonin. We have also found that at least some of the omega-CgTX receptors have complex carbohydrate moieties that are recognized by WGA, together with evidence of heterogeneity of receptor glycosylation. Additionally, we have been able to use the solubilized, partially purified receptors in cross-linking experiments to tentatively identify the molecular weights of the omega-CgTX targets from rat brain. A large peptide of approximately 300 kDa, similar to that identified in photoaffinity studies, is very clearly labeled by the chemical incorporation of [125I]omega-CgTX into partially purified receptor preparations, but some ambiguity remains because of the faint labeling of peptides in the 120-170-kDa range. The approximately 300-kDa peptide is much larger than any single peptide component of DHP receptors from skeletal muscle, and it may be related to a molecular combination of the 170-kDa and 135-kDa subunits of the DHP receptor. Because [125I]omega-CgTX presumably labels both N- and L-type neuronal Ca channels, both channel types will probably be found in the purified preparations. Thus, at some time, it will be necessary to separate DHP-sensitive L-type channels from preparations of L- and N-type channels identified by omega-CgTX binding.

The functional reconstitution of the voltage-regulated Na channel purified from the electroplax of Electrophorus electricus is described. Reconstitution was achieved by removing detergent with Bio-Beads SM-2 followed by freeze-thaw-sonication in the presence of added liposomes. This preparation displayed heat-stable binding of 3H-labeled tetrodotoxin (TTX) (Kd = 33 nM). 22Na+ influx was stimulated 2- to 5-fold by alkaloid neurotoxins and blocked by TTX. Veratridine activated Na+ influx with a K1/2 of 18 microM, and this activation was blocked by TTX precisely in parallel with specific [3H]TTX binding. Batrachotoxin stimulated 22Na+ flux more effectively than did veratridine. No effect of the peptide anemone toxin II was found. Insertion of the Na channel into membranes resulted in 60-70% of the TTX-binding sites facing the vesicle exterior. Thus, external TTX partially inhibited flux, whereas blockade was complete when TTX was also equilibrated with the vesicle interior. The lipid-soluble local anesthetics tetracaine and dibucaine inhibited flux completely. QX-222, a charged derivative of lidocaine, blocked only a fraction of the channels, apparently those oriented inside-out. Purified samples were predominantly composed of the Mr 260,000-300,000 glycopeptide but contained variable quantities of smaller peptides. Veratridine-dependent flux and peptide compositions were determined on fractions across a gel filtration column profile. Stimulated flux codistributed only with the large peptide.

AK+ -selective membrane conductance channel from rabbit sarcoplasmic reticulum (SR) is studied in an artificial planar phospholipid bilayer. Membranes containing many such channels display voltage-dependent conductance, which is well described by a two-state conformational equilibrium with a free energy term linearly dependent on applied voltage. Pronase-derived alkaline proteinase b (APb), when added to the side of the membrane opposite to the SR vesicles (trans side), reduces the voltage dependence of the K+ conductance. Single-channel fluctuation experiments show that after APb treatment, the channel is still able to undergo transitions between its open and closed states, but that the probability of forming the open state is only slightly voltage-dependent. In terms of the conformational model, the enzyme's primary effect is to reduce the effective gating charge of the opening process by over 80%; a second effect of APb is to reduce the internal free energy of opening from +1.2 to +0.4 kcal/mol. The kinetics of APb action are strongly voltage-dependent, so as to indicate that the enzyme can react only with the channel's open state. The results imply that the channel contains a highly charged polypeptide region which moves in the direction perpendicular to the membrane plane when transitions between the open and closed states occur. A lysine or arginine residue in this region becomes exposed to the trans aqueous solution when the channel is in its open conformation.

Electrophysiological recordings were used to analyze single calcium channels in planar lipid bilayers after membranes from bovine cardiac sarcolemmal vesicles had been incorporated into the bilayer. In these cell-free conditions, channels in the bilayer showed unitary barium or calcium conductances, gating kinetics, and pharmacological responses that were similar to dihydropyridine-sensitive calcium channels in intact cells. The open channel current varied in a nonlinear manner with voltage under asymmetric (that is, physiological) ionic conditions. However, with identical solutions on both sides of the bilayer, the current-voltage relation was linear. In matched experiments, calcium channels from skeletal muscle T-tubules differed significantly from cardiac calcium channels in their conductance properties and gating kinetics.

Measures of support, stress, distress, and cardiac symptoms were obtained from a cohort of 73 male cardiac patients at hospitalization and at 3, 6, and 12 months thereafter. Sets of general and alternative hypotheses regarding the direction of causality among these variables were drawn from the literature on cardiac rehabilitation, stress, and support. Structural equation modeling was used to evaluate the stability and duration of these hypotheses over three time-lags. The results showed strong support for the general hypotheses and minimal support for the alternative hypotheses. Support ameliorated the subsequent experience of stress and distress and had opposing effects to these variables on cardiac symptoms. Support was more influential in the first half of the year than it was in the second half, however, whereas stress was predominant causally in the second half. Implications of this pattern for clinical intervention are drawn and directions for further research are proposed.

The open-channel conductance properties of a voltage-gated channel from sarcoplasmic reticulum were studied in planar phospholipid membranes. The channel is ideally selective for K+ over Cl- and for K+ over Ca++. In symmetrical 1 M solutions, the single-channel conductance (in pmho) falls in the order: K+ (214) > NH4+ (157) > Rb+ (125) > Na+ (72) > La+ (8.1) > Cs+ (< 3). In neutral bilayers, the channel conductance saturates with ion activity according to a rectangular hyperbolic relation, with half-saturation activities of 54 mM for K+ and 34 mM for Na+. Under symmetrical salt conditions, the K+:Na+ channel conductance ratio increases with salt activity, but the permeability ratio, measured by single-channel bi-ionic potentials, is constant between 20 mM and 2.5 M salt; the permeability ratio is equal to the conductance ratio in the limit of low-salt concentration. The channel conductance varies < 5% in the voltage range -100 to +70 mV. The maximum conductance varies K+ and Na+ is only weakly temperature dependent (delta H++ = 4.6 and 5.3 kcal/mol, respectively), but that of Li+ varies strongly with temperature (delta H++ = 13 kcal/mol). The channel's K+ conductance is blocked asymmetrically by Cs+, and this block is competitive with K+. The results are consistent with an Eyring-type barriers as it permeates the channel. The data conform to Lüger's (1973. Biochem. Biophys. Acta. 311:423-441) predictions for a "pure" single-ion channel.