Inwardly rectifying K(+) (Kir) channels antagonize ictal-like epileptiform activity in area CA1 of the rat hippocampus.

Reactive glial cells, for example, from patients with temporal lope epilepsy have a reduced density of inward rectifying K(+) (Kir) channels and thus a reduced K(+) buffering capacity. Evidence is accumulating that this downregulation of Kir channels could be implicated in epileptogenesis. In rat hippocampal brain slices, prolonged exposure to the nonselective Kir channel antagonist, Cs(+) (5 mM), gives rise to an epileptiform field potential (Cs-FP) in area CA1 composed of an initial positive (interictal-like) phase followed by a prolonged negative (ictal-like) phase.

Flow-induced [Ca2+]i increase depends on nucleotide release and subsequent purinergic signaling in the intact nephron.

Flow induces cytosolic Ca(2+) increases ([Ca(2+)](i)) in intact renal tubules, but the mechanism is elusive. Mechanical stimulation in general is known to promote release of nucleotides (ATP/UTP) and trigger auto- and paracrine activation of P2 receptors in renal epithelia. It was hypothesized that the flow-induced [Ca(2+)](i) response in the renal tubule involves mechanically stimulated nucleotide release.

Importance of Leu99 in transmembrane segment M1 of the Na+, K+ -ATPase in the binding and occlusion of K+.

Twenty-six point mutations were introduced into the N-terminal and middle parts of transmembrane segment M1 of the Na+, K+ -ATPase and its cytosolic extension. None of the alterations to charged and polar residues in the N-terminal part of M1 and its cytosolic extension had any major effect on the cation binding properties, thus rejecting the hypothesis that these residues are involved in cation selectivity. By contrast, specific residues in the middle part of M1, particularly Leu(99), were found critical to K+ interaction of the enzyme.

Antiphase oscillations of endothelium and smooth muscle [Ca2+]i in vasomotion of rat mesenteric small arteries.

The mechanisms leading to vasomotion in the presence of noradrenaline and inhibitors of the sarcoplasmic/endoplasmic reticulum calcium ATPase were investigated in isolated rat mesenteric small arteries. Isobaric diameter and isometric force were measured together with membrane potential in endothelial cells and smooth muscle cells (SMC). Calcium in the endothelial cells and SMC was imaged with confocal microscopy.

ATP-binding modes and functionally important interdomain bonds of sarcoplasmic reticulum Ca2+-ATPase revealed by mutation of glycine 438, glutamate 439, and arginine 678.

ATP binds to sarcoplasmic reticulum Ca(2+)-ATPase both in a phosphorylating (catalytic) mode and in a nonphosphorylating (modulatory) mode, the latter leading to acceleration of phosphoenzyme turnover (Ca(2)E(1)P --> E(2)P and E(2)P --> E(2) reactions) and Ca(2+) binding (E(2) --> Ca(2)E(1)). In some of the Ca(2+)-ATPase crystal structures, Arg(678) and Glu(439) seem to be involved in the binding of nucleotide or an associated Mg(2+) ion. We have replaced Arg(678), Glu(439), and Gly(438) with alanine to examine their importance for the enzyme cycle and the modulatory effects of ATP and MgATP.

Effects of extracellular HCO3(-) on fatigue, pHi, and K+ efflux in rat skeletal muscles.

Elevated plasma HCO(3)(-) can improve exercise endurance in humans. This effect has been related to attenuation of the work-induced reduction in muscle pH, which is suggested to improve performance via at least two mechanisms: 1) less inhibition of muscle enzymes and 2) reduced opening of muscle K(ATP) channels with less ensuing reduction in excitability. Aiming at determining whether the ergogenic effect of HCO(3)(-) is related to effects on muscles, we examined the effect of elevating extracellular HCO(3)(-) from 25 to 40 mM (pH from 7.

Distal colonic Na(+) absorption inhibited by luminal P2Y(2) receptors.

Luminal P2 receptors are ubiquitously expressed in transporting epithelia. In steroid-sensitive epithelia (e.g.

Interaction between Na+/K+-pump and Na+/Ca2+-exchanger modulates intercellular communication.

Ouabain, a specific inhibitor of the Na(+)/K(+)-pump, has previously been shown to interfere with intercellular communication. Here we test the hypothesis that the communication between vascular smooth muscle cells is regulated through an interaction between the Na(+)/K(+)-pump and the Na(+)/Ca(2+)-exchanger leading to an increase in the intracellular calcium concentration ([Ca(2+)](i)) in discrete areas near the plasma membrane. [Ca(2+)](i) in smooth muscle cells was imaged in cultured rat aortic smooth muscle cell pairs (A7r5) and in rat mesenteric small artery segments simultaneously with force.

Additive protective effects of the addition of lactic acid and adrenaline on excitability and force in isolated rat skeletal muscle depressed by elevated extracellular K+.

During strenuous exercise, extracellular K(+) ([K(+)](o)) is increased, which potentially can reduce muscle excitability and force production. In addition, exercise leads to accumulation of lactate and H(+) and increased levels of circulating catecholamines. Individually, reduced pH and increased catecholamines have been shown to counteract the depressing effect of elevated K(+).

Role of cholinergic-activated KCa1.1 (BK), KCa3.1 (SK4) and KV7.1 (KCNQ1) channels in mouse colonic Cl- secretion.

Aim: Colonic crypts are the site of Cl- secretion. Basolateral K+ channels provide the driving force for luminal cystic fibrosis transmembrane regulator-mediated Cl- exit. Relevant colonic epithelial K+ channels are the intermediate conductance Ca2+-activated K(Ca)3.

Modulation of FXYD interaction with Na,K-ATPase by anionic phospholipids and protein kinase phosphorylation.

FXYD10 is a 74 amino acid small protein which regulates the activity of shark Na,K-ATPase. The lipid dependence of this regulatory interaction of FXYD10 with shark Na,K-ATPase was investigated using reconstitution into DOPC/cholesterol liposomes with or without the replacement of 20 mol % DOPC with anionic phospholipids. Specifically, the effects of the cytoplasmic domain of FXYD10, which contains the phosphorylation sites for protein kinases, on the kinetics of the Na,K-ATPase reaction were investigated by a comparison of the reconstituted native enzyme and the enzyme where 23 C-terminal amino acids of FXYD10 had been cleaved by mild, controlled trypsin treatment.

Post-tetanic potentiation of GABAergic IPSCs in cultured hippocampal neurons is exclusively time-dependent.

We have previously shown that post-tetanic potentiation (PTP) of GABAergic IPSCs in cultured hippocampal neurons involves activation of L-type Ca(2+) channels. Although there is little Ca(2+) entry by this route, it is possible that L-type Ca(2+) channels mediate an increase in probability of release (Pr) by a mechanism that remains dormant in the absence of stimulation. We have tested this hypothesis in the present study using dual whole-cell patch clamp recordings.

The importance of limitations in aerobic metabolism, glycolysis, and membrane excitability for the development of high-frequency fatigue in isolated rat soleus muscle.

We investigated the role of limitations in aerobic metabolism, glycolysis, and membrane excitability for development of high-frequency fatigue in isolated rat soleus muscle. Muscles mounted on force transducers were incubated in buffer bubbled with 5% CO(2) and either 95% O(2) (oxygenated) or 95% N(2) (anoxic) and stimulated at 60 Hz continuously for 30-120 s or intermittently for 120 s. Cyanide (2 mM) and 2-deoxyglucose (10 mM) were used to inhibit aerobic metabolism and both glycolysis and aerobic metabolism, respectively.

Modulation of extrasynaptic THIP conductances by GABAA-receptor modulators in mouse neocortex.

THIP is a hypnotic drug, which displays a unique pharmacological profile, because it activates a subset of extrasynaptic gamma-aminobutyric acid type A (GABA(A)) receptors containing delta-subunits. It is important to study the physiology and pharmacology of these extrasynaptic receptors and to determine how THIP interacts with other hypnotics and anesthetics. Here, we study the modulation of the extrasynaptic response to THIP using three classes of GABA(A)-receptor ligands.

Modulation of protein kinase C by curcumin; inhibition and activation switched by calcium ions.

Background And Purpose: Previous studies have identified the natural polyphenol curcumin as a protein kinase C (PKC) inhibitor. In contrast, we found significant stimulation of PKC activity following curcumin treatment. Thus, the mechanism of curcumin interaction with PKC was investigated.

Energy conservation attenuates the loss of skeletal muscle excitability during intense contractions.

High-frequency stimulation of skeletal muscle has long been associated with ionic perturbations, resulting in the loss of membrane excitability, which may prevent action potential propagation and result in skeletal muscle fatigue. Associated with intense skeletal muscle contractions are large changes in muscle metabolites. However, the role of metabolites in the loss of muscle excitability is not clear.

Beta3-adrenoceptor agonist stimulation of the Na+, K+ -pump in rat skeletal muscle is mediated by beta2- rather than beta3-adrenoceptors.

Background And Purpose: In cardiac muscle, BRL 37344, a selective beta3-adrenoceptor agonist, activates the Na+, K+ -pump via NO signalling. This study investigated whether BRL 37344 also activates the Na+, K+ -pump via beta3-adrenoceptors in skeletal muscle.

Experimental Approach: Isolated rat soleus muscles were incubated between 1 and 60 min in buffer.

Cation binding in Na,K-ATPase, investigated by 205Tl solid-state NMR spectroscopy.

Cation binding to Na,K-ATPase is characterized in native membranes at room temperature by solid-state NMR spectroscopy using the K(+) congener (205)Tl. It has been demonstrated that the signals from occluded Tl(+) and nonspecifically bound Tl(+) can be detected and distinguished by NMR. Effects of dipole-dipole coupling between (1)H and (205)Tl in the occlusion sites show that the ions are rigidly bound, rather than just occluded.

Gamma-hydroxybutyrate--a drug of abuse.

Gamma-hydroxybutyrate (GHB) is a drug of abuse that causes euphoria, anxiolysis, and hypnosis. The recent rise in the recreational intake of GHB, as well as its association with 'drug rape', has turned the attention to GHB in acute hospital settings. Acutely admitted GHB intoxicated patients may display various levels of sedation or coma, but may also show paradoxical agitation, combativeness, or self-injurious behaviors.

Mutational analysis of the conserved TGES loop of sarcoplasmic reticulum Ca2+-ATPase.

Crystal structures have shown that the conserved TGES loop of the Ca2+-ATPase is isolated in the Ca2E1 state but becomes inserted in the catalytic site in E2 states. Here, we have examined the kinetics of the partial reaction steps of the transport cycle and the binding of the phosphoryl analogs BeF, AlF, MgF, and vanadate in mutants with alterations to the TGES residues. The mutations encompassed variation of size, polarity, and charge of the side chains.

Postnatal development of a new type of epileptiform activity in the rat hippocampus.

Long-term application of Cs(+) (5 mM) induces an epileptiform field potential (Cs-FP) in area CA1 of the rat hippocampus, which is independent of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors and gamma-aminobutyric acid (GABA)(A) receptors. To gain insight into possible mechanisms for the induction of the Cs-FP, we investigated the postnatal development of the response. In brain slices prepared from rats at different ages, the Cs-FP was evoked by stimulation of the Schaffer-collateral-commisural pathway.

The structural basis for coupling of Ca2+ transport to ATP hydrolysis by the sarcoplasmic reticulum Ca2+-ATPase.

Recently, a series of structure determinations has nearly completed a structural description of the transport cycle of the sarcoplasmic reticulum Ca(2+)-ATPase, especially those steps concerned with the phosphorylation by ATP and the dephosphorylation reaction. From these structures Ca(2+)-ATPase emerges as a molecular machine, where globular cytosolic domains and transmembrane helices work in concert like a mechanical pump, as can be vividly demonstrated in animated versions of the pump cycle. The structures show that both ATP phosphorylation and dephosphorylation at Asp351 take place as nucleophilic SN2 reactions, which are associated with Ca(2+) and H(+) occluded states, respectively.

Mutations Phe785Leu and Thr618Met in Na+,K+-ATPase, associated with familial rapid-onset dystonia parkinsonism, interfere with Na+ interaction by distinct mechanisms.

The Na(+),K(+)-ATPase plays key roles in brain function. Recently, missense mutations in the Na(+),K(+)-ATPase were found associated with familial rapid-onset dystonia parkinsonism (FRDP). Here, we have characterized the functional consequences of FRDP mutations Phe785Leu and Thr618Met.

Lipid-protein interactions with the Na,K-ATPase.

Studies of lipid interactions with membranous Na,K-ATPase by using electron spin resonance spectroscopy in conjunction with spin-labelled lipids are reviewed. The lipid stoichiometry, selectivity and exchange dynamics at the lipid-protein interface can be determined, in addition to information on the configuration and rotational dynamics of the protein-associated lipid chains. These parameters, particularly the stoichiometry and selectivity, are related directly to the intramembranous structure of the Na,K-ATPase, and can be used to check the integrity of extensively trypsinised preparations.

Na+-K+ pump stimulation improves contractility in damaged muscle fibers.

Skeletal muscles have a high content of Na+-K+-ATPase, an enzyme that is identical to the Na+-K+ pump, a transport system mediating active extrusion of Na+ from the cells and accumulation of K+ in the cells. The major function of the Na+-K+ pumps is to maintain the concentration gradients for Na+ and K+ across the plasma membrane. This generates the resting membrane potential, allowing the propagation of action potentials, excitation-contraction coupling and force development.