Glutamate is required to maintain the steady-state potassium pool in Salmonella typhimurium.

In many bacteria, accumulation of K+ at high external osmolalities is accompanied by accumulation of glutamate. To determine whether there is an obligatory relationship between glutamate and K+ pools, we studied mutant strains of Salmonella typhimurium with defects in glutamate synthesis. Enteric bacteria synthesize glutamate by the combined action of glutamine synthetase and glutamate synthase (GS/GOGAT cycle) or the action of biosynthetic glutamate dehydrogenase (GDH). Activity of the GS/GOGAT cycle is required under nitrogen-limiting conditions and is decreased at high external ammonium/ammonia ((NH4)+) concentrations by lowered synthesis of GS and a decrease in its catalytic activity due to covalent modification (adenylylation by GS adenylyltransferase). By contrast, GDH functions efficiently only at high external (NH4)+ concentrations, because it has a low affinity for (NH4)+. When grown at low concentrations of (NH4)+ (< or = 2 mM), mutant strains of S. typhimurium that lack GOGAT and therefore are dependent on GDH have a low glutamate pool and grow slowly; we now demonstrate that they have a low K+ pool. When subjected to a sudden (NH4)+ upshift, strains lacking GS adenylyltransferase drain their glutamate pool into glutamine and grow very slowly; we now find that they also drain their K+ pool. Restoration of the glutamate pool in these strains at late times after shift was accompanied by restoration of the K+ pool and a normal growth rate. Taken together, the results indicate that glutamate is required to maintain the steady-state K+ pool -- apparently no other anion can substitute as a counter-ion for free K+ -- and that K+ glutamate is required for optimal growth.

N-methyl-D-aspartate receptor-induced proteolytic conversion of postsynaptic class C L-type calcium channels in hippocampal neurons.

Ca2+ influx controls multiple neuronal functions including neurotransmitter release, protein phosphorylation, gene expression, and synaptic plasticity. Brain L-type Ca2+ channels, which contain either alpha 1C or alpha 1D as their pore-forming subunits, are an important source of calcium entry into neurons. Alpha 1C exists in long and short forms, which are differentially phosphorylated, and C-terminal truncation of alpha 1C increases its activity approximately 4-fold in heterologous expression systems. Although most L-type calcium channels in brain are localized in the cell body and proximal dendrites, alpha 1C subunits in the hippocampus are also present in clusters along the dendrites of neurons. Examination by electron microscopy shows that these clusters of alpha 1C are localized in the postsynaptic membrane of excitatory synapses, which are known to contain glutamate receptors. Activation of N-methyl-D-aspartate (NMDA)-specific glutamate receptors induced the conversion of the long form of alpha 1C into the short form by proteolytic removal of the C terminus. Other classes of Ca2+ channel alpha1 subunits were unaffected. This proteolytic processing reaction required extracellular calcium and was blocked by inhibitors of the calcium-activated protease calpain, indicating that calcium entry through NMDA receptors activated proteolysis of alpha1C by calpain. Purified calpain catalyzed conversion of the long form of immunopurified alpha 1C to the short form in vitro, consistent with the hypothesis that calpain is responsible for processing of alpha 1C in hippocampal neurons. Our results suggest that NMDA receptor-induced processing of the postsynaptic class C L-type Ca2+ channel may persistently increase Ca2+ influx following intense synaptic activity and may influence Ca2+-dependent processes such as protein phosphorylation, synaptic plasticity, and gene expression.

Evidence that free polyunsaturated fatty acids modify Na+ channels by directly binding to the channel proteins.

The effects of free polyunsaturated fatty acids (PUFA) on the binding of ligands to receptors on voltage-sensitive Na+ channels of neonatal rat cardiac myocytes were assessed. The radioligand was [benzoyl-2,5-(3)H] batrachotoxinin A 20alpha-benzoate ([(3)H]BTXB), a toxin that binds to the Na+ channel. The PUFA that have been shown to be antiarrhythmic, including eicosapentaenoic acid (EPA; C20:5n-3), docosahexaenoic acid (DHA; C22:6n-3), eicosatetraynoic acid (ETYA), linolenic acid (C18:3n-3), and linoleic acid (C18:2n-6), inhibited [(3)H]BTXB binding in a dose-dependent fashion with IC50 values of 28-35 microM, whereas those fatty acids that have no antiarrhythmic effects including saturated fatty acid (stearic acid, C18:0), monounsaturated fatty acid (oleic acid; C18:1n-9), and EPA methyl ester did not have a significant effect on [(3)H]BTXB binding. Enrichment of the myocyte membrane with cholesterol neither affected [(3)H]BTXB binding when compared with control cells nor altered the inhibitory effects of PUFA on [(3)H]BTXB binding. Scatchard analysis of [(3)H]BTXB binding showed that EPA reduced the maximal binding without altering the Kd for [(3)H]BTXB binding, indicating allosteric inhibition. The inhibition by EPA of [(3)H]BTXB binding was reversible (within 30 min) when delipidated bovine serum albumin was added. The binding of the PUFA to this site on the Na+ channel is reversible and structure-specific and occurs at concentrations close to those required for apparent antiarrhythmic effects and a blocking effect on the Na+ current, suggesting that binding of the PUFA at this site relates to their antiarrhythmic action.

Zn2+ interaction with Alzheimer amyloid beta protein calcium channels.

The Alzheimer disease 40-residue amyloid beta protein (AbetaP[1-40]) forms cation-selective channels across acidic phospholipid bilayer membranes with spontaneous transitions over a wide range of conductances ranging from 40 to 4000 pS. Zn2+ has been reported to bind to AbetaP[1-40] with high affinity, and it has been implicated in the formation of amyloid plaques. We now report the functional consequences of such Zn2+ binding for the AbetaP[1-40] channel. Provided the AbetaP[1-40] channel is expressed in the low conductance (<400 pS) mode, Zn2+ blocks the open channel in a dose- dependent manner. For AbetaP[1-40] channels in the giant conductance mode (>400 pS), Zn2+ doses in the millimolar range were required to exert substantial blockade. The Zn2+ chelator o-phenanthroline reverses the blockade. We also found that Zn2+ modulates AbetaP[1-40] channel gating and conductance only from one side of the channel. These data are consistent with predictions of our recent molecular modeling studies on AbetaP[1-40] channels indicating asymmetric Zn(2+)-AbetaP[1-40] interactions at the entrance to the pore.

Latent N-methyl-D-aspartate receptors in the recurrent excitatory pathway between hippocampal CA1 pyramidal neurons: Ca(2+)-dependent activation by blocking A1 adenosine receptors.

When performed at increased external [Ca2+]/[Mg2+] ratio (2.5 mM/0.5 mM), temporary block of A1 adenosine receptors in hippocampus [by 8-cyclopentyltheophylline (CPT)] leads to a dramatic and irreversible change in the excitatory postsynaptic current (EPSC) evoked by Schaffer collateral/commissural (SCC) stimulation and recorded by in situ patch clamp in CA1 pyramidal neurons. The duration of the EPSC becomes stimulus dependent, increasing with increase in stimulus strength. The later occurring component of the EPSC is carried through N-methyl-D-aspartate (NMDA) receptor-operated channels but disappears under either the NMDA antagonist 2-amino-5-phosphonovaleric acid (APV) or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). These findings indicate that the late component of the SCC-evoked EPSC is polysynaptic: predominantly non-NMDA receptor-mediated SCC inputs excite CA1 neurons that recurrently excite each other by predominantly NDMA receptor-mediated synapses. These recurrent connections are normally silent but become active after CPT treatment, leading to enhancement of the late component of the EPSC. The activity of these connections is maintained for at least 2 hr after CPT removal. When all functional NMDA receptors are blocked by dizocilpine maleate (MK-801), subsequent application of CPT leads to a partial reappearance of NMDA receptor-mediated EPSCs evoked by SCC stimulation, indicating that latent NMDA receptors are recruited. Altogether, these findings indicate the existence of a powerful system of NMDA receptor-mediated synaptic contacts in SCC input to hippocampal CA1 pyramidal neurons and probably also in reciprocal connections between these neurons, which in the usual preparation are kept latent by activity of A1 receptors.

Primary structure and functional expression of a cGMP-gated potassium channel.

Cyclic nucleotides modulate potassium (K) channel activity in many cells and are thought to act indirectly by inducing channel protein phosphorylation. Herein we report the isolation from rabbit of a gene encoding a K channel (Kcn1) that is specifically activated by cGMP and not by cAMP. Analysis of the deduced amino acid sequence (725 amino acids) indicates that, in addition to a core region that is highly homologous to Shaker K channels, Kcn1 also contains a cysteine-rich region similar to that of ligand-gated ion channels and a cyclic nucleotide-binding region. Northern blot analysis detects gene expression in kidney, aorta, and brain. Kcn1 represents a class of K channels that may be specifically regulated by cGMP and could play an important role in mediating the effects of substances, such as nitric oxide, that increase intracellular cGMP.

Blocking effects of polyunsaturated fatty acids on Na+ channels of neonatal rat ventricular myocytes.

Recent evidence indicates that polyunsaturated long-chain fatty acids (PUFAs) prevent lethal ischemia-induced cardiac arrhythmias in animals and probably in humans. To increase understanding of the mechanism(s) of this phenomenon, the effects of PUFAs on Na+ currents were assessed by the whole-cell patch-clamp technique in cultured neonatal rat ventricular myocytes. Extracellular application of the free 5,8,11,14,17-eicosapentaenoic acid (EPA) produced a concentration-dependent suppression of ventricular, voltage-activated Na+ currents (INa). After cardiac myocytes were treated with 5 or 10 microM EPA, the peak INa (elicited by a single-step voltage change with pulses from -80 to -30 mV) was decreased by 51% +/- 8% (P < 0.01; n = 10) and 64% +/- 5% (P < 0.001; n = 21), respectively, within 2 min. Likewise, the same concentrations of 4,7,10,16,19-docosahexaenoic acid produced the same inhibition of INa. By contrast, 5 and 10 microM arachidonic acid (AA) caused less inhibition of INa, but both n - 6 and n - 3 PUFAs inhibited INa significantly. A monounsaturated fatty acid and a saturated fatty acid did not. After washing out EPA, INa returned to the control level. Raising the concentration of EPA to 40 microM completely blocked INa. The IC50 of EPA was 4.8 microM. The inhibition of this Na+ channel was found to be dose and time, but not use dependent. Also, the EPA-induced inhibition of INa was voltage dependent, since 10 microM EPA produced 83% +/- 7% and 29% +/- 5% inhibition of INa elicited by pulses from -80 to -30 mV and from -150 to -30 mV, respectively, in single-step voltage changes. A concentration of 10 microM EPA shifted the steady-state inactivation curve of INa by -19 +/- 3 mV (n = 7; P < 0.01). These effects of PUFAs on INa may be important for their antiarrhythmic effect in vivo.

Sensitivity to abscisic acid of guard-cell K+ channels is suppressed by abi1-1, a mutant Arabidopsis gene encoding a putative protein phosphatase.

Abscisic acid (ABA) modulates the activities of three major classes of ion channels--inward- and outward-rectifying K+ channels (IK,in and IK,out, respectively) and anion channels--at the guard-cell plasma membrane to achieve a net efflux of osmotica and stomatal closure. Disruption of ABA sensitivity in wilty abi1-1 mutants of Arabidopsis and evidence that this gene encodes a protein phosphatase suggest that protein (de)-phosphorylation contributes to guard-cell transport control by ABA. To pinpoint the role of ABI1, the abi1-1 dominant mutant allele was stably transformed into Nicotiana benthamiana and its influence on IK,in, IK,out, and the anion channels was monitored in guard cells under voltage clamp. Compared with guard cells from wild-type and vector-transformed control plants, expression of the abi1-1 gene was associated with 2- to 6-fold reductions in IK,out and an insensitivity of both IK,in and IK,out to 20 microM ABA. In contrast, no differences between control and abi1-1 transgenic plants were observed in the anion current or its response to ABA. Parallel measurements of intracellular pH (pHi) using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) in every case showed a 0.15- to 0.2-pH-unit alkalinization in ABA, demonstrating that the transgene was without effect on the pHi signal that mediates in ABA-evoked K+ channel control. In guard cells from the abi1-1 transformants, normal sensitivity of both K+ channels to and stomatal closure in ABA was recovered in the presence of 100 microM H7 and 0.5 microM staurosporine, both broad-range protein kinase antagonists. These results demonstrate an aberrant K+ channel behavior--including channel insensitivity to ABA-dependent alkalinization of pHi--as a major consequence of abi1-1 action and implicate AB11 as part of a phosphatase/kinase pathway that modulates the sensitivity of guard-cell K+ channels to ABA-evoked signal cascades.

A structural motif for the voltage-gated potassium channel pore.

Mutation studies have identified a region of the S5-S6 loop of voltage-gated K+ channels (P region) responsible for teraethylammonium (TEA) block and permeation/selectivity properties. We previously modeled a similar region of the Na+ channel as four beta-hairpins with the C strands from each of the domains forming the external vestibule and with charged residues at the beta-turns forming the selectivity filter. However, the K+ channel P region amino acid composition is much more hydrophobic in this area. Here we propose a structural motif for the K+ channel pore based on the following postulates (Kv2.1 numbering). (i) The external TEA binding site is formed by four Tyr-380 residues; P loop residues participating in the internal TEA binding site are four Met-371 and Thr-372 residues. (ii) P regions form extended hairpins with beta-turns in sequence ITMT. (iii) only C ends of hairpins form the inner walls of the pore. (iv) They are extended nonregular strands with backbone carbonyl oxygens of segment VGYGD facing the pore with the conformation BRLRL. (v) Juxtaposition of P loops of the four subunits forms the pore. Fitting the external and internal TEA sites to TEA molecules predicts an hourglass-like pore with the narrowest point (GYG) as wide as 5.5 A, suggesting that selectivity may be achieved by interactions of carbonyls with partially hydrated K+. Other potential cation binding sites also exist in the pore.

Hypertonicity activates nonselective cation channels in mouse cortical collecting duct cells.

We investigated the effect of cell shrinkage on whole-cell currents of M-1 mouse cortical collecting duct cells. Addition of 100 mM sucrose to an isotonic NaCl bath solution induced cell shrinkage and increased whole-cell currents within 5-10 min by approximately 12-fold. The effect was reversible upon return to isotonic solution and could also be elicited by adding 100 mM urea or 50 mM NaCl. Replacement of bath Na+ by K+, Cs+, Li+, or Rb+ did not significantly affect the stimulated inward current, but replacement by N-methyl-D-glucamine reduced it by 88.1 +/- 1.3% (n = 34); this demonstrates that hypertonicity activates a nonselective alkali cation conductance. The activation was independent of extra- and intracellular Ca2+, but 1 or 10 mM ATP in the pipette suppressed it in a concentration-dependent manner, indicating that intracellular ATP levels may modulate the degree of channel activation. Flufenamic acid (0.1 mM) and gadolinium (0.1 mM) inhibited the stimulated current by 68.7 +/- 5.9% (n = 9) and 32.4 +/- 11.7% (n = 6), respectively, whereas 0.1 mM amiloride had no significant effect. During the early phase of hypertonic stimulation single-channel transitions could be detected in whole-cell current recordings, and a gradual activation of 30 and more individual channels with a single-channel conductance of 26.7 +/- 0.4 pS (n = 29) could be resolved. Thus, we identified the nonselective cation channel underlying the shrinkage-induced whole-cell conductance that may play a role in volume regulation.

A potassium channel beta subunit related to the aldo-keto reductase superfamily is encoded by the Drosophila hyperkinetic locus.

Genetic and physiological studies of the Drosophila Hyperkinetic (Hk) mutant revealed defects in the function or regulation of K+ channels encoded by the Shaker (Sh) locus. The Hk polypeptide, determined from analysis of cDNA clones, is a homologue of mammalian K+ channel beta subunits (Kv beta). Coexpression of Hk with Sh in Xenopus oocytes increases current amplitudes and changes the voltage dependence and kinetics of activation and inactivation, consistent with predicted functions of Hk in vivo. Sequence alignments show that Hk, together with mammalian Kv beta, represents an additional branch of the aldo-keto reductase superfamily. These results are relevant to understanding the function and evolutionary origin of Kv beta.

cAMP-dependent, long-lasting inhibition of a K+ current in mammalian neurons.

We report the long-term modulation of K+ channels by cAMP in cultured murine colliculi neurons. A short (1-2 s) application of 8-Br-cAMP induced a long-lasting broadening of the action potential, a loss of after-hyperpolarization, and a reduction in spike accommodation. In agreement with these changes, 8-Br-cAMP produced a long-lasting (2 hr) inhibition of a K+ current. These effects were also observed after a short activation of the pituitary adenylyl cyclase-activating polypeptide, beta-adrenergic, and 5-hydroxytryptamine type 4 (5-HT4) receptors, all known to increase cAMP. A transient activation of the cAMP-dependent protein kinase and a long-lasting inhibition of phosphatases (up to 2 hr) were detected. The blockade of the K+ current resulting from a brief application of 8-Br-cAMP or 5-hydroxytryptamine was prolonged from 2 to 4 hr when protein-serine/threonine phosphatases 1 and 2A were inhibited with 10 nM okadaic acid. The critical steps following the cAMP-dependent protein kinase activation and resulting in a long-term blockade of phosphatases are discussed in this report.