Excitatory amino acids act via receptor subtypes in the mammalian central nervous system (CNS). The receptor selectively activated by N-methyl-D-aspartic acid (NMDA) has been best characterized using voltage-clamp and single-channel recording; the results suggest that NMDA receptors gate channels that are permeable to Na+, K+ and other monovalent cations. Various experiments suggest that Ca2+ flux is also associated with the activation of excitatory amino-acid receptors on vertebrate neurones. Whether Ca2+ enters through voltage-dependent Ca2+ channels or through excitatory amino-acid-activated channels of one or more subtype is unclear. Mg2+ can be used to distinguish NMDA-receptor-activated channels from voltage-dependent Ca2+ channels, because at micromolar concentrations Mg2+ has little effect on voltage-dependent Ca2+ channels while it enters and blocks NMDA receptor channels. Marked differences in the potency of other divalent cations acting as Ca2+ channel blockers compared with their action as NMDA antagonists also distinguish the NMDA channel from voltage-sensitive Ca2+ channels. However, we now directly demonstrate that excitatory amino acids acting at NMDA receptors on spinal cord neurones increase the intracellular Ca2+ activity, measured using the indicator dye arsenazo III, and that this is the result of Ca2+ influx through NMDA receptor channels. Kainic acid (KA), which acts at another subtype of excitatory amino-acid receptor, was much less effective in triggering increases in intracellular free Ca2+.

1. Whole-cell patch clamp recordings of membrane currents and fura-2 measurements of free intracellular calcium concentration ([Ca2+]i) were used to study the biophysical properties of a calcium current activated by depletion of intracellular calcium stores in rat peritoneal mast cells. 2. Calcium influx through an inward calcium release-activated calcium current (ICRAC) was induced by three independent mechanisms that result in store depletion: intracellular infusion of inositol 1,4,5-trisphosphate (InsP3) or extracellular application of ionomycin (active depletion), and intracellular infusion of calcium chelators (ethylene glycol bis-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)) to prevent reuptake of leaked-out calcium into the stores (passive depletion). 3. The activation of ICRAC induced by active store depletion has a short delay (4-14 s) following intracellular infusion of InsP3 or extracellular application of ionomycin. It has a monoexponential time course with a time constant of 20-30 s and, depending on the complementary Ca2+ buffer, a mean normalized amplitude (at 0 mV) of 0.6 pA pF-1 (with EGTA) and 1.1 pA pF-1 (with BAPTA). 4. After full activation of ICRAC by InsP3 in the presence of EGTA (10 mM), hyperpolarizing pulses to -100 mV induced an instantaneous inward current that decayed by 64% within 50 ms. This inactivation is probably mediated by [Ca2+]i, since the decrease of inward current in the presence of the fast Ca2+ buffer BAPTA (10 mM) was only 30%. 5. The amplitude of ICRAC was dependent on the extracellular Ca2+ concentration with an apparent dissociation constant (KD) of 3.3 mM. Inward currents were nonsaturating up to -200 mV. 6. The selectivity of ICRAC for Ca2+ was assessed by using fura-2 as the dominant intracellular buffer (at a concentration of 2 mM) and relating the absolute changes in the calcium-sensitive fluorescence (390 nm excitation) with the calcium current integral. This relationship was almost identical to the one determined for Ca2+ influx through voltage-activated calcium currents in chromaffin cells, suggesting a similar selectivity. Replacing Na+ and K+ by N-methyl-D-glucamine (with Ca2+ ions as exclusive charge carriers) reduced the amplitude of ICRAC by only 9% further suggesting a high specificity for Ca2+ ions. 7. The current amplitude was not greatly affected by variations of external Mg2+ in the range of 0-12 mM. Even at 12 mM Mg2+ the current amplitude was reduced by only 23%. 8. ICRAC was dose-dependently inhibited by Cd2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Ventricular arrhythmias and contractile dysfunction are the main causes of death in human heart failure (HF). In a rabbit HF model reproducing these same aspects of human HF, we demonstrate that a 2-fold functional upregulation of Na(+)-Ca(2+) exchange (NaCaX) unloads sarcoplasmic reticulum (SR) Ca(2+) stores, reducing Ca(2+) transients and contractile function. Whereas beta-adrenergic receptors (beta-ARs) are progressively downregulated in HF, residual beta-AR responsiveness at this critical HF stage allows SR Ca(2+) load to increase, causing spontaneous SR Ca(2+) release and transient inward current carried by NaCaX. A given Ca(2+) release produces greater arrhythmogenic inward current in HF (as a result of NaCaX upregulation), and approximately 50% less Ca(2+) release is required to trigger an action potential in HF. The inward rectifier potassium current (I(K1)) is reduced by 49% in HF, and this allows greater depolarization for a given NaCaX current. Partially blocking I(K1) in control cells with barium mimics the greater depolarization for a given current injection seen in HF. Thus, we present data to support a novel paradigm in which changes in NaCaX and I(K1), and residual beta-AR responsiveness, conspire to greatly increase the propensity for triggered arrhythmias in HF. In addition, NaCaX upregulation appears to be a critical link between contractile dysfunction and arrhythmogenesis.

Maintenance of fluid and electrolyte homeostasis is critical for normal neuromuscular function. Bartter's syndrome is an autosomal recessive disease characterized by diverse abnormalities in electrolyte homeostasis including hypokalaemic metabolic alkalosis; Gitelman's syndrome represents the predominant subset of Bartter's patients having hypomagnesemia and hypocalciuria. We now demonstrate complete linkage of Gitelman's syndrome to the locus encoding the renal thiazide-sensitive Na-Cl cotransporter, and identify a wide variety of non-conservative mutations, consistent with loss of function alleles, in affected subjects. These findings demonstrate the molecular basis of Gitelman's syndrome. We speculate that these mutant alleles lead to reduced sodium chloride reabsorption in the more common heterozygotes, potentially protecting against development of hypertension.

Electroneutral cation-Cl(-) cotransporters compose a family of solute carriers in which cation (Na(+) or K(+)) movement through the plasma membrane is always accompanied by Cl(-) in a 1:1 stoichiometry. Seven well-characterized members include one gene encoding the thiazide-sensitive Na(+)-Cl(-) cotransporter, two genes encoding loop diuretic-sensitive Na(+)-K(+)-2Cl(-) cotransporters, and four genes encoding K(+)-Cl(-) cotransporters. These membrane proteins are involved in several physiological activities including transepithelial ion absorption and secretion, cell volume regulation, and setting intracellular Cl(-) concentration below or above its electrochemical potential equilibrium. In addition, members of this family play an important role in cardiovascular and neuronal pharmacology and pathophysiology. Some of these cotransporters serve as targets for loop diuretics and thiazide-type diuretics, which are among the most commonly prescribed drugs in the world, and inactivating mutations of three members of the family cause inherited diseases such as Bartter's, Gitelman's, and Anderman's diseases. Major advances have been made in the past decade as consequences of molecular identification of all members in this family. This work is a comprehensive review of the knowledge that has evolved in this area and includes molecular biology of each gene, functional properties of identified cotransporters, structure-function relationships, and physiological and pathophysiological roles of each cotransporter.

Sodium ion (Na+) channels, which initiate the action potential in electrically excitable cells, are the molecular targets of local anesthetic drugs. Site-directed mutations in transmembrane segment S6 of domain IV of the Na+ channel alpha subunit from rat brain selectively modified drug binding to resting or to open and inactivated channels when expressed in Xenopus oocytes. Mutation F1764A, near the middle of this segment, decreased the affinity of open and inactivated channels to 1 percent of the wild-type value, resulting in almost complete abolition of both the use-dependence and voltage-dependence of drug block, whereas mutation N1769A increased the affinity of the resting channel 15-fold. Mutation I1760A created an access pathway for drug molecules to reach the receptor site from the extracellular side. The results define the location of the local anesthetic receptor site in the pore of the Na+ channel and identify molecular determinants of the state-dependent binding of local anesthetics.

A technique for the rapid production of large unilamellar vesicles by repeated extrusion under moderate pressures (≤ 500 lb/in²) of multilamellar vesicles through polycarbonate filters (100 nm pore size) is demonstrated. In combination with freeze-thaw protocols where required, this procedure results in unilamellar vesicles with diameters in the range 60-100 nm and with trapped volumes in the region of 1-3 μl/μmol phospholipid. Advantages of this technique include the absence of organic solvents or detergents, the high lipid concentrations (up to 300 μmol/ml) that can be employed and the high trapping efficiencies (up to 30%) that can be achieved. Further, the procedure for generating these 'LUVET's' (large unilamellar vesicles by extrusion techniques) is rapid (≤ min preparation time) and can be employed to generate large unilamellar vesicles from a wide variety of lipid species and mixtures. As a particular illustration of the utility of this vesicle preparation, LUVET systems exhibiting a membrane potential (ΔΨ) in response to a transmembrane Na⁺/K⁺ gradient (K⁺ inside) have been characterized. By employing the lipophilic cation methyltriphenylphosphonium (MTPP⁺) it is shown that a K⁺ of diffusion potential (ΔΨ < -100 mV) forms rapidly in the presence of the K⁺ ionophore valinomycin for soya phosphatidylcholine (soya PC) LUVET's. The values of Δψ obtained correlate well with the K⁺ concentration gradient across the membrane, and it is demonstrated that the decay of Δψ with time depends on the flux of Na+ into the vesicles.

Many important agronomic traits in crop plants, including stress tolerance, are complex traits controlled by quantitative trait loci (QTLs). Isolation of these QTLs holds great promise to improve world agriculture but is a challenging task. We previously mapped a rice QTL, SKC1, that maintained K(+) homeostasis in the salt-tolerant variety under salt stress, consistent with the earlier finding that K(+) homeostasis is important in salt tolerance. To understand the molecular basis of this QTL, we isolated the SKC1 gene by map-based cloning and found that it encoded a member of HKT-type transporters. SKC1 is preferentially expressed in the parenchyma cells surrounding the xylem vessels. Voltage-clamp analysis showed that SKC1 protein functions as a Na(+)-selective transporter. Physiological analysis suggested that SKC1 is involved in regulating K(+)/Na(+) homeostasis under salt stress, providing a potential tool for improving salt tolerance in crops.

The amiloride-sensitive epithelial sodium channel (ENaC) plays a major role in sodium transport in kidney and other epithelia, and in regulating blood pressure. The channel is composed of three subunits (alphabetagamma) each containing two proline-rich sequences (P1 and P2) at its C-terminus. The P2 regions in human beta and gammaENaC, identical to the rat betagammarENaC, were recently shown to be deleted in patients with Liddle's syndrome (a hereditary form of hypertension), leading to hyperactivation of the channel. Using a yeast two-hybrid screen, we have now identified the rat homologue of Nedd4 (rNedd4) as the binding partner for the P2 regions of beta and gammarENaC. rNedd4 contains a Ca2+ lipid binding (CaLB or C2) domain, three WW domains and a ubiquitin ligase (Hect) domain. Our yeast two-hybrid and in vitro binding studies revealed that the rNedd4-WW domains mediate this association by binding to the P2 regions, which include the PY motifs (XPPXY) of either betarENaC (PPPNY) or gammarENaC (PPPRY). SH3 domains were unable to bind these sequences. Moreover, mutations to Ala of Pro616 or Tyr618 within the betarENaC P2 sequence (to PPANY or PPPNA, respectively), recently described in Liddle's patients, led to abrogation of rNedd4-WW binding. Nedd4-WW domains also bound to the proline-rich C-terminus (containing the sequence PPPAY) of alpharENaC, and endogenous Nedd4 co-immunoprecipitated with alpharENaC expressed in MDCK cells. These results demonstrate that the WW domains of rNedd4 bind to the PY motifs deleted from beta or gammaENaC in Liddle's syndrome patients, and suggest that Nedd4 may be a regulator (suppressor) of the epithelial Na+ channel.

Tight junctions seal the paracellular pathway of epithelia but, in leaky tissues, also exhibit specific permeability. In order to characterize the contribution of claudin-2 to barrier and permeability properties of the tight junction in detail, we studied two strains of Madin-Darby canine kidney cells (MDCK-C7 and MDCK-C11) with different tight junctional permeabilities. Monolayers of C7 cells exhibited a high transepithelial resistance (>1 kOhms cm(2)), compared with C11 cells (<100 Ohms cm(2)). Genuine expression of claudin-1 and claudin-2, but not of occludin or claudin-3, was reciprocal to transepithelial resistance. However, confocal microscopy revealed a marked subjunctional localization of claudin-1 in C11 cells, indicating that claudin-1 is not functionally related to the low tight junctional resistance of C11 cells. Strain MDCK-C7, which endogenously does not express junctional claudin-2, was transfected with claudin-2 cDNA. In transfected cells, but not in vector controls, the protein was detected in colocalization with junctional occludin by means of immunohistochemical analyses. Overexpression of claudin-2 in the originally tight epithelium with claudin-2 cDNA resulted in a 5.6-fold higher paracellular conductivity and relative ion permeabilities of Na(+) identical with 1, K(+)=1.02, NMDG(+)=0.79, choline(+)=0.71, Cl(-)=0.12, Br(-)=0.10 (vector control, 1:1.04:0.95:0.94:0.85:0.83). By contrast, fluxes of (radioactively labeled) mannitol and lactulose and (fluorescence labeled) 4 kDa dextran were not changed. Hence, with regular Ringer's, Na(+) conductivity was 0.2 mS cm(-2) in vector controls and 1.7 mS cm(-2) in claudin-2-transfected cells, while Cl(-) conductivity was 0.2 mS cm(-2) in both cells. Thus, presence of junctional claudin-2 causes the formation of cation-selective channels sufficient to transform a 'tight' tight junction into a leaky one.

Single channel currents through Ca2+-activated K+ channels of bovine chromaffin cells were measured to determine the effects of small ions on permeation through the channel. The channel selects strongly for K+ over Na+ and Cs+, and Rb+ carries a smaller current through the channel than K+. Tetraethylammonium ion (TEA+) blocks channel currents when applied to either side of the membrane; it is effective at lower concentrations when applied externally. Millimolar concentrations of internal Na+ reduce the average current through the channel and produce large fluctuations (flicker) in the open channel currents. This flickery block is analyzed by a new method, amplitude distribution analysis, which can measure block and unblock rates in the microsecond time range even though individual blocking events are not time-resolved by the recording system. The analysis shows that the rate of block by Na+ is very voltage dependent, but the unblock rate is voltage independent. These results can be explained easily by supposing that current flow through the channel is diffusion limited, a hypothesis consistent with the large magnitude of the single channel current.

We recently analyzed a series of H5N1 viruses isolated from healthy ducks in southern China since 1999 and found that these viruses had progressively acquired the ability to replicate and cause disease in mice. In the present study, we explored the genetic basis of this change in host range by comparing two of the viruses that are genetically similar but differ in their ability to infect mice and have different pathogenicity in mice. A/duck/Guangxi/22/2001 (DKGX/22) is nonpathogenic in mice, whereas A/duck/Guangxi/35/2001 (DKGX/35) is highly pathogenic. We used reverse genetics to create a series of single-gene recombinants that contained one gene from DKGX/22 and the remaining seven gene segments from DKGX/35. We find that the PA, NA, and NS genes of DKGX/22 could attenuate DKGX/35 virus to some extent, but PB2 of DKGX/22 virus attenuated the DKGX/35 virus dramatically, and an Asn-to-Asp substitution at position 701 of PB2 plays a key role in this function. Conversely, of the recombinant viruses in the DKGX/22 background, only the one that contains the PB2 gene of DKGX/35 was able to replicate in mice. A single amino acid substitution (Asp to Asn) at position 701 of PB2 enabled DKGX/22 to infect and become lethal for mice. These results demonstrate that amino acid Asn 701 of PB2 is one of the important determinants for this avian influenza virus to cross the host species barrier and infect mice, though the replication and lethality of H5N1 influenza viruses involve multiple genes and may result from a constellation of genes. Our findings may help to explain the expansion of the host range and lethality of the H5N1 influenza viruses to humans.

Rhythmic and effective cardiac contraction depends on appropriately timed generation and spread of cardiac electrical activity. The basic cellular unit of such activity is the action potential, which is shaped by specialized proteins (channels and transporters) that control the movement of ions across cardiac cell membranes in a highly regulated fashion. Cardiac disease modifies the operation of ion channels and transporters in a way that promotes the occurrence of cardiac rhythm disturbances, a process called "arrhythmogenic remodeling." Arrhythmogenic remodeling involves alterations in ion channel and transporter expression, regulation and association with important protein partners, and has important pathophysiological implications that contribute in major ways to cardiac morbidity and mortality. We review the changes in ion channel and transporter properties associated with three important clinical and experimental paradigms: congestive heart failure, myocardial infarction, and atrial fibrillation. We pay particular attention to K+, Na+, and Ca2+ channels; Ca2+ transporters; connexins; and hyperpolarization-activated nonselective cation channels and discuss the mechanisms through which changes in ion handling processes lead to cardiac arrhythmias. We highlight areas of future investigation, as well as important opportunities for improved therapeutic approaches that are being opened by an improved understanding of the mechanisms of arrhythmogenic remodeling.

The cone snails (genus Conus) are venomous marine molluscs that use small, structured peptide toxins (conotoxins) for prey capture, defense, and competitor deterrence. Each of the 500 Conus can express approximately 100 different conotoxins, with little overlap between species. An overwhelming majority of these peptides are probably targeted selectively to a specific ion channel. Because conotoxins discriminate between closely related subtypes of ion channels, they are widely used as pharmacological agents in ion channel research, and several have direct diagnostic and therapeutic potential. Large conotoxin families can comprise hundreds or thousands of different peptides; most families have a corresponding ion channel family target (i.e., omega-conotoxins and Ca channels, alpha-conotoxins and nicotinic receptors). Different conotoxin families may have different ligand binding sites on the same ion channel target (i.e., mu-conotoxins and delta-conotoxins to sites 1 and 6 of Na channels, respectively). The individual peptides in a conotoxin family are typically each selectively targeted to a diverse set of different molecular isoforms within the same ion channel family. This review focuses on the targeting specificity of conotoxins and their differential binding to different states of an ion channel.

1. Spinal cord and hippocampal neurones in cell culture were voltage clamped using the tight-seal, whole-cell recording technique. The concentration of sodium and a series of divalent cations in the extracellular media was varied to study permeation through excitatory amino acid receptor channels activated by the selective agonists N-methyl-D-aspartic acid (NMDA), kainic acid and quisqualic acid. 2. On raising the extracellular calcium concentration, with [Na+]o held constant at 105 mM, the reversal potential of responses to NMDA shifted in the depolarizing direction. This shift was adequately described by the extended constant-field equation over the range 0.3-50 mM-calcium. Using ionic activity coefficients we calculate a value of PCa/PNa = 10.6. Under the same experimental conditions the reversal potential of responses to kainic and quisqualic acids was much less affected by raising the calcium concentration, such that PCa/PNa = 0.15. A depolarizing shift of the NMDA reversal potential was also recorded during application of 20 mM-barium, strontium or manganese, suggesting permeation of these ions. The permeability sequence was Ca2+ greater than Ba2+ greater than Sr2+ much greater than Mn2+. No depolarizing shift of the NMDA reversal potential occurred during application of 20 mM-cobalt, magnesium or nickel. 3. In experiments in which the extracellular Na+ concentration was varied the extended constant-field equation was adequate in predicting shifts of the NMDA reversal potential recorded on varying [Na+]o over the range 50-150 mM, but failed to accurately predict the reversal potential of responses to NMDA with 10 mM-[Ca2+]o and only 10 or 20 mM-[Na+]o. These results imply an apparent increase in PCa/PNa on lowering [Na+]o and may result from interaction of permeant ions within the channel. 4. Barium and to a lesser extent calcium, but not strontium (all 20 mM), reduced the slope conductance of responses to NMDA recorded within +/- 15 mV of the reversal potential; over this limited range of membrane potential the current-voltage relationship remained linear in the presence of each of these ions. In contrast manganese produced a strong, voltage-dependent block of responses to NMDA, similar to that produced by magnesium, such that even close to the reversal potential the NMDA current-voltage relationship was highly non-linear. Thus manganese both permeates and blocks the NMDA receptor channel. 5. Raising the extracellular calcium concentration, from 0.1 to 5 mM, had two effects on the conductance mechanism activated by NMDA.(ABSTRACT TRUNCATED AT 400 WORDS)

The timing and number of prehistoric migrations involved in the settlement of the American continent is subject to intense debate. Here, we reanalyze Native American control region mtDNA data and demonstrate that only an appropriate phylogenetic analysis accompanied by an appreciation of demographic factors allows us to discern different migrations and to estimate their ages. Reappraising 574 mtDNA control region sequences from aboriginal Siberians and Native Americans, we confirm in agreement with linguistic, archaeological and climatic evidence that (i) the major wave of migration brought one population, ancestral to the Amerinds, from northeastern Siberia to America 20,000-25,000 years ago and (ii) a rapid expansion of a Beringian source population took place at the end of the Younger Dryas glacial phase approximately 11,300 years ago, ancestral to present Eskimo and Na-Dene populations.

The ionic requirements for electro-responsiveness in thalamic neurones were studied using in vitro slice preparations of the guinea-pig diencephalon. Analysis of the current-voltage relationship in these neurones revealed delayed and anomalous rectification. Substitution of Na+ with choline in the bath or addition of tetrodotoxin (TTX) abolished the fast spikes and the plateau potentials, described in the accompanying paper. Ca2+ conductance blockage with Co2+, Cd2+ or Mn2+, or replacement of Ca2+ by Mg2+ abolished the low-threshold spikes (l.t.s.). Substitution with Ba2+ did not significantly increase the duration of the l.t.s., suggesting that under normal conditions the falling phase of this response is brought about by inactivation of the Ca2+ conductance. The after-hyperpolarization (a.h.p.) following fast spikes was markedly reduced in amplitude and duration by bath application of Cd2+, Co2+ or Mn2+, indicating that a large component of this response is generated by a Ca2+-dependent K+ conductance (gK[Ca]). Following hyperpolarizing current pulses, the membrane potential showed a delayed return to base line. This delay is produced by a transient K+ conductance as it can be modified by changing the drive force for K+. Presumptive intra-dendritic recording demonstrated high-threshold Ca2+ spikes (h.t.s.s.) which activate a gK[Ca]. Such h.t.s.s. were also seen at the somatic level when K+ conductance was blocked with 4-aminopyridine. It is proposed that the intrinsic biophysical properties of thalamic neurones allow them to serve as relay systems and as single cell oscillators at two distinct frequencies, 9-10 and 5-6 Hz. These frequencies coincide with the alpha and theta rhythms of the e.e.g. and, in the latter case, with the frequency of Parkinson's tremor.

Redox status changes exert critical impacts on necrotic/apoptotic and normal cellular processes. We report here a widely expressed Ca2+-permeable cation channel, LTRPC2, activated by micromolar levels of H2O2 and agents that produce reactive oxygen/nitrogen species. This sensitivity of LTRPC2 to redox state modifiers was attributable to an agonistic binding of nicotinamide adenine dinucleotide (beta-NAD+) to the MutT motif. Arachidonic acid and Ca2+ were important positive regulators for LTRPC2. Heterologous LTRPC2 expression conferred susceptibility to death on HEK cells. Antisense oligonucleotide experiments revealed physiological involvement of "native" LTRPC2 in H2O2- and TNFalpha-induced Ca2+ influx and cell death. Thus, LTRPC2 represents an important intrinsic mechanism that mediates Ca2+ and Na+ overload in response to disturbance of redox state in cell death.

By genetically targeting tumorigenesis to specific hypothalamic neurons in transgenic mice using the promoter region of the gonadotropin-releasing hormone (GnRH) gene to express the SV40 T-antigen oncogene, we have produced neuronal tumors and developed clonal, differentiated, neurosecretory cell lines. These cells extend neurites, express the endogenous mouse GnRH mRNA, release GnRH in response to depolarization, have regulatable fast Na+ channels found in neurons, and express neuronal, but not glial, cell markers. These immortalized cells will provide an invaluable model system for study of hypothalamic neurosecretory neurons that regulate reproduction. Significantly, their derivation demonstrates the feasibility of immortalizing differentiated neurons by targeting tumorigenesis in transgenic mice to specific neurons of the CNS.

NHE3 is one of five plasma membrane Na+/H+ exchangers and is encoded by the mouse gene Slc9a3. It is expressed on apical membranes of renal proximal tubule and intestinal epithelial cells and is thought to play a major role in NaCl and HCO3- absorption. As the distribution of NHE3 overlaps with that of the NHE2 isoform in kidney and intestine, the function and relative importance of NHE3 in vivo is unclear. To analyse its physiological functions, we generated mice lacking NHE3 function. Homozygous mutant (Slc9a3-/-) mice survive, but they have slight diarrhoea and blood analysis revealed that they are mildly acidotic. HCO3- and fluid absorption are sharply reduced in proximal convoluted tubules, blood pressure is reduced and there is a severe absorptive defect in the intestine. Thus, compensatory mechanisms must limit gross perturbations of electrolyte and acid-base balance. Plasma aldosterone is increased in NHE3-deficient mice, and expression of both renin and the AE1 (Slc4a1) Cl-/HCO3- exchanger mRNAs are induced in kidney. In the colon, epithelial Na+ channel activity is increased and colonic H+,K+-ATPase mRNA is massively induced. These data show that NHE3 is the major absorptive Na+/H+ exchanger in kidney and intestine, and that lack of the exchanger impairs acid-base balance and Na+-fluid volume homeostasis.

Cellular electrophysiology experiments, important for understanding cardiac arrhythmia mechanisms, are usually performed with channels expressed in non myocytes, or with non-human myocytes. Differences between cell types and species affect results. Thus, an accurate model for the undiseased human ventricular action potential (AP) which reproduces a broad range of physiological behaviors is needed. Such a model requires extensive experimental data, but essential elements have been unavailable. Here, we develop a human ventricular AP model using new undiseased human ventricular data: Ca(2+) versus voltage dependent inactivation of L-type Ca(2+) current (I(CaL)); kinetics for the transient outward, rapid delayed rectifier (I(Kr)), Na(+)/Ca(2+) exchange (I(NaCa)), and inward rectifier currents; AP recordings at all physiological cycle lengths; and rate dependence and restitution of AP duration (APD) with and without a variety of specific channel blockers. Simulated APs reproduced the experimental AP morphology, APD rate dependence, and restitution. Using undiseased human mRNA and protein data, models for different transmural cell types were developed. Experiments for rate dependence of Ca(2+) (including peak and decay) and intracellular sodium ([Na(+)](i)) in undiseased human myocytes were quantitatively reproduced by the model. Early afterdepolarizations were induced by I(Kr) block during slow pacing, and AP and Ca(2+) alternans appeared at rates >200 bpm, as observed in the nonfailing human ventricle. Ca(2+)/calmodulin-dependent protein kinase II (CaMK) modulated rate dependence of Ca(2+) cycling. I(NaCa) linked Ca(2+) alternation to AP alternans. CaMK suppression or SERCA upregulation eliminated alternans. Steady state APD rate dependence was caused primarily by changes in [Na(+)](i), via its modulation of the electrogenic Na(+)/K(+) ATPase current. At fast pacing rates, late Na(+) current and I(CaL) were also contributors. APD shortening during restitution was primarily dependent on reduced late Na(+) and I(CaL) currents due to inactivation at short diastolic intervals, with additional contribution from elevated I(Kr) due to incomplete deactivation.

NADH:quinone oxidoreductase (complex I) pumps protons across the inner membrane of mitochondria or the plasma membrane of many bacteria. Human complex I is involved in numerous pathological conditions and degenerative processes. With 14 central and up to 32 accessory subunits, complex I is among the largest membrane-bound protein assemblies. The peripheral arm of the L-shaped molecule contains flavine mononucleotide and eight or nine iron-sulfur clusters as redox prosthetic groups. Seven of the iron-sulfur clusters form a linear electron transfer chain between flavine and quinone. In most organisms, the seven most hydrophobic subunits forming the core of the membrane arm are encoded by the mitochondrial genome. Most central subunits have evolved from subunits of different hydrogenases and bacterial Na+/H+ antiporters. This evolutionary origin is reflected in three functional modules of complex I. The coupling mechanism of complex I most likely involves semiquinone intermediates that drive proton pumping through redox-linked conformational changes.

Endocrine tumors such as aldosterone-producing adrenal adenomas (APAs), a cause of severe hypertension, feature constitutive hormone production and unrestrained cell proliferation; the mechanisms linking these events are unknown. We identify two recurrent somatic mutations in and near the selectivity filter of the potassium (K(+)) channel KCNJ5 that are present in 8 of 22 human APAs studied. Both produce increased sodium (Na(+)) conductance and cell depolarization, which in adrenal glomerulosa cells produces calcium (Ca(2+)) entry, the signal for aldosterone production and cell proliferation. Similarly, we identify an inherited KCNJ5 mutation that produces increased Na(+) conductance in a Mendelian form of severe aldosteronism and massive bilateral adrenal hyperplasia. These findings explain pathogenesis in a subset of patients with severe hypertension and implicate loss of K(+) channel selectivity in constitutive cell proliferation and hormone production.

Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG). The formation and stabilization of DNA G-quadruplexes in the human telomeric sequence have been shown to inhibit the activity of telomerase, thus the telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. However, knowledge of the intact human telomeric G-quadruplex structure(s) formed under physiological conditions is a prerequisite for structure-based rational drug design. Here we report the folding structure of the human telomeric sequence in K+ solution determined by NMR. Our results demonstrate a novel, unprecedented intramolecular G-quadruplex folding topology with hybrid-type mixed parallel/antiparallel G-strands. This telomeric G-quadruplex structure contains three G-tetrads with mixed G-arrangements, which are connected consecutively with a double-chain-reversal side loop and two lateral loops, each consisting of three nucleotides TTA. This intramolecular hybrid-type telomeric G-quadruplex structure formed in K+ solution is distinct from those reported on the 22 nt Tel22 in Na+ solution and in crystalline state in the presence of K+, and appears to be the predominant conformation for the extended 26 nt telomeric sequence Tel26 in the presence of K+, regardless of the presence or absence of Na+. Furthermore, the addition of K+ readily converts the Na+-form conformation to the K+-form hybrid-type G-quadruplex. Our results explain all the reported experimental data on the human telomeric G-quadruplexes formed in the presence of K+, and provide important insights for understanding the polymorphism and interconversion of various G-quadruplex structures formed within the human telomeric sequence, as well as the effects of sequence and cations. This hybrid-type G-quadruplex topology suggests a straightforward pathway for the secondary structure formation with effective packing within the extended human telomeric DNA. The hybrid-type telomeric G-quadruplex is most likely to be of pharmacological relevance, and the distinct folding topology of this G-quadruplex suggests that it can be specifically targeted by G-quadruplex interactive small molecule drugs.