Stimulation of Na,K-ATPase by low potassium requires reactive oxygen species

The signaling pathway that transduces the stimulatory effect of low K⁺ on the biosynthesis of Na,K-ATPase remains largely unknown. The present study was undertaken to examine whether reactive oxygen species (ROS) mediated the effect of low K⁺ in Madin-Darby canine kidney (MDCK) cells. Low K⁺ increased ROS activity in a time- and dose-dependent manner, and this effect was abrogated by catalase and N-acetylcysteine (NAC). To determine the role of ROS in low-K⁺-induced gene expression, the cells were first stably transfected with expression constructs in which the reporter gene chloramphenicol acetyl transferase (CAT) was under the control of the avian Na,K-ATPase -subunit 1.9 kb and 900-bp 5'-flanking regions that have a negative regulatory element. Low K⁺ increased the CAT expression in both constructs. Catalase or NAC inhibited the effect of low K⁺. To determine whether the increased CAT activity was mediated through releasing the repressive effect or a direct stimulation of the promoter, the cells were transfected with a CAT expression construct directed by a 96-bp promoter fragment that has no negative regulatory element. Low K⁺ also augmented the CAT activity expressed by this construct. More importantly, both catalase and NAC abolished the effect of low K⁺. Moreover, catalase and NAC also inhibited low-K⁺-induced increases in the Na,K-ATPase ₁- and ₁-subunit protein abundance and ouabain binding sites. The antioxidants had no significant effect on the basal levels of CAT activity, protein abundance, or ouabain binding sites. In conclusion, low K⁺ enhances the Na,K-ATPase gene expression by a direct stimulation of the promoter activity, and ROS mediate this stimulation and also low-K⁺-induced increases in the Na,K-ATPase protein contents and cell surface molecules. Madin-Darby canine kidney cells; N-acetylcysteine; catalase     

Cd2+-induced swelling-contraction dynamics in isolated kidney cortex mitochondria: role of Ca2+ uniporter, K+ cycling, and protonmotive force

The nephrotoxic metal Cd²⁺ causes mitochondrial damage and apoptosis of kidney proximal tubule cells. A K⁺ cycle involving a K⁺ uniporter and a K⁺/H⁺ exchanger in the inner mitochondrial membrane (IMM) is thought to contribute to the maintenance of the structural and functional integrity of mitochondria. In the present study, we have investigated the effect of Cd²⁺ on K⁺ cycling in rat kidney cortex mitochondria. Cd²⁺ (EC₅₀ 19 µM) induced swelling of nonenergized mitochondria suspended in isotonic salt solutions according to the sequence KCl = NaCl > LiCl >> choline chloride. Cd²⁺-induced swelling of energized mitochondria had a similar EC₅₀ value and showed the same cation dependence but was followed by a spontaneous contraction. Mitochondrial Ca²⁺ uniporter (MCU) blockers, but not permeability transition pore inhibitors, abolished swelling, suggesting the need for Cd²⁺ influx through the MCU for swelling to occur. Complete loss of mitochondrial membrane potential (_m) induced by K⁺ influx did not prevent contraction, but addition of the K⁺/H⁺ exchanger blocker, quinine (1 mM), or the electroneutral protonophore nigericin (0.4 µM), abolished contraction, suggesting the mitochondrial pH gradient (pH_m) driving contraction. Accordingly, a quinine-sensitive partial dissipation of pH_m was coincident with the swelling-contraction phase. The data indicate that Cd²⁺ enters the matrix through the MCU to activate a K⁺ cycle. Initial K⁺ load via a Cd²⁺-activated K⁺ uniporter in the IMM causes osmotic swelling and breakdown of _m and triggers quinine-sensitive K⁺/H⁺ exchange and contraction. Thus Cd²⁺-induced activation of a K⁺ cycle contributes to the dissipation of the mitochondrial protonmotive force. bongkrekic acid; cyclosporin A; lanthanum; Ru360; ruthenium red     

Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase

Ouabain binding toNa⁺/K⁺-ATPase activates Src/epidermal growthfactor receptor (EGFR) to initiate multiple signal pathways thatregulate growth. In cardiac myocytes and the intact heart, the earlyouabain-induced pathways that cause rapid activations of ERK1/2 alsoregulate intracellular Ca²⁺ concentration([Ca²⁺]_i) and contractility. The goal of thisstudy was to explore the role of caveolae in these early signalingevents. Subunits of Na⁺/K⁺-ATPase were detectedby immunoblot analysis in caveolae isolated from cardiac myocytes,cardiac ventricles, kidney cell lines, and kidney outer medulla byestablished detergent-free procedures. Isolated rat cardiac caveolaecontained Src, EGFR, ERK1/2, and 20-30% of cellular contents of₁- and ₂-isoforms ofNa⁺/K⁺-ATPase, along with nearly all ofcellular caveolin-3. Immunofluorescence microscopy of adult cardiacmyocytes showed the presence of caveolin-3 and -isoforms inperipheral sarcolemma and T tubules and suggested their partialcolocalization. Exposure of contracting isolated rat hearts to apositive inotropic dose of ouabain and analysis of isolated cardiaccaveolae showed that ouabain caused 1) no change in totalcaveolar ERK1/2, but a two- to threefold increase in caveolarphosphorylated/activated ERK1/2; 2) no change in caveolar ₁-isoform and caveolin-3; and 3) 50-60%increases in caveolar Src and ₂-isoform. These findings,in conjunction with previous observations, show that components of thepathways that link Na⁺/K⁺-ATPase to ERK1/2 and[Ca²⁺]_i are organized within cardiac caveolaemicrodomains. They also suggest that ouabain-induced recruitments ofSrc and ₂-isoform to caveolae are involved in themanifestation of the positive inotropic effect of ouabain.

     

A voltage-gated K(+) current in renal inner medullary collecting duct cells

We studied the K⁺-selective conductances in primary cultures of rat renal inner medullary collecting duct (IMCD) using perforated-patch and conventional whole cell techniques. Depolarizations above –20 mV induced a time-dependent outward K⁺ current (I_vto) similar to a delayed rectifier. I_vto showed a half-maximal activation around 5.6 mV with a slope factor of 6.8 mV. Its K⁺/Na⁺ selectivity ratio was 11.7. It was inhibited by tetraethylammonium, quinidine, 4-aminopyridine, and Ba²⁺ and was not Ca²⁺ dependent. The delayed rectifying characteristics of I_vto prompted us to screen the expression of Kv1 and Kv3 families by RT-PCR. Analysis of RNA isolated from cell cultures revealed the presence of three Kv -subunits (Kv1.1, Kv1.3, and Kv1.6). Western blot analysis with Kv -subunit antibodies for Kv1.1 and Kv1.3 showed labeling of 70-kDa proteins from inner medulla plasmatic and microsome membranes. Immunocytochemical analysis of cell culture and kidney inner medulla showed that Kv1.3 is colocalized with the Na⁺-K⁺-ATPase at the basolateral membrane, although it is also in the cytoplasm. This is the first evidence of recording, protein expression, and localization of a voltage-gated Kv1 in the kidney IMCD cells. kidney; Kv1.3; potassium channel; potassium transport; whole cell clamp; immunocytochemistry; confocal microscopy     

Urea transport in MDCK cells that are stably transfected with UT-A1

Progress in understanding the cell biology of urea transporter proteins has been hampered by the lack of an appropriate cell culture system. The goal of this study was to create a polarized epithelial cell line that stably expresses the largest of the rat renal urea transporter UT-A isoforms, UT-A1. The gene for UT-A1 was cloned into pcDNA5/FRT and transfected into Madin-Darby canine kidney (MDCK) cells with an integrated Flp recombination target site. The cells from a single clone were grown to confluence on collagen-coated membranes until the resistance was >1,500 ·cm². Transepithelial [¹⁴C]urea fluxes were measured at 37°C in a HCO₃^–/CO₂ buffer, pH 7.4, with 5 mM urea. The baseline fluxes were not different between unstimulated UT-A1-transfected MDCK cells and nontransfected or sham-transfected MDCK cells. However, only in the UT-A1-transfected cells was UT-A1 protein expressed (as measured by Western blot analysis) and urea transport stimulated by forskolin or arginine vasopressin. Forskolin and arginine vasopressin also increased the phosphorylation of UT-A1. Thionicotinamide, dimethylurea, and phloretin inhibited the forskolin-stimulated [¹⁴C]urea fluxes in the UT-A1-transfected MDCK cells. These characteristics mimic those seen in rat terminal inner medullary collecting ducts. This new polarized epithelial cell line stably expresses UT-A1 and reproduces several of the physiological responses observed in rat terminal inner medullary collecting ducts. urea transporter-A1; arginine vasopressin; collecting duct; Madin-Darby canine kidney cells     

Shrinkage-induced activation of Na+/H+ exchange in rat renal mesangial cells

Using thepH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF),we examined the effect of hyperosmolar solutions, which presumablycaused cell shrinkage, on intracellular pH(pH_i) regulation in mesangialcells (single cells or populations) cultured from the rat kidney. Thecalibration of BCECF is identical in shrunken and unshrunken mesangialcells if the extracellular K⁺concentration ([K⁺])is adjusted to match the predicted intracellular[K⁺]. ForpH_i values between ~6.7 and~7.4, the intrinsic buffering power in shrunken cells (600 mosmol/kgH₂O) is threefold larger than in unshrunken cells (~300mosmol/kgH₂O). In the nominalabsence ofCO₂/HCO₃,exposing cell populations to a HEPES-buffered solution supplementedwith ~300 mM mannitol (600 mosmol/kgH₂O) causes steady-statepH_i to increase by ~0.4. The pH_i increase is due to activationofNa⁺/H⁺exchange because, in single cells, it is blocked in the absence ofexternal Na⁺ or in the presence of50 µM ethylisopropylamiloride (EIPA). Preincubating cells in aCl-free solution for atleast 14 min inhibits the shrinkage-induced pH_i increase by 80%. Wecalculated the pH_i dependence oftheNa⁺/H⁺exchange rate in cell populations under normosmolar and hyperosmolar conditions by summing 1) thepH_i dependence of the totalacid-extrusion rate and 2) thepH_i dependence of theEIPA-insensitive acid-loading rate. Shrinkage alkali shifts thepH_i dependence ofNa⁺/H⁺exchange by ~0.7 pH units.     

Potassium Channels at Chara Plasmalemma

Exposure to high K⁺ medium transforms Chara plasmalemma into[K⁺]_osensitive state (K⁺ state). The current-voltage (I/V)characteristicsunder such conditions display a negative conductance region.This feature results from the complex time and voltage dependenceof K⁺ channel opening At potentials more negative than a thresholdp.d. the channels are closed and the I/V characteristics becomelinear with a low slope conductance of 0.8 S m² and only a weakdependence on [K⁺]_o. Such behaviour is usually associated witha non-specific leak current The threshold level for K⁺ channelclosing depends on [K⁺]_o. In 2.0 mol m^–3 and 5.0 mol m^–3K⁺ medium the membrane resting p.d. follows E_K, but hyperpolarizesgradually if the [K⁺]_o is lowered. The proton pump thus appearsto be non-operative, while the cell is in the K⁺ state, andrecovers slowly as the cell is returned to a low K⁺ medium.Excitation currents decline if the cells are kept in K⁺ statefor some hours. Key words: K⁺ channels, Chara corallina, Proton pump, Current/, oltage characteristics, Conductance     

K+ channel KVLQT1 located in the basolateral membrane of distal colonic epithelium is not essential for activating Cl- secretion

The cellular mechanism for Cl^– and K⁺ secretion in the colonic epithelium requires K⁺ channels in the basolateral and apical membranes. Colonic mucosa from guinea pig and rat were fixed, sectioned, and then probed with antibodies to the K⁺ channel proteins K_VLQT1 (Kcnq1) and minK-related peptide 2 (MiRP2, Kcne3). Immunofluorescence labeling for Kcnq1 was most prominent in the lateral membrane of crypt cells in rat colon. The guinea pig distal colon had distinct lateral membrane immunoreactivity for Kcnq1 in crypt and surface cells. In addition, Kcne3, an auxiliary subunit for Kcnq1, was detected in the lateral membrane of crypt and surface cells in guinea pig distal colon. Transepithelial short-circuit current (I_sc) and transepithelial conductance (G_t) were measured for colonic mucosa during secretory activation by epinephrine (EPI), prostaglandin E₂ (PGE₂), and carbachol (CCh). HMR1556 (10 µM), an inhibitor of Kcnq1 channels (Gerlach U, Brendel J, Lang HJ, Paulus EF, Weidmann K, Brüggemann A, Busch A, Suessbrich H, Bleich M, and Greger R. J Med Chem 44: 3831–3837, 2001), partially (50%) inhibited Cl^– secretory I_sc and G_t activated by PGE₂ and CCh in rat colon with an IC₅₀ of 55 nM, but in guinea pig distal colon Cl^– secretory I_sc and G_t were unaltered. EPI-activated K⁺-secretory I_sc and G_t also were essentially unaltered by HMR1556 in both rat and guinea pig colon. Although immunofluorescence labeling with a Kcnq1 antibody supported the basolateral membrane presence in colonic epithelium of the guinea pig as well as the rat, the Kcnq1 K⁺ channel is not an essential component for producing Cl^– secretion. Other K⁺ channels present in the basolateral membrane presumably must also contribute directly to the K⁺ conductance necessary for K⁺ exit during activation of Cl^– secretion in the colonic mucosa. HMR1556; K⁺ secretion; epinephrine; prostaglandin E₂; cholinergic     

Effects of lactic acid and catecholamines on contractility in fast-twitch muscles exposed to hyperkalemia

Intensive exercise is associated with a pronounced increase in extracellular K⁺ ([K⁺]_o). Because of the ensuing depolarization and loss of excitability, this contributes to muscle fatigue. Intensive exercise also increases the level of circulating catecholamines and lactic acid, which both have been shown to alleviate the depressing effect of hyperkalemia in slow-twitch muscles. Because of their larger exercise-induced loss of K⁺, fast-twitch muscles are more prone to fatigue caused by increased [K⁺]_o than slow-twitch muscles. Fast-twitch muscles also produce more lactic acid. We therefore compared the effects of catecholamines and lactic acid on the maintenance of contractility in rat fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) muscles. Intact muscles were mounted on force transducers and stimulated electrically to evoke short isometric tetani. Elevated [K⁺]_o (11 and 13 mM) was used to reduce force to 20% of control force at 4 mM K⁺. In EDL, the ₂-agonist salbutamol (10^–5 M) restored tetanic force to 83 ± 2% of control force, whereas in soleus salbutamol restored tetanic force to 93 ± 1%. In both muscles, salbutamol induced hyperpolarization (5–8 mV), reduced intracellular Na⁺ content and increased Na⁺-K⁺ pump activity, leading to an increased K⁺ tolerance. Lactic acid (24 mM) restored force from 22 ± 4% to 58 ± 2% of control force in EDL, an effect that was significantly lower than in soleus muscle. These results amplify and generalize the concept that the exercise-induced acidification and increase in plasma catecholamines counterbalance fatigue arising from rundown of Na⁺ and K⁺ gradients. muscle fatigue; Na⁺-K⁺ pump; membrane potential     

Intracellular acidification induced by passive and active transport of ammonium ions in astrocytes

We describe anunconventional response of intracellular pH toNH₄Cl in mouse cerebralastrocytes. Rapid alkalinization reversed abruptly to be replaced by anintense sustained acidification in the continued presence ofNH₄Cl. We hypothesize thathigh-velocity NH⁺₄ influx persisted after thedistribution of ammonia attained steady state. From the initial rate ofacidification elicited by 1 mMNH₄Cl in bicarbonate-bufferedsolution, we estimate that NH⁺₄ entered at avelocity of at least 31.5 nmol · min¹ · mgprotein¹. This rateincreased with NH₄Clconcentration, not saturating at up to 20 mMNH₄Cl. Acidification wasattenuated by raising or lowering extracellularK⁺ concentration.Ba²⁺ (50 µM) inhibited theacidification rate by 80.6%, suggesting inwardly rectifyingK⁺ channels as the primaryNH⁺₄ entry pathway. Acidification was 10-foldslower in rat hippocampal astrocytes, consistent with the differencereported for K⁺ flux in vitro. Thecombination of Ba²⁺ and bumetanideprevented net acidification by 1 mMNH₄Cl, identifying theNa⁺-K⁺-2Clcotransporter as a second NH⁺₄ entry route.NH⁺₄ entry viaK⁺ transport pathways could impact"buffering" of ammonia by astrocytes and could initiate theelevation of extracellular K⁺concentration and astrocyte swelling observed in acute hyperammonemia.

     

Mutational analysis of Glu-327 of Na+-K+-ATPase reveals stimulation of 86Rb+ uptake by external K+

A competition assay of⁸⁶Rb⁺uptake in HeLa cells transfected with ouabain-resistantNa⁺-K⁺-ATPasemutants revealed a stimulation of⁸⁶Rb⁺uptake at low external concentrations (1 mM) of competitor(K⁺). Of the models that weretested, those that require that two K⁺ be bound before transportoccurs gave the worst fits. Random and ordered binding schemesdescribed the data equally well. General models in which both bindingand transport were allowed to be cooperative yielded parameter errorslarger than the parameters themselves and could not be utilized. Modelsthat assumed noncooperative transport always showed positivecooperativity in binding. E327Q and E327L mutated forms of rat₂ had lower apparent affinities for the first K⁺ bound than didwild-type rat ₂ modified to beouabain resistant. The mutations did not affect the apparent affinityof the second K⁺ bound. Modelsthat assumed noncooperativity in binding always showed positivelycooperative transport, i.e., enzymes with two K⁺ bound had a higher flux thanthose with one K⁺ bound. Increasesin external Na⁺ decreased theapparent affinity for K⁺ for allmodels and decreased the ratio of the apparent influx rate constantsfor E327L.

     

Dietary cholesterol alters Na+/K+ selectivity at intracellular Na+/K+ pump sites in cardiac myocytes

A modest diet-induced increase in serum cholesterol in rabbits increases the sensitivity of the sarcolemmal Na⁺/K⁺ pump to intracellular Na⁺, whereas a large increase in cholesterol levels decreases the sensitivity to Na⁺. To examine the mechanisms, we isolated cardiac myocytes from controls and from rabbits with diet-induced increases in serum cholesterol. The myocytes were voltage clamped with the use of patch pipettes that contained osmotically balanced solutions with Na⁺ in a concentration of 10 mM and K⁺ in concentrations ([K⁺]_pip) ranging from 0 to 140 mM. There was no effect of dietary cholesterol on electrogenic Na⁺/K⁺ current (I_p) when pipette solutions were K⁺ free. A modest increase in serum cholesterol caused a [K⁺]_pip-dependent increase in I_p, whereas a large increase caused a [K⁺]_pip-dependent decrease in I_p. Modeling suggested that pump stimulation with a modest increase in serum cholesterol can be explained by a decrease in the microscopic association constant K_K describing the backward reaction E₁ + 2K⁺ E₂(K⁺)₂, whereas pump inhibition with a large increase in serum cholesterol can be explained by an increase in K_K. Because hypercholesterolemia upregulates angiotensin II receptors and because angiotensin II regulates the Na⁺/K⁺ pump in cardiac myocytes in a [K⁺]_pip-dependent manner, we blocked angiotensin synthesis or angiotensin II receptors in vivo in cholesterol-fed rabbits. This abolished cholesterol-induced pump inhibition. Because the -isoform of protein kinase C (PKC) mediates effects of angiotensin II on the pump, we included specific PKC-blocking peptide in patch pipette filling solutions. The peptide reversed cholesterol-induced pump inhibition. partial reactions; protein kinase C; angiotensin converting enzyme inhibitors; arteriosclerosis; insulin resistance     

Gastric parietal cell secretory membrane contains PKA- and acid-activated Kir2.1 K+ channels

Our objective was to identify and localize a K⁺ channel involved in gastric HCl secretion at the parietal cell secretory membrane and to characterize and compare the functional properties of native and recombinant gastric K⁺ channels. RT-PCR showed that mRNA for Kir2.1 was abundant in rabbit gastric mucosa with lesser amounts of Kir4.1 and Kir7.1, relative to -actin. Kir2.1 mRNA was localized to parietal cells of rabbit gastric glands by in situ RT-PCR. Resting and stimulated gastric vesicles contained Kir2.1 by Western blot analysis at 50 kDa as observed with in vitro translation. Immunoconfocal microscopy showed that Kir2.1 was present in parietal cells, where it colocalized with H⁺-K⁺-ATPase and ClC-2 Cl^- channels. Function of native K⁺ channels in rabbit resting and stimulated gastric mucosal vesicles was studied by reconstitution into planar lipid bilayers. Native gastric K⁺ channels exhibited a linear current-voltage relationship and a single-channel slope conductance of 11 pS in 400 mM K₂SO₄. Channel open probability (P_o) in stimulated vesicles was high, and that of resting vesicles was low. Reduction of extracellular pH plus PKA treatment increased resting channel P_o to 0.5 as measured in stimulated vesicles. Full-length rabbit Kir2.1 was cloned. When stably expressed in Chinese hamster ovary (CHO) cells, it was activated by reduced extracellular pH and forskolin/IBMX with no effects observed in nontransfected CHO cells. Cation selectivity was K⁺ = Rb⁺ >> Na⁺ = Cs⁺ = Li⁺ = NMDG⁺. These findings strongly suggest that the Kir2.1 K⁺ channel may be involved in regulated gastric acid secretion at the parietal cell secretory membrane. H⁺-K⁺-ATPase; hydrogen chloride secretion; parietal cell K⁺ channel     

Distinct K+ conductive pathways are required for Cl- and K+ secretion across distal colonic epithelium

Secretion of Cl^– and K⁺ in the colonic epithelium operates through a cellular mechanism requiring K⁺ channels in the basolateral and apical membranes. Transepithelial current [short-circuit current (I_sc)] and conductance (G_t) were measured for isolated distal colonic mucosa during secretory activation by epinephrine (Epi) or PGE₂ and synergistically by PGE₂ and carbachol (PGE₂ + CCh). TRAM-34 at 0.5 µM, an inhibitor of K_Ca3.1 (IK, Kcnn4) K⁺ channels (H. Wulff, M. J. Miller, W. Hänsel, S. Grissmer, M. D. Cahalan, and K. G. Chandy. Proc Natl Acad Sci USA 97: 8151–8156, 2000), did not alter secretory I_sc or G_t in guinea pig or rat colon. The presence of K_Ca3.1 in the mucosa was confirmed by immunoblot and immunofluorescence detection. At 100 µM, TRAM-34 inhibited I_sc and G_t activated by Epi (4%), PGE₂ (30%) and PGE₂ + CCh (60%). The IC₅₀ of 4.0 µM implicated involvement of K⁺ channels other than K_Ca3.1. The secretory responses augmented by the K⁺ channel opener 1-EBIO were inhibited only at a high concentration of TRAM-34, suggesting further that K_Ca3.1 was not involved. Sensitivity of the synergistic response (PGE₂ + CCh) to a high concentration TRAM-34 supported a requirement for multiple K⁺ conductive pathways in secretion. Clofilium (100 µM), a quaternary ammonium, inhibited Cl^– secretory I_sc and G_t activated by PGE₂ (20%) but not K⁺ secretion activated by Epi. Thus Cl^– secretion activated by physiological secretagogues occurred without apparent activity of K_Ca3.1 channels but was dependent on other types of K⁺ channels sensitive to high concentrations of TRAM-34 and/or clofilium. epinephrine; prostaglandin E₂; cholinergic; Kcnn4; TRAM-34; clofilium     

K+ Channel in the Chora Plasmalemma: Estimations of K+ Channel Density and Single K+ Channel Conductance

The electromotive force E and the conductance G of the Characorallina plasmalemma were measured under voltage clamp conditions.In the depolarized voltage range less negative than –60mV, E changed according to the Nerhst equation for K⁺, and Gincreased with the external K⁺ concentration [K⁺]_o and alsowith the depolarization of the membrane potential. This is attributedto the voltage-dependent opening of the K⁺ channels in the largelydepolarized voltage region. The voltage-dependent increase ofG was due to the increase of the number of open K⁺ channelsper unit area. The density of the total K⁺ channels in the C. corallina plasmalemmawas estimated to be about 6.50/(10 µm)2. The single K⁺channel conductance _K changed with the external [K⁺]_o; it was79.3, 86.1, 105.9, 119.0 pS for external [K⁺]_o of 0.2, 0.5,2.0 and 5.0 mu respectively. (Received May 22, 1986; Accepted August 22, 1986)     

An electrogenic Na(+)-HCO(-)(3) cotransporter (NBC) with a novel COOH-terminus, cloned from rat brain

We screened rat brain cDNA libraries and used 5'rapid amplification of cDNA ends to clone two electrogenicNa⁺-HCO₃ cotransporter(NBC) isoforms from rat brain (rb1NBC and rb2NBC). At the amino acidlevel, one clone (rb1NBC) is 96% identical to human pancreas NBC. Theother clone (rb2NBC) is identical to rb1NBC except for 61 uniqueCOOH-terminal amino acids, the result of a 97-bp deletion near the3' end of the open-reading frame. Using RT-PCR, we confirmed thatmRNA from rat brain contains this 97-bp deletion. Furthermore, wegenerated rabbit polyclonal antibodies that distinguish between theunique COOH-termini of rb1NBC (rb1NBC) and rb2NBC (rb2NBC).rb1NBC labels an ~130-kDa protein predominantly from kidney, andrb2NBC labels an ~130-kDa protein predominantly from brain.rb2NBC labels a protein that is more highly expressed in corticalneurons than astrocytes cultured from rat brain; rb1NBC exhibits theopposite pattern. In expression studies, applying 1.5%CO₂/10 mM HCO₃ toXenopus oocytes injected with rb2NBC cRNA causes 1)pH_i to recover from the initial CO₂-inducedacidification and 2) the cell to hyperpolarize. Subsequently,removing external Na⁺ reverses the pH_i increaseand elicits a rapid depolarization. In the presence of 450 µM DIDS,removing external Na⁺ has no effect on pH_i andelicits a small hyperpolarization. The rate of the pH_idecrease elicited by removing Na⁺ is insensitive toremoving external Cl. Thus rb2NBC is aDIDS-sensitive, electrogenic NBC that is predominantly expressed inbrain of at least rat.

     

Mechanism and role of high-potassium-induced reduction of intracellular Ca2+ concentration in rat osteoclasts

Osteoclasts are multinucleated, bone-resorbing cells that show structural and functional differences between the resorbing and nonresorbing (motile) states during the bone resorption cycle. In the present study, we measured intracellular Ca²⁺ concentration ([Ca²⁺]_i) in nonresorbing vs. resorbing rat osteoclasts. Basal [Ca²⁺]_i in osteoclasts possessing pseudopodia (nonresorbing/motile state) was around 110 nM and significantly higher than that in actin ring-forming osteoclasts (resorbing state, around 50 nM). In nonresorbing/motile osteoclasts, exposure to high K⁺ reduced [Ca²⁺]_i, whereas high K⁺ increased [Ca²⁺]_i in resorbing state osteoclasts. In nonresorbing/motile cells, membrane depolarization and hyperpolarization applied by the patch-clamp technique decreased and increased [Ca²⁺]_i, respectively. Removal of extracellular Ca²⁺ or application of 300 µM La³⁺ reduced [Ca²⁺]_i to 50 nM in nonresorbing/motile osteoclasts, and high-K⁺-induced reduction of [Ca²⁺]_i could not be observed under these conditions. Neither inhibition of intracellular Ca²⁺ stores or plasma membrane Ca²⁺ pumps nor blocking of L- and N-type Ca²⁺ channels significantly reduced [Ca²⁺]_i. Exposure to high K⁺ inhibited the motility of nonresorbing osteoclasts and reduced the number of actin rings and pit formation in resorbing osteoclasts. These results indicate that in nonresorbing/motile osteoclasts, a La³⁺-sensitive Ca²⁺ entry pathway is continuously active under resting conditions, keeping [Ca²⁺]_i high. Changes in membrane potential regulate osteoclastic motility by controlling the net amount of Ca²⁺ entry in a "reversed" voltage-dependent manner, i.e., depolarization decreases and hyperpolarization increases [Ca²⁺]_i. membrane depolarization; resorbing and motile activities; bone resorbing cycle     

Effect of K+, its Counter Anion, and pH on Sodium Efflux from Barley Root Tips

Sodium efflux from ²²Na⁺-loaded root tips root tips of Hordeumvulgare L. was markedly increased by replacing 10mM Na₂SO₄ inthe washing solution by K₂SO₄ with the same electrical conductivity.This increase was inhibited by both an uncoupler and an inhibitorof oxidative phosphorylation but not by ouabain. Potassium ionsdid not enhance Na⁺ efflux in the presence of a rapidly absorbedcounter anion, such as Cl^–, instead of . Efflux of ²²Na⁺ could also be enhanced by a low pH in theabsence of K⁺; this was prevented by uncouplers, but not byan inhibitor of the mitochondrial ATPase. It seems that K⁺ indirectly enhances Na⁺ efflux. It is suggestedthat metabolic K⁺ uptake in excess of the counter anion resultsin a proton gradient across the plasmalemma (acid outside) inducingH⁺/Na⁺ antiport.     

K+ channel KV3.1 associates with OSP/claudin-11 and regulates oligodendrocyte development

K⁺ channels are differentially expressed throughout oligodendrocyte (Olg) development. K_V1 family voltage-sensitive K⁺ channels have been implicated in proliferation and migration of Olg progenitor cell (OPC) stage, and inward rectifier K+ channels (K_IR)4.1 are required for OPC differentiation to myelin-forming Olg. In this report we have identified a Shaw family K⁺ channel, K_V3.1, that is involved in proliferation and migration of OPC and axon myelination. Application of anti-K_V3.1 antibody or knockout of Kv3.1 gene decreased the sustained K⁺ current component of OPC by 50% and 75%, respectively. In functional assays block of K_V3.1-specific currents or knockout of Kv3.1 gene inhibited proliferation and migration of OPC. Adult Kv3.1 gene-knockout mice had decreased diameter of axons and decreased thickness of myelin in optic nerves compared with age-matched wild-type littermates. Additionally, K_V3.1 was identified as an associated protein of Olg-specific protein (OSP)/claudin-11 via yeast two-hybrid analysis, which was confirmed by coimmunoprecipitation and coimmunohistochemistry. In summary, the K_V3.1 K⁺ current accounts for a significant component of the total K⁺ current in cells of the Olg lineage and, in association with OSP/claudin-11, plays a significant role in OPC proliferation and migration and myelination of axons. membrane potential; tight junction; myelin; progenitor cell     

The Effects of Salinity upon Ion Content and Ion Transport of the Marine RedAlga Porphyra purpurea(Roth) C.Ag.

Ion contents and concentrations (K⁺, Na⁺, Cl^–, Ca²⁺, Mg²⁺,SO^2–₄, NO^–₃, HPOJ^2–₄, amino and organic acids)of P. purpurea have been studied in relation to salinity variation.Cells were shown to accumulate large amounts of K⁺ and Cl^–against their respective gradients of electrochemical potentialin all dilute and concentrated seawater media. Active influxof SOJ^2–₄, NO^–₃, and HPOJ^2–₄ is also suggested,while Na⁺ is actively excluded from cells under hyposaline andhypersaline conditions. The relative proportions of individualcomponents of the internal osmotic potential were found to changeaccording to the external salt concentration. KCL forms themajor fraction of _j} in concentrated seawater media while K⁺-aminoacids form the major fraction in dilute seawaters. Other intracellularsolutes comprise less than 15% oft_j, in all media. Unidirectional fluxes of K⁺ and Cl^– were studied by radioisotopicmeans. Fluxes of K⁺ and Cl^– are reduced in hyposalinemedia, as is absolute KCL content per cell. Intracelhilar KCLcontent was also found to be markedly dependent upon externalK⁺ concentration, rather than water potential. Changes in KC1levels induced by salinity variation occur over a 6 h period.