Molecular simulations and free-energy calculations suggest conformation-dependent anion binding to a cytoplasmic site as a mechanism for Na+/K+-ATPase ion selectivity.

Na+/K+-ATPase transports Na+ and K+ ions across the cell membrane via an ion-binding site becoming alternatively accessible to the intra- and extracellular milieu by conformational transitions that confer marked changes in ion-binding stoichiometry and selectivity. To probe the mechanism of these changes, we used molecular simulation and free-energy perturbation approaches to identify probable protonation states of Na+- and K+-coordinating residues in E1P and E2P conformations of Na+/K+-ATPase. Analysis of these simulations revealed a molecular mechanism responsible for the change in protonation state: the conformation-dependent binding of an anion (a chloride ion in our simulations) to a previously unrecognized cytoplasmic site in the loop between transmembrane helices 8 and 9, which influences the electrostatic potential of the crucial Na+-coordinating residue Asp926 This mechanistic model is consistent with experimental observations and provides a molecular-level picture of how E1P to E2P enzyme conformational transitions are coupled to changes in ion-binding stoichiometry and selectivity.

Identification of electric-field-dependent steps in the Na(+),K(+)-pump cycle.

The charge-transporting activity of the Na(+),K(+)-ATPase depends on its surrounding electric field. To isolate which steps of the enzyme's reaction cycle involve charge movement, we have investigated the response of the voltage-sensitive fluorescent probe RH421 to interaction of the protein with BTEA (benzyltriethylammonium), which binds from the extracellular medium to the Na(+),K(+)-ATPase's transport sites in competition with Na(+) and K(+), but is not occluded within the protein. We find that only the occludable ions Na(+), K(+), Rb(+), and Cs(+) cause a drop in RH421 fluorescence. We conclude that RH421 detects intramembrane electric field strength changes arising from charge transport associated with conformational changes occluding the transported ions within the protein, not the electric fields of the bound ions themselves. This appears at first to conflict with electrophysiological studies suggesting extracellular Na(+) or K(+) binding in a high field access channel is a major electrogenic reaction of the Na(+),K(+)-ATPase. All results can be explained consistently if ion occlusion involves local deformations in the lipid membrane surrounding the protein occurring simultaneously with conformational changes necessary for ion occlusion. The most likely origin of the RH421 fluorescence response is a change in membrane dipole potential caused by membrane deformation.

Membrane potential-dependent inhibition of the Na+,K+-ATPase by para-nitrobenzyltriethylammonium bromide.

Membrane potential (V(M))-dependent inhibitors of the Na(+),K(+)-ATPase are a new class of compounds that may have inherent advantages over currently available drugs targeting this enzyme. However, two questions remain unanswered regarding these inhibitors: (1) what is the mechanism of V(M)-dependent Na(+),K(+)-ATPase inhibition, and (2) is their binding affinity high enough to consider them as possible lead compounds? To address these questions, we investigated how a recently synthesized V(M)-dependent Na(+),K(+)-ATPase inhibitor, para-nitrobenzyltriethylamine (pNBTEA), binds to the enzyme by measuring the extracellular pNBTEA concentration and V(M) dependence of ouabain-sensitive transient charge movements in whole-cell patch-clamped rat cardiac ventricular myocytes. By analyzing the kinetics of charge movements and the steady-state distribution of charge, we show that the V(M)-dependent properties of pNBTEA binding differ from those for extracellular Na(+) and K(+) binding, even though inhibitor binding is competitive with extracellular K(+). The data were also fit to specific models for pNBTEA binding to show that pNBTEA binding is a rate-limiting V(M)-dependent reaction that, in light of homology models for the Na(+),K(+)-ATPase, we interpret as a transfer reaction of pNBTEA from a peripheral binding site in the enzyme to a site near the known K(+) coordination sites buried within the transmembrane helices of the enzyme. These models also suggest that binding occurs with an apparent affinity of 7 µM. This apparent binding affinity suggests that high-affinity V(M)-dependent Na(+),K(+)-ATPase inhibitors should be feasible to design and test as specific enzyme inhibitors.

Evaluating spatial and network properties of NMDA-dependent neuronal connectivity in mixed cortical cultures.

A technique combining fluorescence imaging with Ca2+ indicators and single-cell laser scanning photostimulation of caged glutamate (LSPS) allowed identification of functional connections between individual neurons in mixed cultures of rat neocortical cells as well as observation of synchronous spontaneous activity among neurons. LSPS performed on large numbers of neurons yielded maps of functional connections between neurons and allowed calculation of neuronal network parameters. LSPS also provided an indirect measure of excitability of neurons targeted for photostimulation. By repeating LSPS sessions with the same neurons, stability of connections and change in the number and strength of connections were also determined. Experiments were conducted in the presence of bicuculline to study in detail the properties of excitatory neurotransmission. The AMPA receptor inhibitor, 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), abolished synchronous neuronal activity but had no effect on connections mapped by LSPS. In contrast, the NMDA receptor inhibitor, 2-Amino-5-phosphono-pentanoic acid (APV), dramatically decreased the number of functional connections between neurons while also affecting synchronous spontaneous activity. Functional connections were also decreased by increasing extracellular Mg2+ concentration. These data demonstrated that LSPS mapping interrogates NMDA receptor-dependent connectivity between neurons in the network. In addition, a GluN2A-specific inhibitor, NVP-AAM077, decreased the number and strength of connections between neurons as well as neuron excitability. Conversely, the GluN2A-specific positive modulator, GNE-0723, increased these same properties. These data showed that LSPS can be used to directly study perturbations in the properties of NMDA receptor-dependent connectivity in neuronal networks. This approach should be applicable in a wide variety of in vitro and in vivo experimental preparations.

Quaternary benzyltriethylammonium ion binding to the Na,K-ATPase: a tool to investigate extracellular K+ binding reactions.

This study examined how the quaternary organic ammonium ion, benzyltriethylamine (BTEA), binds to the Na,K-ATPase to produce membrane potential (V(M))-dependent inhibition and tested the prediction that such a V(M)-dependent inhibitor would display electrogenic binding kinetics. BTEA competitively inhibited K(+) activation of Na,K-ATPase activity and steady-state (86)Rb(+) occlusion. The initial rate of (86)Rb(+) occlusion was decreased by BTEA to a similar degree whether it was added to the enzyme prior to or simultaneously with Rb(+), a demonstration that BTEA inhibits the Na,K-ATPase without being occluded. Several BTEA structural analogues reversibly inhibited Na,K-pump current, but none blocked current in a V(M)-dependent manner except BTEA and its para-nitro derivative, pNBTEA. Under conditions that promoted electroneutral K(+)-K(+) exchange by the Na,K-ATPase, step changes in V(M) elicited pNBTEA-activated ouabain-sensitive transient currents that had similarities to those produced with the K(+) congener, Tl(+). pNBTEA- and Tl(+)-dependent transient currents both displayed saturation of charge moved at extreme negative and positive V(M), equivalence of charge moved during and after step changes in V(M), and similar apparent valence. The rate constant (k(tot)) for Tl(+)-dependent transient current asymptotically approached a minimum value at positive V(M). In contrast, k(tot) for pNBTEA-dependent transient current was a "U"-shaped function of V(M) with a minimum value near 0 mV. Homology models of the Na,K-ATPase alpha subunit suggested that quaternary amines can bind to two extracellularly accessible sites, one of them located at K(+) binding sites positioned between transmembrane helices 4, 5, and 6. Altogether, these data revealed important information about electrogenic ion binding reactions of the Na,K-ATPase that are not directly measurable during ion transport by this enzyme.

Quaternary organic amines inhibit Na,K pump current in a voltage-dependent manner: direct evidence of an extracellular access channel in the Na,K-ATPase.

The effects of organic quaternary amines, tetraethylammonium (TEA) chloride and benzyltriethylammonium (BTEA) chloride, on Na,K pump current were examined in rat cardiac myocytes superfused in extracellular Na(+)-free solutions and whole-cell voltage-clamped with patch electrodes containing a high Na(+)-salt solution. Extracellular application of these quaternary amines competitively inhibited extracellular K(+) (K(+)(o)) activation of Na,K pump current; however, the concentration for half maximal inhibition of Na,K pump current at 0 mV (K(0)(Q)) by BTEA, 4.0 +/- 0.3 mM, was much lower than the K(0)(Q) for TEA, 26.6 +/- 0.7 mM. Even so, the fraction of the membrane electric field dissipated during K(+)(o) activation of Na,K pump current (lambda(K)), 39 +/- 1%, was similar to lambda(K) determined in the presence of TEA (37 +/- 2%) and BTEA (35 +/- 2%), an indication that the membrane potential (V(M)) dependence for K(+)(o) activation of the Na,K pump current was unaffected by TEA and BTEA. TEA was found to inhibit the Na,K pump current in a V(M)-independent manner, i.e., inhibition of current dissipated 4 +/- 2% of the membrane electric field. In contrast, BTEA dissipated 40 +/- 5% of the membrane electric field during inhibition of Na,K pump current. Thus, BTEA inhibition of the Na,K-ATPase is V(M)-dependent. The competitive nature of inhibition as well as the similar fractions of the membrane electric field dissipated during K(+)(o)-dependent activation and BTEA-dependent inhibition of Na,K pump current suggest that BTEA inhibits the Na,K-ATPase at or very near the enzyme's K(+)(o) binding site(s) located in the membrane electric field. Given previous findings that organic quaternary amines are not occluded by the Na,K-ATPase, these data clearly demonstrate that an ion channel-like structure provides access to K(+)(o) binding sites in the enzyme.

Effect of acute stretch injury on action potential and network activity of rat neocortical neurons in culture.

The basis for acute seizures following traumatic brain injury (TBI) remains unclear. Animal models of TBI have revealed acute hyperexcitablility in cortical neurons that could underlie seizure activity, but studying initiating events causing hyperexcitability is difficult in these models. In vitro models of stretch injury with cultured cortical neurons, a surrogate for TBI, allow facile investigation of cellular changes after injury but they have only demonstrated post-injury hypoexcitability. The goal of this study was to determine if neuronal hyperexcitability could be triggered by in vitro stretch injury. Controlled uniaxial stretch injury was delivered to a spatially delimited region of a spontaneously active network of cultured rat cortical neurons, yielding a region of stretch-injured neurons and adjacent regions of non-stretched neurons that did not directly experience stretch injury. Spontaneous electrical activity was measured in non-stretched and stretch-injured neurons, and in control neuronal networks not subjected to stretch injury. Non-stretched neurons in stretch-injured cultures displayed a three-fold increase in action potential firing rate and bursting activity 30-60 min post-injury. Stretch-injured neurons, however, displayed dramatically lower rates of action potential firing and bursting. These results demonstrate that acute hyperexcitability can be observed in non-stretched neurons located in regions adjacent to the site of stretch injury, consistent with reports that seizure activity can arise from regions surrounding the site of localized brain injury. Thus, this in vitro procedure for localized neuronal stretch injury may provide a model to study the earliest cellular changes in neuronal function associated with acute post-traumatic seizures.

Direct effects of recurrent hypoglycaemia on adrenal catecholamine release.

In Type 1 and advanced Type 2 diabetes mellitus, elevation of plasma epinephrine plays a key role in normalizing plasma glucose during hypoglycaemia. However, recurrent hypoglycaemia blunts this elevation of plasma epinephrine. To determine whether recurrent hypoglycaemia affects peripheral components of the sympatho-adrenal system responsible for epinephrine release, male rats were administered subcutaneous insulin daily for 3 days. These recurrent hypoglycaemic animals showed a smaller elevation of plasma epinephrine than saline-injected controls when subjected to insulin-induced hypoglycaemia. Electrical stimulation of an adrenal branch of the splanchnic nerve in recurrent hypoglycaemic animals elicited less release of epinephrine and norepinephrine than in controls, without a change in adrenal catecholamine content. Responsiveness of isolated, perfused adrenal glands to acetylcholine and other acetylcholine receptor agonists was also unchanged. These results indicate that recurrent hypoglycaemia compromised the efficacy with which peripheral neuronal activity stimulates adrenal catecholamine release and demonstrate that peripheral components of the sympatho-adrenal system were directly affected by recurrent hypoglycaemia.

Na,K-pump reaction kinetics at the tip of a patch electrode: derivation of reaction kinetics for electrogenic and electrically silent reactions during ion transport by the Na,K-ATPase.

Patch-clamp electrophysiological techniques allow manipulations of electrochemical driving forces for ion transport by the Na,K-ATPase. For this reason, this technique has been used to study steady-state ion transport properties of the Na,K-ATPase. High temporal resolution during these manipulations also permits rapid reactions, such as extracellular ion-binding reactions, to be measured as charge movements when the enzyme is engaged in electroneutral ion exchange modes. Just as useful, but less widely recognized, is the ease with which electrophysiological techniques can be used to critically study reaction steps that do not directly involve ion binding. Three studies are briefly presented to show how pre-steady-state and/or steady-state electrophysiological techniques can be used to study ion-binding reactions in a novel fashion and the kinetics of electrically silent reaction steps of this enzyme. The reaction kinetics derived from each of these studies can be used to attain detailed mechanistic information about ion transport by the Na,K-ATPase.

Hypothalamic S-nitrosylation contributes to the counter-regulatory response impairment following recurrent hypoglycemia.

AIMS: Hypoglycemia is a severe side effect of intensive insulin therapy. Recurrent hypoglycemia (RH) impairs the counter-regulatory response (CRR) which restores euglycemia. During hypoglycemia, ventromedial hypothalamus (VMH) production of nitric oxide (NO) and activation of its receptor soluble guanylyl cyclase (sGC) are critical for the CRR. Hypoglycemia also increases brain reactive oxygen species (ROS) production. NO production in the presence of ROS causes protein S-nitrosylation. S-nitrosylation of sGC impairs its function and induces desensitization to NO. We hypothesized that during hypoglycemia, the interaction between NO and ROS increases VMH sGC S-nitrosylation levels and impairs the CRR to subsequent episodes of hypoglycemia. VMH ROS production and S-nitrosylation were quantified following three consecutive daily episodes of insulin-hypoglycemia (RH model). The CRR was evaluated in rats in response to acute insulin-induced hypoglycemia or via hypoglycemic-hyperinsulinemic clamps. Pretreatment with the anti-oxidant N-acetyl-cysteine (NAC) was used to prevent increased VMH S-nitrosylation. RESULTS: Acute insulin-hypoglycemia increased VMH ROS levels by 49±6.3%. RH increased VMH sGC S-nitrosylation. Increasing VMH S-nitrosylation with intracerebroventricular injection of the nitrosylating agent S-nitroso-L-cysteine (CSNO) was associated with decreased glucagon secretion during hypoglycemic clamp. Finally, in RH rats pre-treated with NAC (0.5% in drinking water for 9 days) hypoglycemia-induced VMH ROS production was prevented and glucagon and epinephrine production was not blunted in response to subsequent insulin-hypoglycemia. CONCLUSION: These data suggest that NAC may be clinically useful in preventing impaired CRR in patients undergoing intensive-insulin therapy.

Voltage-dependent modulation of L-type calcium currents by intracellular magnesium in rat ventricular myocytes.

Effects of changing cytosolic free Mg(2+) concentration on L-type Ca(2+) (I(Ca)) and Ba(2+) currents (I(Ba)) were investigated in rat ventricular myocytes voltage-clamped with pipettes containing 0.2 or 1.8mM [Mg(2+)] ([Mg(2+)](p)) buffered with 30mM citrate and 10mM ATP. Increasing [Mg(2+)](p) from 0.2 to 1.8mM reduced current amplitude and accelerated its decay under a variety of experimental conditions. To investigate the mechanism for these effects, steady-state and instantaneous current-voltage relationships were studied with two-pulse and tail current (I(T)) protocols, respectively. Increasing [Mg(2+)](p) shifted the V(M) for half inactivation by -20mV but dramatically decreased I(Ca) amplitude at all potentials tested, consistent with a change in gating kinetics that decreases channel availability. This conclusion was supported by analysis of I(T) amplitude, but these latter experiments also suggested that, in the millimolar concentration range, [Mg(2+)](p) might also inhibit permeation through open Ca(2+) channels at positive V(M).

Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration.

Previous studies have shown that inhibition of L-type Ca(2+) current (I(Ca)) by cytosolic free Mg(2+) concentration ([Mg(2+)](i)) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I(Ca), rat cardiac myocytes and tsA201 cells expressing L-type Ca(2+) channels were whole cell voltage-clamped with patch pipettes in which [Mg(2+)] ([Mg(2+)](p)) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca(2+) channels (alpha(1C)/beta(2A)/alpha(2)delta), increasing [Mg(2+)](p) from 0.2 mM to 1.8 mM decreased peak I(Ca) by 76 +/- 4.5% (n = 7). Mg(2+)-dependent modulation of I(Ca) was also observed in cells loaded with ATP-gamma-S. With 0.2 mM [Mg(2+)](p), manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP(2A)) produced large changes in I(Ca) amplitude; however, with 1.8 mM [Mg(2+)](p), these same manipulations had no significant effect on I(Ca). With mutant channels lacking principal PKA phosphorylation sites (alpha(1C/S1928A)/beta(2A/S478A/S479A)/alpha(2)delta), increasing [Mg(2+)](p) had only small effects on I(Ca). However, when channel open probability was increased by alpha(1C)-subunit truncation (alpha(1CDelta1905)/beta(2A/S478A/S479A)/alpha(2)delta), increasing [Mg(2+)](p) greatly reduced peak I(Ca). Correspondingly, in myocytes voltage-clamped with pipette PP(2A) to minimize channel phosphorylation, increasing [Mg(2+)](p) produced a much larger reduction in I(Ca) when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg(2+) modulates the extent to which channel phosphorylation regulates I(Ca). This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation.

Ca2+ and voltage dependence of cardiac ryanodine receptor channel block by sphingosylphosphorylcholine.

The effect of sphingosylphosphorylcholine (SPC) on the cytoplasmic Ca(2+) and voltage dependence of channel gating by cardiac ryanodine receptors (RyR) was examined in lipid bilayer experiments. Micromolar concentrations of the lysosphingolipid SPC added to cis solutions rapidly and reversibly decreased the single-channel open probability (P(o)) of reconstituted RyR channels. The SPC-induced decrease in P(o) was marked by an increase in mean closed time and burst-like channel gating. Gating kinetics during intraburst periods were unchanged from those observed in the absence of the sphingolipid, although SPC induced a long-lived closed state that appeared to explain the observed decrease in channel P(o). SPC effects were observed over a broad range of cis [Ca(2+)] but were not competitive with Ca(2+). Interestingly, the sphingolipid-induced, long-lived closed state displayed voltage-dependent kinetics, even though other channel gating kinetics were not sensitive to voltage. Assuming SPC effects represent channel blockade, these results suggest that the blocking rate is independent of voltage whereas the unblocking rate is voltage dependent. Together, these results suggest that SPC binds directly to the cytoplasmic side of the RyR protein in a location in or near the membrane dielectric, but distinct from cytoplasmic Ca(2+) binding sites on the protein.

Regulation of L-type calcium current by intracellular magnesium in rat cardiac myocytes.

The effects of changing cytosolic [Mg(2+)] ([Mg(2+)](i)) on L-type Ca(2+) currents were investigated in rat cardiac ventricular myocytes voltage-clamped with patch pipettes containing salt solutions with defined [Mg(2+)] and [Ca(2+)]. To control [Mg(2+)](i) and cytosolic [Ca(2+)] ([Ca(2+)](i)), the pipette solution included 30 mM citrate and 10 mM ATP along with 5 mM EGTA (slow Ca(2+) buffer) or 15 mM EGTA plus 5 mM BAPTA (fast Ca(2+) buffer). With pipette [Ca(2+)] ([Ca(2+)](p)) set at 100 nM using a slow Ca(2+) buffer and pipette [Mg(2+)] ([Mg(2+)](p)) set at 0.2 mM, peak l-type Ca(2+) current density (I(Ca)) was 17.0 +/- 2.2 pA pF(-1). Under the same conditions, but with [Mg(2+)](p) set to 1.8 mM, I(Ca) was 5.6 +/- 1.0 pA pF(-1), a 64 +/- 2.8% decrease in amplitude. This decrease in I(Ca) was accompanied by an acceleration and a -8 mV shift in the voltage dependence of current inactivation. The [Mg(2+)](p)-dependent decrease in I(Ca) was not significantly different when myocytes were preincubated with 10 microM forskolin and 300 microM 3-isobutyl-L-methylxanthine and voltage-clamped with pipettes containing 50 microM okadaic acid, to maximize Ca(2+) channel phosphorylation. However, when myocytes were voltage-clamped with pipettes containing protein phosphatase 2A, to promote channel dephosphorylation, I(Ca) decreased only 25 +/- 3.4% on changing [Mg(2+)](p) from 0.2 to 1.8 mM. In the presence of 0.2 mM[Mg(2+)](p), changing channel phosphorylation conditions altered I(Ca) over a 4-fold range; however, with 1.8 mM[Mg(2+)](p), these same manoeuvres had a much smaller effect on I(Ca). These data suggest that [Mg(2+)](i) can antagonize the effects of phosphorylation on channel gating kinetics. Setting [Ca(2+)](p) to 1, 100 or 300 nM also showed that the [Mg(2+)](p)-induced reduction of I(Ca) was smaller at the lowest [Ca(2+)](p), irrespective of channel phosphorylation conditions. This interaction between [Ca(2+)](i) and [Mg(2+)](i) to modulate I(Ca) was not significantly affected by ryanodine, fast Ca(2+) buffers or inhibitors of calmodulin, calmodulin-dependent kinase and calcineurin. Thus, physiologically relevant [Mg(2+)](i) modulates I(Ca) by counteracting the effects of Ca(2+) channel phosphorylation and by an unknown [Ca(2+)](i)-dependent mechanism. The magnitude of these effects suggests that changes in [Mg(2+)](i) could be critical in regulating L-type channel gating.