Importance of intramembrane carboxylic acids for occlusion of K+ ions at equilibrium in renal Na,K-ATPase

Site-directed mutagenesis and assay of Rb+ and Tl+ occlusion in recombinant Na,K-ATPase from yeast were combined to establish structure-function relationships of amino acid side chains involved in high-affinity occlusion of K+ in the E2[2K] form. The wild-type yeast enzyme was capable of occluding 2 Rb+ or Tl+ ions/ouabain binding site or alpha 1 beta 1 unit with high apparent affinity (Kd(Tl+) = 7 +/- 2 microM), like the purified Na,K-ATPase from pig kidney. Mutations of Glu327(Gln,Asp), Asp804(Asn, Glu), Asp808(Asn, Glu) and Glu779(Asp) abolished high-affinity occlusion of Rb+ or Tl+ ions. The substitution of Glu779 for Gln reduced the occlusion capacity to 1 Tl+ ion/alpha 1 beta 1-unit with a 3-fold decrease of the apparent affinity for the ion (Kd(Tl+) = 24 +/- 8 microM). These effects on occlusion were closely correlated to effects of the mutations on K0.5(K+) for K+ displacement of ATP binding. Each of the four carboxylate residues Glu327, Glu779, and Asp804 or Asp808 in transmembrane segments 4, 5, and 6 is therefore essential for high-affinity occlusion of K+ in the E2[2K] form. These residues either may engage directly in cation coordination or they may be important for formation or stability of the occlusion cavity.     

The multi-ion nature of the pore in Shaker K+ channels

We have investigated some of the permeation properties of the pore in Shaker K channels. We determined the apparent permeability ratio of K+, Rb+, and NH4+ ions and block of the pore by external Cs+ ions. Shaker channels were expressed with the baculovirus/Sf9 expression system and the channel currents measured with the whole-cell variant of the patch clamp technique. The apparent permeability ratio, PRb/PK, determined in biionic conditions with internal K+, was a function of external Rb+ concentration. A large change in PRb/PK occurred with reversed ionic conditions (internal Rb+ and external K+). These changes in apparent permeability were not due to differences in membrane potential. With internal K+, PNH4/PK was not a function of external NH4+ concentration (at least over the range 50-120 mM). We also investigated block of the pore by external Cs+ ions. At a concentration of 20 mM, Cs+ block had a voltage dependence equivalent to that of an ion with a valence of 0.91; this increased to 1.3 at 40 mM Cs+. We show that a 4-barrier, 3-site permeation model can simulate these and many of the other known properties of ion permeation in Shaker channels.     

Carbachol-induced increase of Na+/H+ antiport and recruitment of Na+,K(+)-ATPase in rabbit lacrimal acini

Parallel arrays of Na+/H+ and Cl-/HCO3- antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na+ and Cl- influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na+/H+ antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H+ gradients accelerated 22Na+ uptake, and amiloride inhibited 96% of the H+ gradient-dependent 22Na+ flux. Amiloride-sensitive 22Na+ influx was half-maximal at an extravesicular Na+ concentration of 14 mM. In vitro stimulation of isolated lacrimal acini with 10 microM carbachol for 30 min increased Na+/H+ antiport activity of a subsequently isolated basolateral membrane sample 2.5-fold, but it did not significantly affect Na+/H+ antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na+,K(+)-ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na+,K(+)-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na+,K(+)-ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Na+/H+ antiport activity remains unclear.     

Charge translocation by the Na+/K+-ATPase investigated on solid supported membranes: cytoplasmic cation binding and release

In the preceding publication (. Biophys. J. 76:000-000) a new technique was described that was able to produce concentration jumps of arbitrary ion species at the surface of a solid supported membrane (SSM). This technique can be used to investigate the kinetics of ion translocating proteins adsorbed to the SSM. Charge translocation of the Na+/K+-ATPase in the presence of ATP was investigated. Here we describe experiments carried out with membrane fragments containing Na+/K+-ATPase from pig kidney and in the absence of ATP. Electrical currents are measured after rapid addition of Na+. We demonstrate that these currents can be explained only by a cation binding process on the cytoplasmic side, most probably to the cytoplasmic cation binding site of the Na+/K+-ATPase. An electrogenic reaction of the protein was observed only with Na+, but not with other monovalent cations (K+, Li+, Rb+, Cs+). Using Na+ activation of the enzyme after preincubation with K+ we also investigated the K+-dependent half-cycle of the Na+/K+-ATPase. A rate constant for K+ translocation in the absence of ATP of 0.2-0.3 s-1 was determined. In addition, these experiments show that K+ deocclusion, and cytoplasmic K+ release are electroneutral.     

E2P phosphoforms of Na,K-ATPase. II. Interaction of substrate and cation-binding sites in Pi phosphorylation of Na,K-ATPase

In this investigation the effects of alkali cations on the transient kinetics of Na,K-ATPase phosphoenzyme formation from either ATP (E2P) or Pi (E'2P) were characterized by chemical quench methods as well as by stopped-flow RH421 fluorescence experiments. By combining the two methods it was possible to characterize the kinetics of Na, K-ATPase from two sources, shark rectal glands and pig kidney. The rate of the spontaneous dephosphorylation of E2P and E'2P was identical with a rate constant of about 1.1 s-1 at 20 degreesC. However, whereas dephosphorylation of E2P formed from ATP was strongly stimulated by K+, dephosphorylation of E'2P formed from Pi in the absence of alkali cations was K+-insensitive, although in pig renal enzyme K+ binding to E'2P could be demonstrated with RH421 fluorescence. It appears, therefore, that in pig kidney enzyme the rapid binding of K+ to E'2P was followed by a slow transition to a nonfluorescent form. For shark enzyme the K+-induced decrease of RH421 fluorescence of Pi phosphorylated enzyme was due to K+ binding to the dephosphoenzyme (E1), thus shifting the equilibrium away from E'2P. When Pi phosphorylation was performed with enzyme equilibrated with K+ or its congeners Tl+, Rb+, and Cs+ but not with Na+ or Li+, both the phosphorylation and the dephosphorylation rates were considerably increased. This indicates that binding of cations modifies the substrate site in a cation-specific way, suggesting an allosteric interaction between the conformation of the cation-binding sites and the phosphorylation site of the enzyme.     

Calcium-activated potassium conductances on cultured nontransformed dog pancreatic duct epithelial cells

Pancreatic duct epithelial cells (PDECs) mediate the pancreatic secretion of fluid and electrolytes. Membrane K+ channels on these cells regulate intracellular K+ concentration; in combination with the Na+/H+ antiport and Na+,K+ adenosine triphosphatase (ATPase), they may also mediate serosal H+ secretion, balancing luminal HCO3- secretion. We describe the K+ conductances on well-differentiated and functional nontransformed cultured dog PDECs. Through 86Rb+ efflux studies, we demonstrated Ca(2+)-activated K+ channels that were stimulated by A23187, thapsigargin, and 1-ethyl-2-benzimidazolinone, but not forskolin. These conductances also were localized on the basolateral membrane because 86Rb+ efflux was directed toward the serosal compartment. Of the K+ channel blockers, BaCl2, charybdotoxin, clotrimazole, and quinidine, but not 4-aminopyridine, apamin, tetraethylammonium, or iberiotoxin, inhibited 86Rb+ efflux. This efflux was not inhibited by amiloride, ouabain, and bumetanide, inhibitors of the Na+/H+ antiport, the Na+,K(+)-ATPase pump, and the Na+,K+,2Cl- cotransporter, respectively. When apically permeabilized PDEC monolayers were mounted in Ussing chambers with a luminal-to-serosal K+ gradient, A23187 and 1-ethyl-2-benzimidazolinone stimulated a charybdotoxin-sensitive short-circuit current (Isc) increase. Characterization of K+ channels on these cultured PDECs, along with previous identification of Cl- channels (1), further supports the importance of these cells as models for pancreatic duct secretion.     

Stimulation of ouabain-sensitive 86Rb+ uptake and Na+,K+-ATPase alpha-subunit phosphorylation by a cAMP-dependent signalling pathway in intact cells from rat kidney cortex

We investigated in intact cortical kidney tubules the role of PKA-mediated phosphorylation in the short-term control of Na+,K+-ATPase activity. The phosphorylation level of Na+,K+-ATPase was evaluated after immunoprecipitation of the enzyme from 32P-labelled cortical tubules and the cation transport activity of Na+,K+-ATPase was measured by ouabain-sensitive 86Rb+ uptake. Incubation of cells with cAMP analogues (8-bromo-cAMP, dibutyryl-cAMP) or with forskolin plus 3-isobutyl-1-methylxanthine increased the phosphorylation level of the Na+,K+-ATPase alpha-subunit and stimulated ouabain-sensitive 86Rb+ uptake. Inhibition of PKA by H-89 blocked the effects of dibutyryl-cAMP on both phosphorylation and 86Rb+ uptake processes. The results suggest that phosphorylation by PKA stimulates the Na+,K+-ATPase activity.     

Stop-flow studies of solute uptake in rat lungs

Stop-flow studies were used to characterize solute uptake in isolated rat lungs. These lungs were perfused at 8 or 34 ml/min for 10-28 s with solutions containing 125I-albumin and two or more of the following diffusible indicators: [3H]mannitol, [14C]urea, 3HOH, 201Tl+, or 86Rb+. After this loading period, flow was stopped for 10-300 s and then resumed to flush out the perfusate that remained in the pulmonary vasculature during the stop interval. Concentrations of 201Tl+ and 86Rb+ in the venous outflow decreased after the stop interval, indicating uptake from exchange vessels during the stop interval. The amount of these K+ analogs lost from the circulation during the stop interval was greater when the intervals were longer. However, losses of 201T1+ at 90 s approached those at 300 s. Because extraction continued after the vasculature had been flushed, vascular levels had presumably fallen to negligible levels during the stop interval. By 90 s of stop flow the vascular volume that was cleared of 201T1+ averaged 0.657 +/- 0.034 (SE) ml in the experiments perfused at 8 ml/min and 0.629 +/- 0.108 ml in those perfused at 34 ml/min. Increases in perfusate K+ decreased the cleared volumes of 201T1+ and 86Rb+. Uptake of [3H]mannitol, [14C]urea, and 3HOH during the stop intervals was observed only when the lungs were loaded at high flow for short intervals. Decreases in 201T1+ and 86Rb+ concentrations in the pulmonary outflow can be used to identify the fraction of the collected samples that were within exchange vessels of the lung during the stop interval and may help determine the distribution of solute and water exchange along the pulmonary vasculature.     

Na+,K+-ATPase was found to be the membrane component responsible for the hydrophobic behavior of the brain membrane tubulin

We have previously described that the tubulin isolated from brain membranes as a hydrophobic compound by partitioning into Triton X-114 is a peripheral membrane protein [corrected]. The hydrophobic behavior of this tubulin is due to its interaction with membrane protein(s) and the interaction occurs principally with the acetylated tubulin isotype. In the present work we identified the membrane protein that interacts with tubulin as the Na+,K+-ATPase alpha subunit by amino acid sequencing. Using purified brain Na+,K+-ATPase we were able to isolate part of the total hydrophilic tubulin as a hydrophobic compound which contains a high proportion of the acetylated tubulin isotype.     

Pathways of Rb+ influx and their relation to intracellular [Na+] in the perfused rat heart. A 87Rb and 23Na NMR study

The aims of this study were to characterize the routes of influx of the K+ congener, Rb+, into cardiac cells in the perfused rat heart and to evaluate their links to the intracellular Na+ concentration ([Na+]i) using 87Rb and 23Na nuclear magnetic resonance (NMR) spectroscopy. The rate constant for Rb+ equilibration in the extracellular space was 8.5 times higher than that for the intracellular space. The sensitivity of the rate of Rb+ accumulation in the intracellular space of the perfused rat heart to the inhibitors of the K+ and Na+ transport systems has been analyzed. The Rb+ influx rates were measured in both beating and arrested hearts: both procaine (5 mmol/L) and lidocaine (1 mmol/L) halved the Rb+ influx rate. In procaine-arrested hearts, the Na+,K(+)-ATPase inhibitor ouabain (0.6 mmol/L) decreased Rb+ influx by 76 +/- 24% relative to that observed in untreated but arrested hearts. Rb+ uptake was insensitive to the K+ channel blocker 4-aminopyridine (1 mmol/L). The inhibitor of Na+/K+/2 Cl- cotransport bumetanide (30 mumol/L) decreased Rb+ uptake only slightly (by 9 +/- 8%). Rb+ uptake was dependent on [Na+]i: it increased by 58 +/- 34% when [Na+]i was increased with the Na+ ionophore monensin (1 mumol/L) and decreased by 48 +/- 9% when [Na+]i was decreased by the Na+ channel blockers procaine and lidocaine. Dimethylamiloride (15 to 20 mumol/L), an inhibitor of the Na+/H+ exchanger, slightly reduced [Na+]i and Rb+ entry into the cardiomyocytes (by 15 +/- 5%). 31P NMR spectroscopy was used to monitor the energetic state and intracellular pH (pHi) in a parallel series of hearts. Treatment of the hearts with lidocaine, 4-aminopyridine, dimethylamiloride, or bumetanide for 15 to 20 minutes at the same concentrations as used for the Rb+ and Na+ experiments did not markedly affect the levels of the phosphate metabolites or pHi. These data show that under normal physiological conditions, Rb+ influx occurs mainly through Na+,K(+)-ATPase; the contribution of the Na+/K+/2 Cl- cotransporter and K+ channels to Rb+ influx is small. The correlation between Rb+ influx and [Na+bdi during infusion of drugs that affect [Na+]i indicates that, in rat hearts at 37 degrees C, Rb+ influx can serve as a measure of Na+ influx. We estimate that, at normothermia, at least 50% of the Na+ entry into beating cardiac cells is provided by the Na+ channels, with only minor contributions (< 15%) from the Na+/K+/2 Cl- cotransporter and the Na+/H+ exchanger.     

Functional activity of Na+,K(+)-pump in normal and pathological tissues

Na+,K(+)-ATPase, supporting the ionic homeostasis of the cell, is under control of Na+, K+, Mg2+, and ATP. The regulating effect of Mg2+ is rather unclear, whereas the Na+/K+ ratio in the cytoplasm is a potent regulatory factor, especially for osmotic balance in excitable cells. We have demonstrated two possibilities for regulation of ion pumping activity: First, via the number of Na+,K(+)-ATPase molecules under operation, and second, via changes in the turnover rate of the active molecules. In the presence of low ATP concentration, which is typical for cells with membrane damage (ischemic cardiac myocytes, tumor cells, fatigued muscles) Na+,K(+)-ATPase is transformed to a regime of the decreased efficiency. Radiation inactivation study demonstrates the weakening of the interprotein interactions in the enzyme complexes during ATP deficiency. Thus, measurements of ATPase activity of the purified enzyme under optimal conditions in vitro may be useless for the discrimination of pathological from normal tissues. In such a case, the estimation of lipid composition and microviscosity of the membranes under study could be important. This review briefly discusses several basic mechanisms of the regulation of Na+,K(+)-ATPase--an integral protein of the outer cell membranes.     

Characterization of electrogenic Na/K pump in rat neostriatal neurons

The electrogenic Na/K pump current (Ip) was studied in the dissociated neostriatal neurons of the rat by using the nystatin-perforated patch recording mode. The Ip was activated by external K+ in a concentration-dependent manner with an EC50 of 0.7 mM at a holding potential (VH) of -40 mV. Other monovalent cations also caused Ip and the order of potency was Tl+>K+, Rb+>NH4+, Cs+>Li+. The Ip decreased with membrane hyperpolarization in an external solution containing 150 mM Na+, while the Ip did not show such voltage dependency without external Na+. Ouabain showed a steady-state inhibition of Ip in a concentration- and temperature-dependent manner at a VH of -40 mV. The IC50 values at 20 and 30 degrees C were 7.1 x 10(-6) and 1.3 x 10(-6) M, respectively. The decay of Ip after adding ouabain well fitted with a single exponential function. At a VH of -40 Mv, the association (k+1) and dissociation (k-1) rate constants estimated from the time constant of the current decay at 20 degrees C were 4.0 x10(2) s-1 M-1 and 6.3 x 10(-3) s-1, respectively. At 30 degrees C, k+1 increased to 2.8 x 10(3) s-1 M-1 while k-1 showed no such change with a value of 1.8 x 10(-3) s-1. A continuous Na+ influx was demonstrated by both the Na+-dependent leakage current and tetrodotoxin-sensitive Na+ current, which resulted in the continuous activation of the Na/K pump. It was thus concluded that the Na/K pump activity was well-maintained in the dissociated rat neostriatal neurons with distinct functional properties and that the activity of the pump was tightly connected with Na+ influxes.     

A novel crystallization method for visualizing the membrane localization of potassium channels

The high permeability of K+ channels to monovalent thallium (Tl+) ions and the low solubility of thallium bromide salt were used to develop a simple yet very sensitive approach to the study of membrane localization of potassium channels. K+ channels (Kir1.1, Kir2.1, Kir2.3, Kv2.1), were expressed in Xenopus oocytes and loaded with Br ions by microinjection. Oocytes were then exposed to extracellular thallium. Under conditions favoring influx of Tl+ ions (negative membrane potential under voltage clamp, or high concentration of extracellular Tl+), crystals of TlBr, visible under low-power microscopy, formed under the membrane in places of high density of K+ channels. Crystals were not formed in uninjected oocytes, but were formed in oocytes expressing as little as 5 microS K+ conductance. The number of observed crystals was much lower than the estimated number of functional channels. Based on the pattern of crystal formation, K+ channels appear to be expressed mostly around the point of cRNA injection when injected either into the animal or vegetal hemisphere. In addition to this pseudopolarized distribution of K+ channels due to localized microinjection of cRNA, a naturally polarized (animal/vegetal side) distribution of K+ channels was also frequently observed when K+ channel cRNA was injected at the equator. A second novel "agarose-hemiclamp" technique was developed to permit direct measurements of K+ currents from different hemispheres of oocytes under two-microelectrode voltage clamp. This technique, together with direct patch-clamping of patches of membrane in regions of high crystal density, confirmed that the localization of TlBr crystals corresponded to the localization of functional K+ channels and suggested a clustered organization of functional channels. With appropriate permeant ion/counterion pairs, this approach may be applicable to the visualization of the membrane distribution of any functional ion channel.     

Cations affect [3H]mazindol and [3H]WIN 35,428 binding to the human dopamine transporter in a similar fashion

The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn2+, Mg2+, Hg2+, Li+, K+, and Na+ were assessed on the binding of [3H]mazindol and [3H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21 degrees C. Zn2+ (30-100 microM) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [3H]mazindol; Mg2+ (0.1-100 microM) had no effect; Hg2+ at approximately 3 microM stimulated binding to membranes, with a relatively smaller effect for [3H]mazindol than [3H]WIN 35,428 at 0 degrees C, and at 30-100 microM inhibited both intact cell and membrane binding; Li+ and K+ substitution (30-100 mM) inhibited binding to membranes more severely than to intact cells; and Na+ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21 degrees C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn2+ at 0 and 21 degrees C and Hg2+ at 0 degrees C.     

Time-irreversible subconductance gating associated with Ba2+ block of large conductance Ca2+-activated K+ channels

Ba2+ block of large conductance Ca2+-activated K+ channels was studied in patches of membrane excised from cultures of rat skeletal muscle using the patch clamp technique. Under conditions in which a blocking Ba2+ ion would dissociate to the external solution (150 mM N-methyl-D-glucamine+o, 500 mM K+i, 10 microM Ba2+i, +30 mV, and 100 microM Ca2+i to fully activate the channel), Ba2+ blocks with a mean duration of approximately 2 s occurred, on average, once every approximately 100 ms of channel open time. Of these Ba2+ blocks, 78% terminated with a single step in the current to the fully open level and 22% terminated with a transition to a subconductance level at approximately 0.26 of the fully open level (preopening) before stepping to the fully open level. Only one apparent preclosing was observed in approximately 10,000 Ba2+ blocks. Thus, the preopenings represent Ba2+-induced time-irreversible subconductance gating. The fraction of Ba2+ blocks terminating with a preopening and the duration of preopenings (exponentially distributed, mean = 0.75 ms) appeared independent of changes in [Ba2+]i or membrane potential. The fractional conductance of the preopenings increased from 0.24 at +10 mV to 0.39 at +90 mV. In contrast, the average subconductance level during normal gating in the absence of Ba2+ was independent of membrane potential, suggesting different mechanisms for preopenings and normal subconductance levels. Preopenings were also observed with 10 mM Ba2+o and no added Ba2+i. Adding K+, Rb+, or Na+ to the external solution decreased the fraction of Ba2+ blocks with preopenings, with K+ and Rb+ being more effective than Na+. These results are consistent with models in which the blocking Ba2+ ion either induces a preopening gate, and then dissociates to the external solution, or moves to a site located on the external side of the Ba2+ blocking site and acts directly as the preopening gate.     

Effect of angiotensin-(1-7) on ATPase activities in several tissues

The present investigation was undertaken to determine whether Ang-(1-7) is able to modify ATPase activities in membrane fractions prepared from several tissues. In the presence of 10(-6) M Ang-(1-7), total (Na , K+, Mg2+)-ATPase activity decreased 31% in rat atrium and 13% in sheep atrium but was unmodified in sheep liver, rat ventricle or crude brain membranes. In rat brain synaptosomal membranes, Ang-(1-7) at 10(-8) and 10(-7) M concentrations activated Na+, K+-ATPase 20 and 24%, respectively. Rat kidney Na+, K+-ATPase activity decreased roughly 40-70% with 10(-10)-10(-6) M Ang-(1-7)), but increased 22% with 10(-12) M peptide concentration, thus indicating a biphasic effect. Our findings showing that ATPase from several tissues responds differently to Ang-(1-7) are attributable to enzyme tissue specificity.     

Localisation of potassium pumps in Drosophila ovarian follicles

It has been shown previously that, in Drosophila oogenesis, potassium ions are important for bioelectric phenomena as well as for other physiological and developmental processes. In the present study we determined the spatial distribution and activity of the Na+,K+)-pump and of ouabain-insensitive K+ pumps in plasma membranes of vitellogenic ovarian follicles (stage 10). We used the light microscopic anthroylouabain method as well as the cytochemical lead and cerium precipitation methods in combination with electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS). (Na+,K+)-ATPase activity was predominantly observed on the oolemma as well as on the membranes of the columnar follicle cells covering the oocyte, whereas on the membranes of the nurse cells and of the squamous follicle cells covering the nurse cells the activity was very low. The highest activity of the (Na+,K+)-pump was found at the anterior and posterior ends of the oocyte, and this on the oolemma as well as on the membranes of the follicle cells located here. Strong activity of the ouabain-insensitive K+-pumps was observed on most of the oolemma (except at the anterior of the oocyte) and on the membranes of some nurse cells located next to the oocyte, whereas less activity was found on the other nurse cell membranes and on the membranes of all follicle cells. The suitability of the different methods used for determining the localisation as well as the activity of K+-pumps is discussed. We further discuss the nature of the ouabain-insensitive K+ pumps and the relevance of the observed distribution of K+-pumps for K+ uptake, extrafollicular ionic current flow, intercellular signalling and other developmental processes in Drosophila oogenesis.     

Analysis of leak current properties in the lobster stretch receptor neurone

Experiments were performed to characterize the so-called leak current of the slowly adapting stretch receptor neurone of the European lobster with respect to its ionic basis, its kinetics and its pharmacology. Estimates of the leak current were obtained by subtraction of a Na-K pump current and of an unspecific impalement current from a non-dynamic ('instantaneous') current, recorded in a voltage range from approximately -120 to approximately -30 mV, after blockage of spike-generating currents and a hyperpolarization-activated inwardly rectifying current (Q-current). The leak current, estimated in this way, was seen to reverse direction at the cell's K+ equilibrium voltage, thus indicating that it is carried by K+ passing through channels which, also, proved to be permeable to Rb+ and NH4+, but not permeable to Na+ or Cl- to any significant extent. Kinetically, the leak current was found to be characterized by being enhanced by increases in extracellular K+ and by being subject to outward rectification, most distinctly at elevated extracellular [K+]. In quantitative terms, these kinetic properties could be accounted for by a mathematical model comprising (1) a one-site two-barrier Eyring formulation describing ion permeation through membrane channels and (2) an ordinary dose-response relationship describing the channel-opening effect of K+ at an extracellular regulatory site. Pharmacologically, the leak current proved to be distinguished by being reversibly blockable, in a non-voltage dependent manner, by CO2+ (Kd = 0.9 mM, Hill coefficient 1.1) and procaine, but not by Ba2+, Gd3+, bupivacaine (a local anesthetic), or other K+ channel blockers such as TEA, 4-AP and Cs+. It is concluded that, in native unimpaled cells, the K+ carried leak current (1) is setting the resting voltage together with the (mainly) Na(+)-carried Q-current and the Na-K pump current, (2) is determining the cell's firing threshold, together with the spike generating currents, and (3) is also stabilizing the cell's membrane excitability in conditions of varying extracellular [K+], by virtue of its K+ sensitivity.     

Glucose decreases Na+,K+-ATPase activity in pancreatic beta-cells. An effect mediated via Ca2+-independent phospholipase A2 and protein kinase C-dependent phosphorylation of the alpha-subunit

In the pancreatic beta-cell, glucose-induced membrane depolarization promotes opening of voltage-gated L-type Ca2+ channels, an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i), and exocytosis of insulin. Inhibition of Na+,K+-ATPase activity by ouabain leads to beta-cell membrane depolarization and Ca2+ influx. Because glucose-induced beta-cell membrane depolarization cannot be attributed solely to closure of ATP-regulated K+ channels, we investigated whether glucose regulates other transport proteins, such as the Na+,K+-ATPase. Glucose inhibited Na+,K+-ATPase activity in single pancreatic islets and intact beta-cells. This effect was reversible and required glucose metabolism. The inhibitory action of glucose was blocked by pretreatment of the islets with a selective inhibitor of a Ca2+-independent phospholipase A2. Arachidonic acid, the hydrolytic product of this phospholipase A2, also inhibited Na+, K+-ATPase activity. This effect, like that of glucose, was blocked by nordihydroguaiaretic acid, a selective inhibitor of the lipooxygenase metabolic pathway, but not by inhibitors of the cyclooxygenase or cytochrome P450-monooxygenase pathways. The lipooxygenase product 12(S)-HETE (12-S-hydroxyeicosatetranoic acid) inhibited Na+,K+-ATPase activity, and this effect, as well as that of glucose, was blocked by bisindolylmaleimide, a specific protein kinase C inhibitor. Moreover, glucose increased the state of alpha-subunit phosphorylation by a protein kinase C-dependent process. These results demonstrate that glucose inhibits Na+, K+-ATPase activity in beta-cells by activating a distinct intracellular signaling network. Inhibition of Na+,K+-ATPase activity may thus be part of the mechanisms whereby glucose promotes membrane depolarization, an increase in [Ca2+]i, and thereby insulin secretion in the pancreatic beta-cell.