Phosphoregulation of the Na–K–2Cl and K–Cl cotransporters by the WNK kinases

Precise regulation of the intracellular concentration of chloride [Cl?]i is necessary for proper cell volume regulation, transepithelial transport, and GABA neurotransmission. The Na–K–2Cl (NKCCs) and K–Cl (KCCs) cotransporters, related SLC12A transporters mediating cellular chloride influx and efflux, respectively, are key determinants of [Cl?]i in numerous cell types, including red blood cells, epithelial cells, and neurons. A common “chloride/volume-sensitive kinase”, or related system of kinases, has long been hypothesized to mediate the reciprocal but coordinated phosphoregulation of the NKCCs and the KCCs, but the identity of these kinase(s) has remained unknown. Recent evidence suggests that the WNK (with no lysine = K) serine–threonine kinases directly or indirectly via the downstream Ste20-type kinases SPAK/OSR1, are critical components of this signaling pathway. Hypertonic stress (cell shrinkage), and possibly decreased [Cl?]i, triggers the phosphorylation and activation of specific WNKs, promoting NKCC activation and KCC inhibition via net transporter phosphorylation. Silencing WNK kinase activity can promote NKCC inhibition and KCC activation via net transporter dephosphorylation, revealing a dynamic ability of the WNKs to modulate [Cl?]. This pathway is essential for the defense of cell volume during osmotic perturbation, coordination of epithelial transport, and gating of sensory information in the peripheral system. Commiserate with their importance in serving these critical roles in humans, mutations in WNKs underlie two different Mendelian diseases, pseudohypoaldosteronism type II (an inherited form of salt-sensitive hypertension), and hereditary sensory and autonomic neuropathy type 2. WNKs also regulate ion transport in lower multicellular organisms, including Caenorhabditis elegans, suggesting that their functions are evolutionarily-conserved. An increased understanding of how the WNKs regulate the Na–K–2Cl and K–Cl cotransporters may provide novel opportunities for the selective modulation of these transporters, with ramifications for common human diseases like hypertension, sickle cell disease, neuropathic pain, and epilepsy.     

ATP Dependence of Na+-Driven Cl–HCO3 Exchange in Squid Axons

Squid giant axons recover from acid loads by activating a Na(+)-driven Cl-HCO(3) exchanger. We internally dialyzed axons to an intracellular pH (pH( i )) of 6.7, halted dialysis and monitored the pH(i) recovery (increase) in the presence of ATP or other nucleotides, using cyanide to block oxidative phosphorylation. We computed the equivalent acid-extrusion rate (J(H)) from the rate of pH(i) increase and intracellular buffering power. In experimental series 1, we used dialysis to vary [ATP](i), finding that Michaelis-Menten kinetics describes J (H) vs. [ATP](i), with an apparent V(max) of 15.6 pmole cm(-2 )s(-1) and K (m) of 124 microM. In series 2, we examined ATP gamma S, AMP-PNP, AMP-PCP, AMP-CPP, GMP-PNP, ADP, ADP beta S and GDP beta S to determine if any, by themselves, could support transport. Only ATP gamma S (8 mM) supported acid extrusion; ATP gamma S also supported the HCO (3)(-) -dependent (36)Cl efflux expected of a Na(+)-driven Cl-HCO(3) exchanger. Finally, in series 3, we asked whether any nucleotide could alter J (H) in the presence of a background [ATP](i) of approximately 230 microM (control J (H) = 11.7 pmol cm(-2 )s(-1)). We found J (H) was decreased modestly by 8 mM AMP-PNP (J (H) = 8.0 pmol cm(-2 )s(-1)) but increased modestly by 1 mM ADP beta S (J (H) = 16.0 pmol cm(-2 )s(-1)). We suggest that ATP gamma S leads to stable phosphorylation of the transporter or an essential activator.     

Half-sandwich ruthenium–arene complexes with thiosemicarbazones: synthesis and biological evaluation of [(η⁶-p-cymene)Ru(piperonal thiosemicarbazones)Cl]Cl complexes

The synthesis and characterization of a number of organometallic ruthenium(II) complexes containing a series of bidentate thiosemicarbazone ligands derived from piperonal is reported. The structure of compounds have been confirmed by spectroscopic analysis (IR and NMR) as well as X-ray crystallographic analysis of [(η⁶-p-cymene)Ru(pPhTSC)Cl]Cl (4) (pPhTSC is piperonal-N(4)-phenylthiosemicarbazone). The interaction of the complexes ([(η⁶-p-cymene)Ru(pEtTSC)Cl]Cl) (3) (pEtTSC is piperonal-N(4)-ethylthiosemicarbazone) and 4 with calf thymus DNA, human serum albumin (HSA) and pBR322 plasmid DNA were studied by spectroscopic, gel electrophoresis and hydrodynamic methods. The apparent binding constant for the interaction with DNA was determined to be 3.97 × 10³ M^− 1 and 4.07 × 10³ M^− 1 at 293 K for 3 and 4 respectively. The complexes bind strongly to HSA with binding constants of 2.94 × 10⁴ M^− 1 and 12.2 × 10⁴ M^− 1 at 296 K for 3 and 4 respectively. The in vitro anticancer activity of 3 and 4 has been evaluated against two human colon cancer cell line (HCT-116 and Caco-2) with IC₅₀ values in the range of 26-150 μM. Both 3 and 4 show good activity as a catalytic inhibitor of human topoisomerase II at concentrations as low as 20 μM. The proficiency of 3 and 4 to act as antibacterial agents was also evaluated against six pathogenic bacterial strains with the best activity seen against Gram-positive strains.     

Current-voltage curve of electrogenic Cl− pump predicts voltage-dependent Cl− efflux inAcetabularia

Summary The current-voltage relationship of carrier-mediated, passive and active ion transport systems with one charge-carrying pathway can exactly be described by a simple reaction kinetic model. This model consists of two carrier states (one inside, one outside) and two pairs (forwards and backwards) of rate constants: a voltage-dependent one, describing the transport of charge and a voltage-insensitive one, summarizing all the other (voltage-independent) reactions. For the electrogenic Cl^– pump inAcetabularia these four rate constants have been determined from electrical measurements of the current-voltage relationship of the pump (Gradmann, Hansen & Slayman, 1981;in: Electrogenic Ion Pumps, Academic Press, New York). The unidirectional Cl^– efflux through the pump can also be calculated by the availiable reaction kinetic parameters.³⁶Cl^– efflux experiments on singleAcetabularia cells with simultaneous electrical stimulation (action potentials) and recording, demonstrate the unidirectional Cl^– efflux to depend on the membrane potential. After subtraction of an efflux portion which bypasses the pump, agreement is found between the measured flux-voltage relationship and the theoretical one as obtained from the reaction kinetic model and its parameters from the electrical data.     

Reconstituted Cl− pump protein: A novel ion(Cl−)-motive ATPase

Cl^– absorption by theAplysia californica foregut is effected through an active Cl^– transport mechanism located in the basolateral membrane of the epithelial absorptive cells. These basolateral membranes contain both Cl^–-stimulated ATPase and ATP-dependent Cl^– transport activities which can be incorporated into liposomes via reconstitution. Utilizing the proteoliposomal preparation, it was demonstrated that ATP, and its subsequent hydrolysis, Mg²⁺, Cl^–, and a pH optimum of 7.8 were required to generate maximal intraliposomal Cl^– accumulation, electrical negativity, and ATPase activity. Additionally, an inwardly-directed valinomycininduced K⁺ diffusion potential, making the liposome interior electrically positive, enhanced both ATP-driven Cl^– accumulation and electrical potential while an outwardly-directed valinomycininduced K⁺ diffusion potential, making the liposome interior electrically negative, decreased both ATP-driven Cl^– accumulation and electrical potential compared with proteoliposomes lacking the ionophore. Either orthovanadate orp-chloromercurobenzene sulfonate inhibited both the ATP-dependent intraliposomal Cl^– accumulation, intraliposomal negative potential difference, and also Cl^–-stimulated ATPase activity. Both aspects of Cl^– pump transport kinetics and its associated catalytic component kinetics were the first obtained utilizing a reconstituted transporter protein. These results strongly support the hypothesis that Cl^–-ATPase actively transports Cl^– by an electrogenic process.     

Excitation-revealed changes in cytoplasmic Cl− concentration in “Cl−-starved”Chara cells

Summary The changes in the cytoplasmic Cl^– concentration, [Cl^–] _c , are monitored at the time of withdrawal (starvation) and subsequent replacement of Cl^– in the outside medium. The measurement technique exploits the involvement of Cl^– inChara excitation. The transient clamp current due to Cl^–,I _Cl, is separated from other excitation transients through Hodgkin-Huxley (HH) equations, which have been adjusted toChara. TheI _Cl amplitude depends on HH parameters, [Cl^–] _c and the maximum membrane conductance to Cl^–, . The results are discussed in terms of these quantities.I _Cl and were found to fall after 6–10 hr of Cl^– starvation, thus supporting the hypothesis that [Cl^– _c decreases in Cl^–-free medium. The best HH fit to starved data was obtained with [Cl^– _c =3.5mm. The time-course forI _Cl decline is considerably slower than the time-course of the rise of the starvation-stimulated influx. As cells starved for periods longer than 24 hr are re-exposed to Cl^–, it is revealed that while [Cl^–] _c remains low during long starvation, increases to values greater than those of the normal cells. Such differences among cells starved for various lengths of time have not been detected previously.     

Chloroquine Stimulates Cl? Secretion by Ca2+ Activated Cl? Channels in Rat Ileum

Chloroquine (CQ), a bitter tasting drug widely used in treatment of malaria, is associated gastrointestinal side effects including nausea or diarrhea. In the present study, we investigated the effect of CQ on electrolyte transport in rat ileum using the Ussing chamber technique. The results showed that CQ evoked an increase in short circuit current (I_SC) in rat ileum at lower concentration (≤5×10⁻⁴ M ) but induced a decrease at higher concentrations (≥10⁻³ M). These responses were not affected by tetrodotoxin (TTX). Other bitter compounds, such as denatoniumbenzoate and quinine, exhibited similar effects. CQ-evoked increase in I_SC was partly reduced by amiloride(10⁻⁴ M), a blocker of epithelial Na⁺ channels. Furosemide (10⁻⁴ M), an inhibitor of Na⁺-K⁺ -2Cl⁻ co-transporter, also inhibited the increased I_SC response to CQ, whereas another Cl⁻ channel inhibitor, CFTR(inh)-172(10⁻⁵M), had no effect. Intriguingly, CQ-evoked increases were almost completely abolished by niflumic acid (10⁻⁴M), a relatively specific Ca²⁺-activated Cl⁻ channel (CaCC) inhibitor. Furthermore, other CaCC inhibitors, such as DIDS and NPPB, also exhibited similar effects. CQ-induced increases in I_SC were also abolished by thapsigargin(10⁻⁶M), a Ca²⁺ pump inhibitor and in the absence of either Cl⁻ or Ca²⁺ from bathing solutions. Further studies demonstrated that T2R and CaCC-TMEM16A were colocalized in small intestinal epithelial cells and the T2R agonist CQ evoked an increase of intracelluar Ca²⁺ in small intestinal epithelial cells. Taken together, these results demonstrate that CQ induces Cl⁻ secretion in rat ileum through CaCC at low concentrations, suggesting a novel explanation for CQ-associated gastrointestinal side-effects during the treatment of malaria.     

Cl− channels in basolateral renal medullary vesicles: V. Comparison of basolateral mTALH Cl− channels with apical Cl− channels from jejunum and trachea

Summary Cl^– channels from basolaterally-enriched rabbit outer renal medullary membranes are activated either by increases in intracellular Cl^– activity or by intracellular protein kinase A (PKA). Phosphorylation by PKA, however, is not obligatory for channel activity since channels can be activated by intracellular Cl^– in the absence of PKA. The PKA requirement for activation of Cl^– channels in certain secretory epithelia is, in contrast, obligatory. In the present studies, we examined the effects of PKA and intracellular Cl^– concentrations on the properties of Cl^– channels obtained either from basolaterally-enriched vesicles derived from highly purified suspensions of mouse medullary thick ascending limb (mTALH) segments, or from apical membrane vesicles obtained from two secretory epithelia, bovine trachea and rabbit small intestine. Our results indicate that the Cl^– channels from mTALH suspensions were virtually identical to those previously described from rabbit outer renal medulla. In particular, an increase in intracellular (trans) Cl^– concentration from 2 to 50 mm increased both channel activity (P _o) and channel conductance (g _Cl, pS). Likewise, trans PKA increased mTALH Cl^– channel activity by increasing the activity of individual channels when the trans solutions were 2 mm Cl. Under the latter circumstance, PKA did not activate quiescent channels, nor did it affect g _Cl. Moreover, when mTALH Cl^– channels were inactivated by reducing cis Cl^– concentrations to 50 mm, cis PKA addition did not affect P _o. These results are consistent with the view that these Cl^– channels originated from basolateral membranes of the mTALH.Cl^– channels from apical vesicles from trachea and small intestine were completely insensitive to alterations in trans Cl^– concentrations and demonstrated markedly different responses to PKA. In the absence of PKA, tracheal Cl^– channels inactivated spontaneously after a mean time of 8 min; addition of PKA to trans solutions reactivated these channels. The intestinal Cl^– channels did not inactivate with time. Trans PKA addition activated new channels with no effect on basal channel activity. Thus the regulation of Cl^– channel activity by both intracellular Cl^– and by PKA differ in basolateral mTALH Cl^– channels compared to apical Cl^– channels from either the tracheal or small intestine.We acknowledge the able technical assistance of Steven D. Chasteen. Clementine M. Whitman provided her customary excellent secretarial assistance. This work was supported by Veterans Administration Merit Review Grants to T.E. Andreoli and to W.B. Reeves. C.J. Winters is a Veterans Administration Associate Investigator.     

Cl− transport in basolateral renal medullary vesicles: I. Cl− transport in intact vesicles

Summary This paper provides the results of studies which characterized conductive³⁶Cl^– flux in basolaterally enriched membrane vesicles prepared from rabbit renal outer medulla. Conductive³⁶Cl^– uptake was studied under two different experimental conditions. In the first,³⁶Cl^– flux was driven by an inside positive voltage created with oppositely directed Cl^– and gluconate gradients. In the second, an inwardly direct K⁺ gradient was used to drive³⁶Cl^– uptake. By these two methods, voltage-sensitive³⁶Cl^– uptake was shown to comprise about 45 and 65%, respectively, of the initial rates of total³⁶Cl^– flux. Separate paired studies demonstrated that the conductive³⁶Cl^– uptake was inhibited by the Cl^– channel blocker diphenylamine-2-carboxylate (DPC) with an IC₅₀ for DPC of 154 m. The voltagedependent³⁶Cl^– uptake had an activation energy of 6.4 kcal/mole. This³⁶Cl^– conductance had an anion selectivity sequence of I^–>Cl^–NO ₃ ^– gluconate.     

Effect of Glycine and Strychnine on Cl–-Activated Mg2+-ATPase from Bream Brain Microsomes (Abramis bramaL.)

The effect of glycine and strychnine on Mg²⁺-ATPase from the microsomal fraction of the bream (Abramis bramaL.) brain was studied. The glycine in the concentration range 10^–7–10^–4M activates the enzyme. The effect of glycine on Mg²⁺-ATPase is obviated by 100 M strychnine. The strychnine in the concentration range 5–90 M activates the basal Mg²⁺-ATPase but decreases the effect of the enzyme activation by 10^–4M glycine. The effect of Cl^–on Mg²⁺-ATPase depends on the substrate concentration (Mg²⁺-ATP) and is not observed in the presence of 100 M strychnine. A receptor-dependent pathway of glycine and strychnine action on Cl^–-activated Mg²⁺-ATPase from bream brain microsomes is proposed.     

Cl− transport in basolateral renal medullary vesicles: II. Cl− channels in planar lipid bilayers

Summary The present studies examined some of the properties of Cl^– channels in renal outer medullary membrane vesicles incorporated into planar lipid bilayers. The predominant channel was anion selective having aP _Cl/P _K ratio of 10 and a unit conductance of 93 pS in symmetric 320mm KCl. In asymmetric KCl solutions, theI-V relations conformed to the Goldman-Hodgkin-Katz equation. Channel activity was voltage-dependent with a gating charge of unity. This voltage dependence of channel activity may account, at least in part, for the striking voltage dependence of the basolateral membrane Cl^– conductance of isolated medullary thick ascending limb segments. The Cl^– channels incorporated into the planar bilayers were asymmetrical: thetrans surface was sensitive to changes in ionized Ca²⁺ concentrations and insensitive to reducing KCl concentrations to 10mm, while thecis side was insensitive to changes in ionized Ca²⁺ concentrations, but was inactivated by reducing KCl concentrations to 50mm.     

Calculation of the Three-Dimensional Structures of Two Antigenic Sequences, Pro–Pro–Pro–Pro–Asn–Pro–Asn–Asp–Pro and Pro–Pro–Pro–Pro–Asn–Pro–Asn–Asp–Pro–Pro–Pro, of the Circumsporozoite Protein From a Malaria-Causing Plasmodium

Using the chain build-up procedure based on the program ECEPP, we have computed the lowest energy structures for two terminally blocked subsequences from the antigenic circumsporozoite protein of Plasmodium berghei, that is known to cause malaria in animals. The full antigenic sequence is an octapeptide proline-rich tandem repeat, (Pro–Pro–Pro–Pro–Asn–Pro–Asn–Asp)₂. We computed the structures for the first octapeptide plus one Pro from the second octapeptide, terminally blocked CH₃CO–Pro–Pro–Pro–Pro–Asn–Pro–Asn–Asp–Pro–NHCH₃ as well as the first octpeptide with an additional three Pro residues from the adjoining unit, i.e., CH₃CO–Pro–Pro–Pro–Pro–Asn–Pro–Asn–Asp–Pro–Pro–Pro–NHCH₃. We find that the first sequence adopts a number of different low energy structures, the most probable of which has a probability of occurrence of 56 %. Addition of two more Pro residues results in the adoption a single, unique lowest energy structure that has a probability of occurrence of over 95 % without solvation effects and 86 % when solvation effects are included in the calculations. We predict that this structure may be the one recognized as a major antigenic determinant.     

The Ethanol-Induced Stimulation of Rat Duodenal Mucosal Bicarbonate Secretion In Vivo Is Critically Dependent on Luminal Cl–

Alcohol may induce metabolic and functional changes in gastrointestinal epithelial cells, contributing to impaired mucosal barrier function. Duodenal mucosal bicarbonate secretion (DBS) is a primary epithelial defense against gastric acid and also has an important function in maintaining the homeostasis of the juxtamucosal microenvironment. The aim in this study was to investigate the effects of the luminal perfusion of moderate concentrations of ethanol in vivo on epithelial DBS, fluid secretion and paracellular permeability. Under thiobarbiturate anesthesia, a ∼30-mm segment of the proximal duodenum with an intact blood supply was perfused in situ in rats. The effects on DBS, duodenal transepithelial net fluid flux and the blood-to-lumen clearance of ⁵¹Cr-EDTA were investigated. Perfusing the duodenum with isotonic solutions of 10% or 15% ethanol-by-volume for 30 min increased DBS in a concentration-dependent manner, while the net fluid flux did not change. Pre-treatment with the CFTR inhibitor CFTR_inh172 (i.p. or i.v.) did not change the secretory response to ethanol, while removing Cl⁻ from the luminal perfusate abolished the ethanol-induced increase in DBS. The administration of hexamethonium (i.v.) but not capsazepine significantly reduced the basal net fluid flux and the ethanol-induced increase in DBS. Perfusing the duodenum with a combination of 1.0 mM HCl and 15% ethanol induced significantly greater increases in DBS than 15% ethanol or 1.0 mM HCl alone but did not influence fluid flux. Our data demonstrate that ethanol induces increases in DBS through a mechanism that is critically dependent on luminal Cl⁻ and partly dependent on enteric neural pathways involving nicotinic receptors. Ethanol and HCl appears to stimulate DBS via the activation of different bicarbonate transporting mechanisms.     

Regulation of K–Cl cotransport in erythrocytes of frog Rana temporaria by commonly used protein kinase and protein phosphatase inhibitors

Recently (Agalakova and Gusev in J Comp Physiol 179:443–450, 2009), we demonstrated that the activity of K–Cl cotransport (KCC) in frog red blood cells is inhibited under stimulation of protein kinase C (PKC) with phorbol ester PMA (12-myristate-13-acetate). Present work was performed to uncover possible implication of protein kinases and protein phosphatases (PPs) in the regulation of baseline and volume-dependent KCC activity in these cells. K⁺ influx was estimated as ⁸⁶Rb uptake by the cells in isotonic or hypotonic media in the presence of ouabain, K⁺ efflux was determined as the difference between K⁺ loss by the cells incubated in parallel in isotonic or hypotonic K⁺-free Cl⁻- and NO₃ ⁻-media. Swelling of the cells in hypotonic medium was accompanied by approximately 50% activation of Cl-dependent K⁺ influx and efflux. Protein tyrosine kinase (PTK) inhibitor genistein (0.1 mM) stably and considerably (up to 89%) suppressed both baseline and volume-dependent KCC activity in each direction. Other PTK blockers (tyrphostin 23 and quercetin) had no influence on KCC activity in frog erythrocytes. PKC inhibitor chelerythrine (20 μM) and both PP inhibitors, fluoride (5 mM) and okadaic acid (1 μM), reduced KCC activity by 25–70%. Neither basal nor swelling-activated KCC in frog erythrocytes was affected by PKC inhibitor staurosporine (1 μM). Based on the previous and present results, we can suggest that the main role in the maintenance of basal and volume-dependent KCC activity in frog erythrocytes belongs to PTKs and PPs, whereas PKC is a negative regulator of this ion system.     

Apical Cl− Channels in A6 Cells

Short-circuit current (I _sc ), transepithelial conductance (G _t ), electrical capacitance (C _T ) and the fluctuation in I _sc were analyzed in polarized epithelial cells from the distal nephron of Xenopus laevis (A6 cell line). Tissues were incubated with Na⁺- and Cl⁻-free solutions on the apical surface. Basolateral perfusate was NaCl-Ringer. Agents that increase cellular cAMP evoked increases in G _t , C _T , I _sc and generated a Lorentzian I _sc -noise. The responses could be related to active, electrogenic secretion of Cl⁻. Arginine-vasotocin and oxytocin caused a typical peak-plateau response pattern. Stimulation with a membrane-permeant nonhydrolyzable cAMP analogue or forskolin showed stable increases in G _t with only moderate peaking of I _sc . Phosphodiesterase inhibitors also stimulated Cl⁻ secretion with peaking responses in G _t and I _sc . All stimulants elicited a spontaneous Lorentzian noise, originating from the activated apical Cl⁻ channel, with almost identical corner frequency (40–50 Hz). Repetitive challenge with the hormones led to a refractory behavior of all parameters. Activation of the cAMP route could overcome this refractoriness. All agents caused C _T , a measure of apical membrane area, to increase in a manner roughly synchronous with G _t . These results suggest that activation of the cAMP-messenger route may, at least partly, involve exocytosis of a vesicular Cl⁻ channel pool. Apical flufenamate depressed Cl⁻ current and conductance and apparently generated blocker-noise. However, blocking kinetics extracted from noise experiments could not be reconciled with those obtained from current inhibition, suggesting the drug does not act as simple open-channel inhibitor. Received: 20 May 1998/Revised: 8 September 1998     

The energetics of Cl− active transport inChara

Summary The rate of Cl^– influx in intactChara was inhibited whenever the ATP concentration was reduced by application of metabolic inhibitors. In perfused cells, however, a net influx of Cl^– against its electrochemical gradient could be observed in the absence of ATP. Addition of ATP to the perfusion medium slightly stimulated Cl^– influx in one experiment but had no effect in another. Addition of ADP, NADH or metabolic inhibitors did not alter the influx rate. Consideration of the potential energy gradients across theChara plasmalemma in the perfused state leads to the conclusion that Cl^– influx occurs by cotransport with H⁺ or OH^–.