A molecular model of cooperative binding of Ca2+ with rhodopsin molecules in photoreceptor membranes]

Kinetics of calcium binding by photoreceptor membranes of cattle retina in concentration Ca2+ 0.5 and 1.0.10(-5) M in 5 mM tris-HCl buffer, pH 7.4 at 37 degrees C has been studied. Such kinetics is of oscillating nature. Analysis of calcium binding process curves by photoreceptor membranes allow to conclude, that crystalline areas of rhodopsin (receptor domains) can be formed in the structure of photoreceptor membranes. Conformation states and structure of rhodopsin molecules Ca-binding sites in receptor domains depend on the presence of Ca2+ in the medium. The structure of rhodopsin molecules Ca-binding sites in receptor domain formed in the presence of Ca2+ in the medium was proposed. According to the Hodgkin and Huxley conception concerning the properties of Na(+)- and K(+)-channels, the receptor domain with such a structure of rhodopsin molecules Ca-binding sites can represent the conjugate system of Na(+)- and K(+)-channels. Molecular mechanisms of photoreceptor and nerve cells excitation was also proposed.     

Memory and potassium channels

The K(+)-channels of the surface membrane play a crucial role in the generation of electrical activity of a neuron. There is a large diversity of the K(+)-channels that depends on a great number (over 200) of genes encoding channels proteins. An evolutionary conservation of channel's proteins is determined. The K(+)-channels were found to have a great importance in the memory processes. It was shown on different model systems that K(+)-current of the surface membrane decreases during the learning. The antagonists of K(+)-channels were found to improve the learning and memory. It was revealed in electrophysiological experiments that K(+)-channels antagonists can either themselves induce a long-term synaptic potentiation or intensify the synaptic potentiation induced by a tetanization. The disfunction of K(+)-channels is believed to be an important link in the mechanisms of memory disturbances. In animal mutants with K(+)-channels disfunction, learning and memory are deficient. In behavioral experiments, the use of K(+)-channels openers make the learning worse. Amnesia caused by cerebral ischemia is explained by strong activity of K(+)-channels which not only inhibits neuronal excitement but also causes neurodegeneration. The question on the K(+)-channels involvement into pathophysiology of Alzheimer's disease is discussed. Neurotoxic peptide beta-amyloid, which is supposed to be involved into mechanisms of Alzheimer's disease, modulates K(+)-channels function. The effect of beta-amyloid depends on the subtype of K(+)-channels: A-channels are inhibited, and KDR-channels, on the contrary, become stronger. The effect of the cognitive enhancers (vinpocetine, piracetam, tacrine, linopirdine) on K(+)-current also depends on the subtype of K(+)-channels. Slow-inactivating K(+)-currents (IDR, IK(Ca), IM) are inhibited in the presence of these drugs, while fast-in-activating K(+)-current (A-current) remains unchanged or even increases.     

Ion pumps in polarized cells: sorting and regulation of the Na+, K+- and H+, K+-ATPases

The physiologic function of an ion transport protein is determined, in part, by its subcellular localization and by the cellular mechanisms that modulate its activity. The Na(+),K(+)-ATPase and the H(+),K(+)-ATPases are closely related members of the P-type family of ion transporting ATPases. Despite their homology, these pumps are sorted to different domains in polarized epithelial cells, and their enzymatic activities are subject to distinct regulatory pathways. The molecular signals responsible for these properties have begun to be elucidated. It appears that a complex array of inter- and intramolecular interactions govern trafficking, distribution, and catalytic capacities of these proteins.     

Developmental clustering of ion channels at and near the node of Ranvier.

Voltage-gated Na(+) and K(+) channels are localized to distinct subcellular domains in mammalian myelinated nerve fibers. Specifically, Na(+) channels are clustered in high densities at nodes of Ranvier, while K(+) channels are found in juxtaparanodal zones just beyond regions of axoglial contact where sequential layers of the myelin sheath terminate. Specific targeting, clustering, and maintenance of these channels in their respective domains are essential to achieve high conduction velocities of action potential propagation. The cellular, molecular, and developmental mechanisms that exist to achieve this neuronal specialization are discussed and reviewed. Current evidence points to a prominent role in channel clustering played by myelinating glial cells, and sites of axoglial contact in particular.     

Na+ shows a markedly higher potential than K+ in DNA compaction in a crowded environment

Whereas many physicochemical investigations have shown that among monovalent cations Na(+) ion possesses minimal potential for DNA binding, biological assays have shown that Na(+) ion (in contrast to K(+) ion) plays a primary role in chromatin compaction and related processes. It is difficult to explain this inverse relationship between the compaction potentials of Na(+) and K(+) and their binding abilities. In this study we sought to resolve this contradiction and emphasize the phenomenological distinction between DNA compaction and DNA binding processes in the case of DNA compaction by monocations. Using polyethylene glycol solutions as a model of a crowded cell environment, we studied DNA compaction by alkali metal salts LiCl, NaCl, KCl, RbCl, and CsCl, and found that all of these monocations promote DNA compaction. Among these monovalent cations Na(+) produces the greatest compaction and the ratio of K(+) cand Na(+) oncentrations for DNA compaction is approximately 1.5-2. A comparative analysis of recent experimental results indicates that a higher binding activity of monocation generally corresponds to a low compaction potential of the corresponding monovalent ion. This inverse relation is explained as a result of partial dehydration of monocations in the compact state.     

Distinguishing roles of the membrane-cytoskeleton and cadherin mediated cell-cell adhesion in generating different Na+,K(+)-ATPase distributions in polarized epithelia

In simple epithelia, the distribution of ion transporting proteins between the apical or basal-lateral domains of the plasma membrane is important for determining directions of vectorial ion transport across the epithelium. In the choroid plexus, Na+,K(+)-ATPase is localized to the apical plasma membrane domain where it regulates sodium secretion and production of cerebrospinal fluid; in contrast, Na+,K(+)-ATPase is localized to the basal-lateral membrane of cells in the kidney nephron where it regulates ion and solute reabsorption. The mechanisms involved in restricting Na+,K(+)-ATPase distribution to different membrane domains in these simple epithelia are poorly understood. Previous studies have indicated a role for E-cadherin mediated cell-cell adhesion and membrane-cytoskeleton (ankyrin and fodrin) assembly in regulating Na+,K(+)-ATPase distribution in absorptive kidney epithelial cells. Confocal immunofluorescence microscopy reveals that in chicken and rat choroid plexus epithelium, fodrin, and ankyrin colocalize with Na+,K(+)-ATPase at the apical plasma membrane, but fodrin, ankyrin, and adducin also localize at the lateral plasma membrane where Na+,K(+)- ATPase is absent. Biochemical analysis shows that fodrin, ankyrin, and Na+,K(+)-ATPase are relatively resistant to extraction from cells in buffers containing Triton X-100. The fractions of Na+,K(+)-ATPase, fodrin, and ankyrin that are extracted from cells cosediment in sucrose gradients at approximately 10.5 S. Further separation of the 10.5 S peak of proteins by electrophoresis in nondenaturing polyacrylamide gels revealed that fodrin, ankyrin, and Na+,K(+)-ATPase comigrate, indicating that these proteins are in a high molecular weight complex similar to that found previously in kidney epithelial cells. In contrast, the anion exchanger (AE2), a marker protein of the basal- lateral plasma membrane in the choroid plexus, did not cosediment in sucrose gradients or comigrate in nondenaturing polyacrylamide gels with the complex of Na+,K(+)-ATPase, ankyrin, and fodrin. Ca(++)- dependent cell adhesion molecules (cadherins) were detected at lateral membranes of the choroid plexus epithelium and colocalized with a distinct fraction of ankyrin, fodrin, and adducin. Cadherins did not colocalize with Na+,K(+)-ATPase and were absent from the apical membrane. The fraction of cadherins that was extracted with buffers containing Triton X-100 cosedimented with ankyrin and fodrin in sucrose gradients and comigrated in nondenaturing gels with ankyrin and fodrin in a high molecular weight complex. Since a previous study showed that E-cadherin is an instructive inducer of Na+,K(+)-ATPase distribution, we examined protein distributions in fibroblasts transfected with B- cadherin, a prominent cadherin expressed in the choroid plexus epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Formation of new high density glycogen-microtubule structures is induced by cardiac steroids

Cardiac steroids (CS), an important class of naturally occurring compounds, are synthesized in plants and animals. The only established receptor for CS is the ubiquitous Na(+),K(+)-ATPase, a major plasma membrane transporter. The binding of CS to Na(+),K(+)-ATPase causes the inhibition of Na(+) and K(+) transport and elicits cell-specific activation of several intracellular signaling mechanisms. It is well documented that the interaction of CS with Na(+),K(+)-ATPase is responsible for numerous changes in basic cellular physiological properties, such as electrical plasma membrane potential, cell volume, intracellular [Ca(2+)] and pH, endocytosed membrane traffic, and the transport of other solutes. In the present study we show that CS induces the formation of dark structures adjacent to the nucleus in human NT2 and ACHN cells. These structures, which are not surrounded by membranes, are clusters of glycogen and a distorted microtubule network. Formation of these clusters results from a relocation of glycogen and microtubules in the cells, two processes that are independent of one another. The molecular mechanisms underlying the formation of the clusters are mediated by the Na(+),K(+)-ATPase, ERK1/2 signaling pathway, and an additional unknown factor. Similar glycogen clusters are induced by hypoxia, suggesting that the CS-induced structural change, described in this study, may be part of a new type of cellular stress response.     

Regulation of Na+-K+-ATPase: effect of Mg and Ca ions

The participation of Mg2+ and Ca2+ in complicated mechanisms of Na+, K(+)-ATPase regulation is discussed in the survey. The regulatory actions of Mg2+ on Na+, K(+)-ATPase such as its participation in phosphorylation and dephosphorylation of the enzyme, ADP/ATP-exchange inhibition, cardiac glycosides and vanadate binding with the enzyme, conformational changes induction during ATPase cycle are reviewed in detail. Some current views of mechanisms of above mentioned Mg2+ regulatory effects are discussed. The experimental evidence of Ca2+ immediate influence on the functional activity of Na+, K(+)-ATPase (catalytic, transport and glycoside-binding) are given. It's noted that these effects are based on the conformational changes in the enzyme and also on the phase transition in membrane induced by Ca2+. Unimmediate action of Ca2+ on Na+, K(+)-ATPase is also discussed, especially due to its effect on other membrane systems functionally linked with Na(+)-pump (for instance, due to Na+/Ca(+)-exchanger activation). It's concluded that Mg2+ and Ca2+ as "universal regulators" of the cell effectively influence the functional activity and conformational states of Na+, K(+)-ATPase.     

The 14-3-3 protein translates the NA+,K+-ATPase {alpha}1-subunit phosphorylation signal into binding and activation of phosphoinositide 3-kinase during endocytosis

Clathrin-dependent endocytosis of Na(+),K(+)-ATPase molecules in response to G protein-coupled receptor signals is triggered by phosphorylation of the alpha-subunit and the binding of phosphoinositide 3-kinase. In this study, we describe a molecular mechanism linking phosphorylation of Na(+),K(+)-ATPase alpha-subunit to binding and activation of phosphoinositide 3-kinase. Co-immunoprecipitation studies, as well as experiments using confocal microscopy, revealed that dopamine favored the association of 14-3-3 protein with the basolateral plasma membrane and its co-localization with the Na(+),K(+)-ATPase alpha-subunit. The functional relevance of this interaction was established in opossum kidney cells expressing a 14-3-3 dominant negative mutant, where dopamine failed to decrease Na(+),K(+)-ATPase activity and to promote its endocytosis. The phosphorylated Ser-18 residue within the alpha-subunit N terminus is critical for 14-3-3 binding. Activation of phosphoinositide 3-kinase by dopamine during Na(+),K(+)-ATPase endocytosis requires the binding of the kinase to a proline-rich domain within the alpha-subunit, and this effect was blocked by the presence of a 14-3-3 dominant negative mutant. Thus, the 14-3-3 protein represents a critical linking mechanism for recruiting phosphoinositide 3-kinase to the site of Na(+),K(+)-ATPase endocytosis.     

Protection of plasma membrane K+ transport by the salt overly sensitive1 Na+-H+ antiporter during salinity stress

Physicochemical similarities between K(+) and Na(+) result in interactions between their homeostatic mechanisms. The physiological interactions between these two ions was investigated by examining aspects of K(+) nutrition in the Arabidopsis salt overly sensitive (sos) mutants, and salt sensitivity in the K(+) transport mutants akt1 (Arabidopsis K(+) transporter) and skor (shaker-like K(+) outward-rectifying channel). The K(+)-uptake ability (membrane permeability) of the sos mutant root cells measured electrophysiologically was normal in control conditions. Also, growth rates of these mutants in Na(+)-free media displayed wild-type K(+) dependence. However, mild salt stress (50 mm NaCl) strongly inhibited root-cell K(+) permeability and growth rate in K(+)-limiting conditions of sos1 but not wild-type plants. Increasing K(+) availability partially rescued the sos1 growth phenotype. Therefore, it appears that in the presence of Na(+), the SOS1 Na(+)-H(+) antiporter is necessary for protecting the K(+) permeability on which growth depends. The hypothesis that the elevated cytoplasmic Na(+) levels predicted to result from loss of SOS1 function impaired the K(+) permeability was tested by introducing 10 mm NaCl into the cytoplasm of a patch-clamped wild-type root cell. Complete loss of AKT1 K(+) channel activity ensued. AKT1 is apparently a target of salt stress in sos1 plants, resulting in poor growth due to impaired K(+) uptake. Complementary studies showed that akt1 seedlings were salt sensitive during early seedling development, but skor seedlings were normal. Thus, the effect of Na(+) on K(+) transport is probably more important at the uptake stage than at the xylem loading stage.     

AtHKT1 facilitates Na+ homeostasis and K+ nutrition in planta

Genetic and physiological data establish that Arabidopsis AtHKT1 facilitates Na(+) homeostasis in planta and by this function modulates K(+) nutrient status. Mutations that disrupt AtHKT1 function suppress NaCl sensitivity of sos1-1 and sos2-2, as well as of sos3-1 seedlings grown in vitro and plants grown in controlled environmental conditions. hkt1 suppression of sos3-1 NaCl sensitivity is linked to higher Na(+) content in the shoot and lower content of the ion in the root, reducing the Na(+) imbalance between these organs that is caused by sos3-1. AtHKT1 transgene expression, driven by its innate promoter, increases NaCl but not LiCl or KCl sensitivity of wild-type (Col-0 gl1) or of sos3-1 seedlings. NaCl sensitivity induced by AtHKT1 transgene expression is linked to a lower K(+) to Na(+) ratio in the root. However, hkt1 mutations increase NaCl sensitivity of both seedlings in vitro and plants grown in controlled environmental conditions, which is correlated with a lower K(+) to Na(+) ratio in the shoot. These results establish that AtHKT1 is a focal determinant of Na(+) homeostasis in planta, as either positive or negative modulation of its function disturbs ion status that is manifested as salt sensitivity. K(+)-deficient growth of sos1-1, sos2-2, and sos3-1 seedlings is suppressed completely by hkt1-1. AtHKT1 transgene expression exacerbates K(+) deficiency of sos3-1 or wild-type seedlings. Together, these results indicate that AtHKT1 controls Na(+) homeostasis in planta and through this function regulates K(+) nutrient status.     

Aldosterone regulation of intestinal Na absorption involves SGK-mediated changes in NHE3 and Na+ pump activity

Aldosterone-induced intestinal Na(+) absorption is mediated by increased activities of apical membrane Na(+)/H(+) exchange (aNHE3) and basolateral membrane Na(+)-K(+)-ATPase (BLM-Na(+)-K(+)-ATPase) activities. Because the processes coordinating these events were not well understood, we investigated human intestinal Caco-2BBE cells where aldosterone increases within 2-4 h of aNHE3 and alpha-subunit of BLM-Na(+)-K(+)-ATPase, but not total abundance of these proteins. Although aldosterone activated Akt2 and serum glucorticoid kinase-1 (SGK-1), the latter through stimulation of phosphatidylinositol 3-kinase (PI3K), only the SGK-1 pathway mediated its effects on Na(+)-K(+)-ATPase. Ouabain inhibition of the early increase in aldosterone-induced Na(+)-K(+)-ATPase activation blocked most of the apical NHE3 insertion, possibly by inhibiting Na(+)-K(+)-ATPase-induced changes in intracellular sodium concentration ([Na](i)). Over the next 6-48 h, further increases in aNHE3 and BLM-Na(+)-K(+)-ATPase activity and total protein expression were observed to be largely mediated by aldosterone-activated SGK-1 pathway. Aldosterone-induced increases in NHE3 mRNA, for instance, could be inhibited by RNA silencing of SGK-1, but not Akt2. Additionally, aldosterone-induced increases in NHE3 promoter activity were blocked by silencing SGK-1 as well as pharmacological inhibition of PI3K. In conclusion, aldosterone-stimulated intestinal Na(+) absorption involves two phases. The first phase involves stimulation of PI3K, which increases SGK-dependent insertion and function of BLM-Na(+)-K(+)-ATPase and subsequent increased membrane insertion of aNHE3. The latter may be caused by Na(+)-K(+)-ATPase-induced changes in [Na] or transcellular Na flux. The second phase involves SGK-dependent increases in total NHE3 and Na(+)-K(+)-ATPase protein expression and activities. The coordination of apical and BLM transporters after aldosterone stimulation is therefore a complex process that requires multiple time- and interdependent cellular processes.     

Arrestins and spinophilin competitively regulate Na+,K+-ATPase trafficking through association with a large cytoplasmic loop of the Na+,K+-ATPase

The activity and trafficking of the Na(+),K(+)-ATPase are regulated by several hormones, including dopamine, vasopressin, and adrenergic hormones through the action of G-protein-coupled receptors (GPCRs). Arrestins, GPCR kinases (GRKs), 14-3-3 proteins, and spinophilin interact with GPCRs and modulate the duration and magnitude of receptor signaling. We have found that arrestin 2 and 3, GRK 2 and 3, 14-3-3 epsilon, and spinophilin directly associate with the Na(+),K(+)-ATPase and that the associations with arrestins, GRKs, or 14-3-3 epsilon are blocked in the presence of spinophilin. In COS cells that overexpressed arrestin, the Na(+),K(+)-ATPase was redistributed to intracellular compartments. This effect was not seen in mock-transfected cells or in cells expressing spinophilin. Furthermore, expression of spinophilin appeared to slow, whereas overexpression of beta-arrestins accelerated internalization of the Na(+),K(+)-ATPase endocytosis. We also find that GRKs phosphorylate the Na(+),K(+)-ATPase in vitro on its large cytoplasmic loop. Taken together, it appears that association with arrestins, GRKs, 14-3-3 epsilon, and spinophilin may be important modulators of Na(+),K(+)-ATPase trafficking.     

Cytoplasmic gatekeepers of K+-channel flux: a structural perspective

Recently, rapid progress in our structural knowledge of K(+)-selective channels has started to provide a basis for comprehending the biophysical machinery underlying their electrophysiological properties. These studies have begun to reveal how a diverse array of distinct, cytoplasmically positioned domains affect the activity of associated channels. Some of these establish functional diversity by selectively mediating channel assembly. More importantly, these cytoplasmic domains couple intracellular signals to the gating of their associated pore. New structural insights are providing a clearer understanding of the fundamental molecular mechanisms of these K(+) channels that, in turn, partly underlie complex neurological phenomena.     

Two Isoenzymes of Na+, K+-ATPase Have Different Kinetics of K+ Dephosphorylation in Normal Cat and Human Brain Cortex

Analysis of purified Na+,K+-ATPase from cat and human cortex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals two large catalytic subunits called alpha (-) (lower molecular weight) and alpha (+) (higher molecular weight). Differences in K+ dephosphorylation of these two molecular forms have been investigated by measuring the phosphorylation level of each protein after their separation on sodium dodecyl sulfate gels. In the presence of Na+, Mg2+, and ATP, both subunits are phosphorylated. Increasing concentrations (from 0 to 3 mM) of K+ induce progressive dephosphorylation of both alpha-subunits, although the phosphoprotein content of alpha (-) is decreased significantly less than that of alpha (+). Ka values of alpha (-) for K+ are 40% and 50% greater in cat and human cortex, respectively, than values of alpha (+). alpha (-) and alpha (+) are thought to be localized in specific cell types of the brain: alpha (-) is the exclusive form of nonneuronal cells (astrocytes), whereas alpha (+) is the only form of axolemma. Our results support the hypothesis that glial and neuronal Na+,K+-ATPases are different molecular entities differing at least by their K+ sensitivity. Results are discussed in relation to the role of glial cells in the regulation of extracellular K+ in brain.     

Positive heterotropic allosteric regulators of dihydropyridine binding increase the Ca2+ affinity of the L-type Ca2+ channel. Stereoselective reversal by the novel Ca2+ antagonist BM 20.1140.

The alpha 1-subunit of the voltage-dependent L-type Ca2+ channel has distinct, allosterically coupled binding domains for drugs from different chemical classes (dihydropyridines, benzothiazepines, phenylalkylamines, diphenylbutylpiperidines). (-)-BM 20.1140 (ethyl-2,2-di-phenyl-4-(1-pyrrolidino)-5-(2-picolyl)- oxyvalerate) is a novel Ca2+ channel blocker which potently stimulates dihydropyridine binding (K0.5 = 2.98 nM) to brain membranes. This property is shared by (+)-cis-diltiazem, (+)-tetrandrine, fostedil and trans-diclofurime, but (-)-BM 20.1140 does not bind in a competitive manner to the sites labeled by (+)-cis-[3H]diltiazem. (+)-cis-Diltiazem and (-)-BM 20.1140 have differential effects on the rate constants of dihydropyridine binding. (+)-BM 20.1140 reverses the stimulation of the positive allosteric regulators (pA2 value for reversal of (-)-BM 20.1140 stimulation = 7.4, slope 0.72). The underlying molecular mechanism of the potentiation of dihydropyridine binding has been clarified. The K0.5 for free Ca2+ to stabilize a high affinity binding domain for dihydropyridines on purified L-type channels from rabbit skeletal muscle is 300 nM. (+)-Tetrandine (10 microM) increases the affinity 8-fold (K0.5 for free Ca2+ = 30.1 nM) and (+)-BM 20.114 (10 microM) inhibits the affinity increase (K0.5 for free Ca2+ = 251 nM). Similar results were obtained with membrane-bound Ca(2+)-channels from brain tissue which have higher affinity for free Ca2+ (K0.5 for free Ca2+ = 132 nM) and for dihydropyridines compared with skeletal muscle. It is postulated that the dihydropyridine and Ca(2+)-binding sites are interdependent on the alpha 1-subunit, that the different positive heterotropic allosteric regulators (by their differential effects on Ca2+ rate constants) optimize coordination for Ca2+ in the channel pore and, in turn, increase affinity for the dihydropyridines.     

Residues within transmembrane domains 4 and 6 of the Na,K-ATPase alpha subunit are important for Na+ selectivity

The Na,K- and H,K-ATPases are plasma membrane enzymes responsible for the active exchange of extracellular K(+) for cytoplasmic Na(+) or H(+), respectively. At present, the structural determinants for the specific function of these ATPases remain poorly understood. To investigate the cation selectivity of these ATPases, we constructed a series of Na,K-ATPase mutants in which residues in the membrane spanning segments of the alpha subunit were changed to the corresponding residues common to gastric H,K-ATPases. Thus, mutants were created with substitutions in transmembrane domains TM1, TM4, TM5, TM6, TM7, and TM8 independently or together (designated TMAll). The function of each mutant was assessed after coexpression with the beta subunit in Sf-9 cells using baculoviruses. The enzymatic properties of TM1, TM7, and TM8 mutants were similar to the wild-type Na,K-ATPase, and while TM5 showed modest changes in apparent affinity for Na(+), TM4, TM6, and TMAll displayed an abnormal activity. This resulted in a Na(+)-independent hydrolysis of ATP, a 2-fold higher K(0.5) for Na(+) activation, and the ability to function at low pH. These results suggest a loss of discrimination for Na(+) over H(+) for the enzymes. In addition, TM4, TM6, and TMAll mutants exhibited a 1.5-fold lower affinity for K(+) and a 4-5-fold decreased sensitivity to vanadate. Altogether, these results provide evidence that residues in transmembrane domains 4 and 6 of the alpha subunit of the Na,K-ATPase play an important role in determining the specific cation selectivity of the enzyme and also its E1/E2 conformational equilibrium.     

GABAB receptor activation inhibits Ca2+-activated potassium channels in synaptosomes: involvement of G-proteins

86Rb-efflux assay from preloaded synaptosomes of rat cerebral cortex was developed to study the effect of GABAB receptor agonist baclofen on Ca2+-activated K+-channels. Depolarization (100 mM K) of 86Rb-loaded synaptosomes in physiological buffer increased Ca2+-activated 86Rb-efflux by 400%. The 86Rb-efflux was blocked by quinine sulphate, tetraethylammonium and La3+ indicating the involvement of Ca2+-activated K+-channels. (-)Baclofen inhibited Ca2+-activated 86Rb-efflux in a stereospecific manner. The inhibitory effect of (-)baclofen was mediated by GABAB receptor activation, since it was blocked by GABAB antagonist phaclofen, but not by bicuculline. Further, pertussis toxin also blocked the ability of baclofen or depolarizing action to affect Ca2+-activated K+-channels. These results suggest that baclofen inhibits Ca2+-activated K+-channels in synaptosomes and these channels are regulated by G-proteins. This assay may provide an ideal in vitro model to study GABAB receptor pharmacology.     

The anion exchanger and Na+K(+)-ATPase interact with distinct sites on ankyrin in in vitro assays

This report demonstrates that the high affinity binding of ankyrin to two well characterized ankyrin-binding proteins, the erythrocyte anion exchanger and kidney Na+K(+)-ATPase, requires interaction of these proteins with unique sites on the ankyrin molecule. Binding of 125I-labeled erythrocyte ankyrin and ankyrin proteolytic domains was measured to the anion exchanger and Na+K(+)-ATPase incorporated into phosphatidylcholine liposomes. 125I-Labeled ankyrin associated with both anion exchanger and Na+K(+)-ATPase liposomes with a high affinity (KD ranging from 10 to 25 nM), and a capacity approaching 1 mol of ankyrin/2 mol of ATPase and 1 mol of ankyrin/8 mol of anion exchanger. The 43 kDa cytoplasmic domain of the erythrocyte anion exchanger inhibited binding of ankyrin to both the anion exchanger and Na+K(+)-ATPase liposomes with a 50% reduction at approximately 90 nM for both proteins. Further binding experiments using proteolytic domains derived from ankyrin demonstrated the following differences between the anion exchanger and Na+K(+)-ATPase in interactions with ankyrin: 1) 125I-Labeled Na+K(+)-ATPase associated with both the 89-kDa domain as well as the spectrin binding domain of ankyrin, while the anion exchanger only associated with the 89-kDa domain. 2) The 125I-labeled 89-kDa domain of ankyrin associated with Na+K(+)-ATPase liposomes with at least a 20-fold lower affinity compared with intact ankyrin while this domain associated with the anion exchanger with a 2-3-fold increase in affinity compared with intact ankyrin. 3) The 125I-labeled spectrin-binding domain of ankyrin associated with the Na+K(+)-ATPase liposomes to at least an 8-fold greater extent than to anion exchanger liposomes. The data are consistent with an independent acquisition of high affinity ankyrin binding activity for the anion exchanger and Na+K(+)-ATPase proteins through a convergent evolutionary process.     

Effect of high-NaCl or high-KCl diet on hepatic Na+- and K+-receptor sensitivity and NKCC1 expression in rats

We previously reported that the bumetanide-sensitive Na(+)-K(+)-2Cl- cotransporter (NKCC1) is involved in the hepatic Na+ and K+ sensor mechanism. In the present study, we examined the effects of a high-NaCl or high-KCl diet on hepatic Na+ and K+ receptor sensitivity and NKCC1 expression in the liver of Sprague-Dawley rats. RT-PCR and Western blots were used to measure NKCC1 mRNA and protein expression, respectively. Infusion of hypertonic NaCl or isotonic KCl + NaCl solutions into the portal vein increased hepatic afferent nerve activity (HANA) in a Na+ or K+ dose-dependent manner. After 4 wk on a high-NaCl or high-KCl diet, HANA responses were attenuated compared with animals fed a normal diet, and NKCC1 expression was reduced. These results show that a high-NaCl or high-KCl diet decreases NKCC1 expression in the liver, and it might cause a reduction in hepatic Na(+)- and K(+)-receptor sensitivity.