Carsten A. Wagner

Mutations in GJB6 cause nonsyndromic autosomal dominant deafness at DFNA3 locus.

factors1. Mutations in the connexin26 gene (GJB2), located on 13q12, are responsible for non-syndromic recessive and dominant forms of deafness2–4. Connexin-31 and connexin-32 have also been implicated in deafness5,6. The identification of deaf families linked to 13q12 but negative for mutations in GJB2 (ref. 7) suggested the presence of other deafness genes in this region. Recently, the mouse connexin-30 gene (Gjb6), which is expressed in cochlea, has been mapped to a region with syntenic homology to human chromosome 13q12 (refs 8,9). To verify if human GJB6 is involved in deafness, we cloned a 1,799-bp cDNA fragment containing an ORF of 261 amino acids (EMBL HSA005585). CX30 protein has a structure similar to that of other connexins10 and shares 93% homology with mouse Cx30 and 76% identity with human CX26. GJB6 is not interrupted by introns and maps to chromosome 13q12, approximately 800 kb centromeric to GJB2. SSCP mutational analysis in 198 deaf patients, including 38 families linked to 13q12, revealed a threonine-to-methionine change at position 5 (T5M) in an Italian family affected by bilateral middle/ high-frequency hearing loss (Fig. 1a–c). Audiograms in T5M family members showed a 20–50-dB decrease at frequencies of 2,000–8,000 Hz (I-2), a progressive impaired threshold above 500 Hz (II-1) and a profound sensorineural deafness (II-2). This variability of hearing impairment can be explained by a different expressivity of the disease, which is almost the rule for dominant deafness. Northern blots, RT-PCR and in situ hybridization on mouse embryos revealed Gjb6 expression in trachea, thyroid, thymus, brain and cochlea, confirming reported expression patterns (refs 8,9,11). The threonine residue at position 5 is evolutionarily conserved and also present in human connexin 26 (Fig. 1d). The T5M substitution abolishes a hydrophilic residue possibly involved in interor Mutations in GJB6 cause nonsyndromic autosomal dominant deafness at DFNA3 locus correspondence

Obligatory Amino Acid Exchange via Systems bo,+-like and y+L-like A TERTIARY ACTIVE TRANSPORT MECHANISM FOR RENAL REABSORPTION OF CYSTINE AND DIBASIC AMINO ACIDS

Mutations in the rBAT gene cause type I cystinuria, a common inherited aminoaciduria of cystine and dibasic amino acids due to their defective renal and intestinal reabsorption (Calonge, M. J., Gasparini, P., Chillarón, J., Chillón, M., Gallucci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barceló, P., Estivill, X., Zorzano, A., Nunes, V., and Palacín, M. (1994) Nat. Genet. 6, 420-426; Calonge, M. J., Volipini, V., Bisceglia, L., Rousaud, F., De Sanctis, L., Beccia, E., Zelante, L., Testar, X., Zorzano, A., Estivill, X., Gasparini, P., Nunes, V., and Palacín, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9667-9671). One important question that remains to be clarified is how the apparently non-concentrative system bo,+-like, associated with rBAT expression, participates in the active renal reabsorption of these amino acids. Several studies have demonstrated exchange of amino acids induced by rBAT in Xenopus oocytes. Here we offer evidence that system bo,+-like is an obligatory amino acid exchanger in oocytes and in the “renal proximal tubular” cell line OK. System bo,+-like showed a 1:1 stoichiometry of exchange, and the hetero-exchange dibasic (inward) with neutral (outward) amino acids were favored in oocytes. Obligatory exchange of amino acids via system bo,+-like fully explained the amino acid-induced current in rBAT-injected oocytes. Exchange via system bo,+-like is coupled enough to ensure a specific accumulation of substrates until the complete replacement of the internal oocyte substrates. Due to structural and functional analogies of the cell surface antigen 4F2hc to rBAT, we tested for amino acid exchange via system y+L-like. 4F2hc-injected oocytes accumulated substrates to a level higher than CAT1-injected oocytes (i.e. oocytes expressing system y+) and showed exchange of amino acids with the substrate specificity of system y+L and L-leucine-induced outward currents in the absence of extracellular sodium. In contrast to L-arginine, system y+L-like did not mediate measurable L-leucine efflux from the oocyte. We propose a role of systems bo,+-like and y+L-like in the renal reabsorption of cystine and dibasic amino acids that is based on their active tertiary transport mechanism and on the apical and basolateral localization of rBAT and 4F2hc, respectively, in the epithelial cells of the proximal tubule of the nephron.

Effects of the Serine/Threonine Kinase SGK1 on the Epithelial Na+ Channel (ENaC) and CFTR: Implications for Cystic Fibrosis

Cystic fibrosis (CF) is characterized by impaired Cl- secretion and increased Na+ reabsorption in several tissues including respiratory epithelium. Many CFTR mutations have been identified over the past years. However, only a poor correlation between the genotype and lung phenotype was found suggesting additional factors influencing the phenotype and course of the disease. The serine/threonine kinase SGK1 has recently been shown to stimulate the activity of the epithelial Na+ channel ENaC. A variety of stimuli such as aldosterone, cell shrinkage, insulin or TGF-β1 stimulate transcription and activate the SGK1 kinase. Here we further examined the effects of SGK1 on ENaC and CFTR which have mutual interactions and we analyzed sgk1 mRNA abundance in lung tissue from CF patients. Coexpression of CFTR and h-SGK1 in Xenopus oocytes increased ENaC currents as previously described. In addition CFTR mediated currents were also stimulated. h-SGK1 accelerated the expression of the amiloride sensitive Na+- current in Xenopus oocytes paralleled by increased ENaC-protein abundance in the oocyte membrane, an effect which was reversed by a h-SGK1K127R mutation lacking the ATP-binding site. The cation selectivity or Na+ affinity were not affected. However, coexpression of h-SGK1 with ENaC altered the sensitivity of the Na+-channel to the inhibitors amiloride and triamterene. The inhibitory effect of CFTR expression on ENaC current was not affected by coexpression of h-SGK1 in Xenopus oocytes. Lung tissue from CF patients strongly expressed the serine/threonine kinase h-sgk1 which was not the case for non-CF lung tissue. Loss of CFTR function itself in a CF lung epithelial cell line did not increase SGK1 expression. In summary, enhanced expression of h-SGK1 in epithelial cells of CF-lung tissue may be a novel pathophysiological factor contributing to increased Na+ channel activity and thus to increased Na+ transport in CF. .

Inhibition of Jurkat-T-lymphocyte Na+/H+-exchanger by CD95(Fas/Apo-1)-receptor stimulation.

Abstract. Mitogenic factors are known to stimulate the Na+/H+-exchanger (NHE), leading to cytosolic alkalinization and/or cell swelling. Conversely, a hallmark of apoptosis is cell shrinkage and CD95-induced apoptosis has been reported to be paralleled by cytosolic acidification. To assess whether the CD95-receptor regulates NHE activity in Jurkat T-lymphocytes, we performed conventional BCECF fluorescence measurements and SNARF flow cytometric analysis (FACS). The recoveries from acidifications following application of butyrate or a NH3 pulse were both abolished by a specific NHE-inhibitor, HOE694, indicating that they fully depend on NHE activity. Thus they were taken as a measure of NHE activity. CD95-receptor stimulation caused a cytosolic acidification and blunted the recovery from acidification following application of butyrate or a NH3 pulse. Moreover, the NHE-dependent alkalinization following osmotic cell shrinkage was almost abolished by CD95-receptor stimulation. As apparent from the effect of osmotic cell shrinkage, inhibition of the NHE by CD95-receptor stimulation was absent in Lck56-deficient J-CaM1.6 cells and restored by retransfection of J-CaM1.6-cells with Lck56. CD95-receptor stimulation led within 4xa0h to a decrease of cellular ATP which could contribute to NHE inhibition. Treatment of Jurkat cells with the NHE inhibitor HOE694 accelerated CD95-induced DNA fragmentation. In conclusion, CD95-receptor stimulation inhibits NHE activity through a mechanism that depends directly or indirectly on the activation of the Src-like kinase Lck56. This effect contributes to CD95-induced cytosolic acidification, DNA fragmentation and cell shrinkage.

Cerebral localization and regulation of the cell volume-sensitive serum- and glucocorticoid-dependent kinase SGK1

The serum- and glucocorticoid-dependent kinase SGK1 is regulated by alterations of cell volume, whereby cell shrinkage increases and cell swelling decreases the transcription, expression and activity of SGK1. The kinase is expressed in all human tissues studied including the brain. The present study was performed to localize the sites of SGK1 transcription in the brain, to elucidate the influence of the hydration status on SGK1 transcription and to explore the functional significance of altered SGK1 expression. Northern blot analysis of human brain showed SGK1 to be expressed in all cerebral structures examined: amygdala, caudate nucleus, corpus callosum, hippocampus, substantia nigra, subthalamic nucleus and thalamus. In situ hybridization and immunohistochemistry in the rat revealed increased expression of SGK1 in neurons of the hippocampal area CA3 after dehydration, compared with similar slices from brains of euvolaemic rats. Additionally, several oligodendrocytes, a few microglial cells, but no astrocytes, were positive for SGK1. The abundance of SGK1 mRNA in the temporal lobe, including hippocampus, was increased by dehydration and SGK1 transcription in neuroblastoma cells was stimulated by an increase of extracellular osmolarity. Co-expression studies in Xenopus laevis oocytes revealed that SGK1 markedly increased the activity of the neuronal K+ channel Kv1.3. As activation of K+ channels modifies excitation of neuronal cells, SGK1 may participate in the regulation of neuronal excitability.

CHLORIDE CONDUCTANCE AND PI TRANSPORT ARE SEPARATE FUNCTIONS INDUCED BY THE EXPRESSION OF NAPI-1 IN XENOPUS OOCYTES

Abstract. Expression of the protein NaPi-1 in Xenopus oocytes has previously been shown to induce an outwardly rectifying Cl− conductance (GCl), organic anion transport and Na+-dependent Pi-uptake. In the present study we investigated the relation between the NaPi-1 induced GCl and Pi-induced currents and transport. NaPi-1 expression induced Pi-transport, which was not different at 1–20 ng/oocyte NaPi-1 cRNA injection and was already maximal at 1–2 days after cRNA injection. In contrast, GCl was augmented at increased amounts of cRNA injection (1–20 ng/oocyte) and over a five day expression period. Subsequently all experiments were performed on oocytes injected with 20 ng/oocytes cRNA. Pi-induced currents (Ip) could be observed in NaPi-1 expressing oocytes at high concentrations of Pi (≥ 1 mm Pi). The amplitudes of Ip correlated well with GCl. Ip was blocked by the Cl− channel blocker NPPB, partially Na+-dependent and completely abolished in Cl− free solution. In contrast, Pi-transport in NaPi-1 expressing oocytes was not NPPB sensitive, stronger depending on extracellular Na+ and weakly affected by Cl− substitution. Endogenous Pi-uptake in water-injected oocytes amounted in all experiments to 30–50% of the Na+-dependent Pi-transport observed in NaPi-1 expressing oocytes. The properties of the endogenous Pi-uptake system (Km for Pi > 1 mm; partial Na+- and Cl−-dependence; lack of NPPB block) were similar to the NaPi-1 induced Pi-uptake, but no Ip could be recorded at Pi-concentrations ≤3 mm. In summary, the present data suggest that Ip does not reflect charge transfer related to Pi-uptake, but a Pi-mediated modulation of GCl.

Transport characteristics of a murine renal Na/Pi

A complementary deoxyribonucleic acid (cDNA) corresponding to a murine renal cortical Na/phosphate-(Na/Pi-) cotransporter was isolated and its transport properties characterized by electrophysiological techniques after expression in Xenopus laevis oocytes. A Na-dependent inward movement of positive charges (“short-circuit current”) was observed upon superfusion with Pi (and with arsenate). Increasing the Na concentration led to a sigmoidal elevation in Pi-induced short-circuit current; the apparent Michaelis constant, Km, (around 40 mM Na) was increased by lowering the pH of the superfusate but was not influenced by altering the Pi concentration. Increasing the Pi (and arsenate) concentration led to a hyperbolic elevation in Na-dependent short-circuit current (apparent Km for Pi at 100 mM Na was around 0.1 mM; apparent Km for arsenate was around 1 mM); lowering the Na concentration decreased the apparent affinity for Pi. The Pi-induced short-circuit current was lower at more acidic pH values (at pH 6.3 it was about 50% of the value at pH 7.8); this pH dependence was similar if the Pi concentration was calculated in total, or if distinction was made between its mono- and divalent forms. Thus, the pH dependence of Na-dependent Pi transport (total Pi) may not be related primarily to a pH-dependent alteration in the availability of divalent Pi, but includes also a competitive interaction of Na with protons. The effect of Pi and Na concentration on the apparent Km values for Na or Pi, respectively, provides evidence for an ordered interaction of “cosubstrate” (Na first) and “substrate” (Pi or arsenate second).

Swelling-induced taurine release without chloride channel activity in Xenopus laevis oocytes expressing anion channels and transporters.

Taurine is an important osmolyte involved in cell volume regulation. During regulatory volume decrease it is released via a volume-sensitive organic osmolyte/anion channel. Several molecules have been suggested as candidates for osmolyte release. In this study, we chose three of these, namely ClC-2, ClC-3 and ICln, because of their expression in rat astrocytes, a cell type which is known to release taurine under hypotonic stress, and their activation by hypotonic shock. As all three candidates were also suggested to be chloride channels, we investigated their permeability for both chloride and taurine under isotonic and hypotonic conditions using the Xenopus laevis oocyte expression system. We found a volume-sensitive increase of chloride permeability in ClC-2-expressing oocytes only. Yet, the taurine permeability was significantly increased under hypotonic conditions in oocytes expressing any of the tested candidates. Further experiments confirmed that the detected taurine efflux does not represent unspecific leakage. These results suggest that ClC-2, ClC-3 and ICln either participate in taurine transport themselves or upregulate an endogenous oocyte osmolyte channel. In either case, the taurine efflux of oocytes not being accompanied by an increased chloride flux suggests that taurine and chloride can be released via two separate pathways.

The heterodimeric amino acid transporter 4F2hc/LAT1 is associated in Xenopus oocytes with a non-selective cation channel that is regulated by the serine/threonine kinase sgk-1.

1 System L is the major Na+‐independent amino acid transporter of mammalian cells. It is constituted of the type II membrane protein 4F2hc (CD98) which is covalently linked to the polytopic membrane protein LAT1 via a disulfide bridge. The transporter is known to be regulated by the mineralcorticoid aldosterone in Xenopus A6 cells. To understand the regulation of the transporter, the 4F2hc/LAT1 heterodimer was functionally expressed in Xenopus laevis oocytes and its transport properties were analysed using flux measurements and the two‐electrode voltage‐clamp technique. 2 Expression of 4F2hc/LAT1 resulted in a rapid increase in a Na+‐independent neutral amino acid antiport activity and simultaneously gave rise to a cation conductance. The cation channel was non‐rectifying and non‐selective, conducting Li+ > Cs+= Na+ > K+. After replacement of Na+ by NMDG, however, the currents were suppressed almost completely. The cation channel was not inhibited by amiloride, Ba2+, TEA, Hoe293B, flufenamic acid or substrates of the system L amino acid transporter. Significant inhibition, however, was observed in the presence of La3+, Gd3+ and quinidine. Channel activity was upregulated by coexpression of 4F2hc/LAT1 with the aldosterone‐regulated protein kinase sgk‐1. 3 The cation conductance was sensitive to changes in the redox potential, being inhibited following incubation of the oocytes with DTE for 30 min. Mutation of either of the disulfide bridge‐constituting cysteines to serine resulted in a loss of ion channel activity whereas amino acid transport was unaffected. 4 It is concluded that the 4F2hc/LAT1 heterodimer regulates a closely associated cation channel or even constitutes a cation channel itself.

Intracellular pH activates membrane-bound Na(+)/H(+) exchanger and vacuolar H(+)-ATPase in human embryonic kidney (HEK) cells.

Mammalian cells regulate their cytosolic pH through a variety of proton extruding and bicarbonate loading mechanisms. The human embryonic kidney cell line HEK 293 is thought to show some characteristics of proximal tubule cells. The present study was performed to investigate the activity of proton extruding mechanisms (i.e. Na⁺/H⁺ exchange and/or V-H⁺-ATPases) and the influence of intracellular pH (pH_i) on the activation of these transport processes. At resting pH_i (7.4) and in the absence of bicarbonate, removal of extracellular Na⁺ did not alter pH_i Intracellular acidification (pH_i ñ6.2) after a NH₄Cl prepulse (20 mM) followed by exposure to a Na⁺ free bath solution led to a slow pH_i recovery (with a delay of 5 min), which was inhibited by the specific vacuolar H⁺-ATPase inhibitor bafilomyocin. There was no Na⁺-dependent pH_i recovery upon exposure to Na⁺ after a short intracellular acidification (less than 5 min). However, when the intracellular acidification phase was extended, the activation of a Na⁺-dependent pH_i recovery was seen. This Na⁺-dependent pH_i recovery was inhibited by 30 µM EIPA but not by 2 µM EIPA or less, suggesting the involvement of NHE3. Western blot analysis confirmed the presence of NHE3 protein in HEK 293 cells. Disruption of the microtubular network by colchicine (20 µM) did not significantly inhibit the rate of Na⁺-independent or –dependent pH_i recovery indicating that activation of both H⁺-ATPase, and the NHE were not due to stimulated trafficking of the transport proteins or some activators to the membrane. We conclude that a) the plasma membrane of HEK293 cells contains both, NHE and H⁺-ATPase and b) both H⁺ extrusion systems are inactive at neutral pH_i, c) a decrease of cytosolic pH to 6.5 activates both transport proteins in a slow, time-dependent manner.