Étude fonctionnelle du cotransporteur Na+/glucose (hSGLT1) : courant de fuite, vitesse de cotransport et modélisation cinétique

Les résultats présentés dans cette thèse précisent certains aspects de la fonction du cotransporteur Na+/glucose (SGLT1), une protéine transmembranaire qui utilise le gradient électrochimique favorable des ions Na+ afin d’accumuler le glucose à l’intérieur des cellules épithéliales de l’intestin grêle et du rein. Nous avons tout d’abord utilisé l’électrophysiologie à deux microélectrodes sur des ovocytes de xénope afin d’identifier les ions qui constituaient le courant de fuite de SGLT1, un courant mesuré en absence de glucose qui est découplé de la stoechiométrie stricte de 2 Na+/1 glucose caractérisant le cotransport. Nos résultats ont démontré que des cations comme le Li+, le K+ et le Cs+, qui n’interagissent que faiblement avec les sites de liaison de SGLT1 et ne permettent pas les conformations engendrées par la liaison du Na+, pouvaient néanmoins générer un courant de fuite d’amplitude comparable à celui mesuré en présence de Na+. Ceci suggère que le courant de fuite traverse SGLT1 en utilisant une voie de perméation différente de celle définie par les changements de conformation propres au cotransport Na+/glucose, possiblement similaire à celle empruntée par la perméabilité à l’eau passive. Dans un deuxième temps, nous avons cherché à estimer la vitesse des cycles de cotransport de SGLT1 à l’aide de la technique de la trappe ionique, selon laquelle le large bout d’une électrode sélective (~100 μm) est pressé contre la membrane plasmique d’un ovocyte et circonscrit ainsi un petit volume de solution extracellulaire que l’on nomme la trappe. Les variations de concentration ionique se produisant dans la trappe en conséquence de l’activité de SGLT1 nous ont permis de déduire que le cotransport Na+/glucose s’effectuait à un rythme d’environ 13 s-1 lorsque le potentiel membranaire était fixé à -155 mV. Suite à cela, nous nous sommes intéressés au développement d’un modèle cinétique de SGLT1. En se servant de l’algorithme du recuit simulé, nous avons construit un schéma cinétique à 7 états reproduisant de façon précise les courants du cotransporteur en fonction du Na+ et du glucose extracellulaire. Notre modèle prédit qu’en présence d’une concentration saturante de glucose, la réorientation dans la membrane de SGLT1 suivant le relâchement intracellulaire de ses substrats est l’étape qui limite la vitesse de cotransport.

Tamizaje neonatal para fibrosis quística en una muestra de la ciudad de Bogotá

La Fibrosis Quística es la enfermedad autosómica recesiva mas frecuente en caucásicos. En Colombia no se conoce la incidencia de la enfermedad, pero investigaciones del grupo de la Universidad del Rosario indican que podría ser relativamente alta. Objetivo: Determinar la incidencia de afectados por Fibrosis Quística en una muestra de recién nacidos de la ciudad de Bogotá. Metodología: Se analizan 8.297 muestras de sangre de cordón umbilical y se comparan tres protocolos de tamizaje neonatal: TIR/TIR, TIR/DNA y TIR/DNA/TIR. Resultados: El presente trabajo muestra una incidencia de 1 en 8.297 afectados en la muestra analizada. Conclusiones: Dada la relativamente alta incidencia demostrada en Bogotá, se justifica la implementación de Tamizaje Neonatal para Fibrosis Quística en Colombia.

Functional analysis of a novel missense NBC1 mutation and of other mutations causing proximal renal tubular acidosis

Mutations in the Na+-HCO3- cotransporter NBC1 cause severe proximal tubular acidosis (pRTA) associated with ocular abnormalities. Recent studies have suggested that at least some NBC1 mutants show abnormal trafficking in the polarized cells. This study identified a new homozygous NBC1 mutation (G486R) in a patient with severe pRTA. Functional analysis in Xenopus oocytes failed to detect the G486R activity due to poor surface expression. In ECV304 cells, however, G486R showed the efficient membrane expression, and its transport activity corresponded to approximately 50% of wild-type (WT) activity. In Madin-Darby canine kidney (MDCK) cells, G486R was predominantly expressed in the basolateral membrane domain as observed for WT. Among the previously identified NBC1 mutants that showed poor surface expression in oocytes, T485S showed the predominant basolateral expression in MDCK cells. On the other hand, L522P was exclusively retained in the cytoplasm in ECV304 and MDCK cells, and functional analysis in ECV304 cells failed to detect its transport activity. These results indicate that G486R, like T485S, is a partial loss of function mutation without major trafficking abnormalities, while L522P causes the clinical phenotypes mainly through its inability to reach the plasma membranes. Multiple experimental approaches would be required to elucidate potential disease mechanism by NBC1 mutations.

Cloning and characterization of a zebrafish homologue of human AQP1: a bifunctional water and gas channel

Chen LM, Zhao J, Musa-Aziz R, Pelletier MF, Drummond IA, Boron WF. Cloning and characterization of a zebrafish homologue of human AQP1: a bifunctional water and gas channel. Am J Physiol Regul Integr Comp Physiol 299: R1163-R1174, 2010. First published August 25, 2010; doi:10.1152/ajpregu.00319.2010.-The mammalian aquaporins AQP1, AQP4, and AQP5 have been shown to function not only as water channels but also as gas channels. Zebrafish have two genes encoding an AQP1 homologue, aqp1a and aqp1b. In the present study, we cloned the cDNA that encodes the zebrafish protein Aqp1a from the 72-h postfertilization (hpf) embryo of Danio rerio, as well as from the swim bladder of the adult. The deduced amino-acid sequence of aqp1a consists of 260 amino acids and is 59% identical to human AQP1. By analyzing the genomic DNA sequence, we identified four exons in the aqp1a gene. By in situ hybridization, aqp1a is expressed transiently in the developing vasculature and in erythrocytes from 16 to 48 h of development. Later, at 72 hpf, aqp1a is expressed in dermal ionocytes and in the swim bladder. Western blot analysis of adult tissues reveals that Aqp1a is most highly expressed in the eye and swim bladder. Xenopus oocytes expressing aqp1a have a channel-dependent (*) osmotic water permeability (P(f)*) that is indistinguishable from that of human AQP1. On the basis of the magnitude of the transient change in surface pH (Delta pHS) that were recorded as the oocytes were exposed to either CO(2) or NH(3), we conclude that zebrafish Aqp1a is permeable to both CO(2) and NH(3). The ratio (Delta pHS*)CO2/P(f)* is about half that of human AQP1, and the ratio (Delta pHS*)NH3/P(f)* is about one-quarter that of human AQP1. Thus, compared with human AQP1, zebrafish Aqp1a has about twice the selectivity for CO(2) over NH(3).

Relative CO(2)/NH(3) selectivities of mammalian aquaporins 0-9

Previous work showed that aquaporin 1 (AQP1), AQP4-M23, and AQP5 each has a characteristic CO(2)/NH(3) and CO(2)/H(2)O permeability ratio. The goal of the present study is to characterize AQPs 0-9, which traffic to the plasma membrane when heterologously expressed in Xenopus oocytes. We use video microscopy to compute osmotic water permeability (P(f)) and microelectrodes to record transient changes in surface pH (ΔpH(S)) caused by CO(2) or NH(3) influx. Subtracting respective values for day-matched, H(2)O-injected control oocytes yields the channel-specific values P(f)* and ΔpH(S)*. We find that P(f)* is significantly >0 for all AQPs tested except AQP6. (ΔpH(S)*)(CO(2)) is significantly >0 for AQP0, AQP1, AQP4-M23, AQP5, AQP6, and AQP9. (ΔpH(S)*)(NH(3)) is >0 for AQP1, AQP3, AQP6, AQP7, AQP8, and AQP9. The ratio (ΔpH(S)*)(CO(2))/P(f)* falls in the sequence AQP6 (∞) > AQP5 > AQP4-M23 > AQP0 ≅ AQP1 ≅ AQP9 > others (0). The ratio (ΔpH(S)*)(NH(3))/P(f)* falls in the sequence AQP6 (∞) > AQP3 ≅ AQP7 ≅ AQP8 ≅ AQP9 > AQP1 > others (0). Finally, the ratio (ΔpH(S)*)(CO(2))/(-ΔpH(S)*)(NH(3)) falls in the sequence AQP0 (∞) ≅ AQP4-M23 ≅ AQP5 > AQP6 > AQP1 > AQP9 > AQP3 (0) ≅ AQP7 ≅ AQP8. The ratio (ΔpH(S)*)(CO(2))/(-ΔpH(S)*)(NH(3)) is indeterminate for both AQP2 and AQP4-M1. In summary, we find that mammalian AQPs exhibit a diverse range of selectivities for CO(2) vs. NH(3) vs. H(2)O. As a consequence, by expressing specific combinations of AQPs, cells could exert considerable control over the movements of each of these three substances

Relative CO2/NH3 permeabilities of human RhAG, RhBG and RhCG

Mammalian glycosylated rhesus (Rh) proteins include the erythroid RhAG and the nonerythroid RhBG and RhCG. RhBG and RhCG are expressed in multiple tissues, including hepatocytes and the collecting duct (CD) of the kidney. Here, we expressed human RhAG, RhBG and RhCG in Xenopus oocytes (vs. H2O-injected control oocytes) and used microelectrodes to monitor the maximum transient change in surface pH (ΔpHS) caused by exposing the same oocyte to 5 % CO2/33 mM HCO3 − (an increase) or 0.5 mM NH3/NH4 + (a decrease). Subtracting the respective values for day-matched, H2O-injected control oocytes yielded channel-specific values (*). (ΔpH∗S)CO2 and (−ΔpH∗S)NH3 were each significantly >0 for all channels, indicating that RhBG and RhCG—like RhAG—can carry CO2 and NH3. We also investigated the role of a conserved aspartate residue, which was reported to inhibit NH3 transport. However, surface biotinylation experiments indicate the mutants RhBGD178N and RhCGD177N have at most a very low abundance in the oocyte plasma membrane. We demonstrate for the first time that RhBG and RhCG—like RhAG—have significant CO2 permeability, and we confirm that RhAG, RhBG and RhCG all have significant NH3 permeability. However, as evidenced by (ΔpH∗S)CO2/(−ΔpH∗S)NH3 values, we could not distinguish among the CO2/NH3 permeability ratios for RhAG, RhBG and RhCG. Finally, we propose a mechanism whereby RhBG and RhCG contribute to acid secretion in the CD by enhancing the transport of not only NH3 but also CO2 across the membranes of CD cells.

Movement of NH3 through the human urea transporter B: a new gas channel

Aquaporins and Rh proteins can function as gas (CO2 and NH3) channels. The present study explores the urea, H2O, CO2, and NH3 permeability of the human urea transporter B (UT-B) (SLC14A1), expressed in Xenopus oocytes. We monitored urea uptake using [14C]urea and measured osmotic water permeability (Pf) using video microscopy. To obtain a semiquantitative measure of gas permeability, we used microelectrodes to record the maximum transient change in surface pH (∆pHS) caused by exposing oocytes to 5% CO2/33 mM HCO3- (pHS increase) or 0.5 mM NH3/NH4+ (pHS decrease). UT-B expression increased oocyte permeability to urea by >20-fold, and Pf by 8-fold vs. H2O-injected control oocytes. UT-B expression had no effect on the CO2-induced ∆pHS but doubled the NH3-induced ∆pHS. Phloretin reduced UT-B-dependent urea uptake (Jurea * ) by 45%, Pf * by 50%, and (- ∆pHS * )NH3 by 70%. p-Chloromercuribenzene sulfonate reduced Jurea * by 25%, Pf * by 30%, and (∆pHS * )NH3 by 100%. Molecular dynamics (MD) simulations of membrane-embedded models of UT-B identified the monomeric UT-B pores as the main conduction pathway for both H2O and NH3 and characterized the energetics associated with permeation of these species through the channel. Mutating each of two conserved threonines lining the monomeric urea pores reduced H2O and NH3 permeability. Our data confirm that UT-B has significant H2O permeability and for the first time demonstrate significant NH3 permeability. Thus the UTs become the third family of gas channels. Inhibitor and mutagenesis studies and results of MD simulations suggest that NH3 and H2O pass through the three monomeric urea channels in UT-B.

Diversity of structure and function of alpha1alpha6beta3delta GABAA receptors: comparison with alpha1beta3delta and alpha6beta3delta receptors

delta subunit-containing gamma-aminobutyric acid, type A (GABA(A))receptors are expressed extrasynaptically and mediate tonic inhibition. In cerebellar granule cells, they often form receptors together with alpha(1) and/or alpha(6) subunits. We were interested in determining the architecture of receptors containing both subunits. We predefined the subunit arrangement of several different GABA(A) receptor pentamers by concatenation. These receptors composed of alpha(1), alpha(6), beta(3), and delta subunits were expressed in Xenopus oocytes. Currents elicited in response to GABA were determined in the presence and absence of 3alpha,21-dihydroxy-5alpha-pregnan-20-one (THDOC) or ethanol, or currents were elicited by 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP). Several subunit configurations formed active channels. We therefore conclude that delta can assume multiple positions in a receptor pentamer made up of alpha(1), alpha(6), beta(3), and delta subunits. The different receptors differ in their functional properties. Functional expression of one receptor type was only evident in the combined presence of the neurosteroid THDOC with the channel agonist GABA. Most, but not all, receptors active with GABA/THDOC responded to THIP. None of the receptors was modulated by ethanol concentrations up to 30 mm. Several observations point to a preferred position of delta subunits between two alpha subunits in alpha(1)alpha(6)beta(3)delta receptors. This property is shared by alpha(1)beta(3)delta and alpha(6)beta(3)delta receptors, but there are differences in the additionally expressed isoforms.

A venom-derived neurotoxin, CsTx-1, from the spider Cupiennius salei exhibits cytolytic activities

CsTx-1, the main neurotoxic acting peptide in the venom of the spider Cupiennius salei, is composed of 74 amino acid residues, exhibits an inhibitory cysteine knot motif, and is further characterized by its highly cationic charged C terminus. Venom gland cDNA library analysis predicted a prepropeptide structure for CsTx-1 precursor. In the presence of trifluoroethanol, CsTx-1 and the long C-terminal part alone (CT1-long; Gly-45-Lys-74) exhibit an α-helical structure, as determined by CD measurements. CsTx-1 and CT1-long are insecticidal toward Drosophila flies and destroys Escherichia coli SBS 363 cells. CsTx-1 causes a stable and irreversible depolarization of insect larvae muscle cells and frog neuromuscular preparations, which seem to be receptor-independent. Furthermore, this membranolytic activity could be measured for Xenopus oocytes, in which CsTx-1 and CT1-long increase ion permeability non-specifically. These results support our assumption that the membranolytic activities of CsTx-1 are caused by its C-terminal tail, CT1-long. Together, CsTx-1 exhibits two different functions; as a neurotoxin it inhibits L-type Ca(2+) channels, and as a membranolytic peptide it destroys a variety of prokaryotic and eukaryotic cell membranes. Such a dualism is discussed as an important new mechanism for the evolution of spider venomous peptides.

A GABA(A) receptor of defined subunit composition and positioning: concatenation of five subunits

We show that the five subunits of a gamma-aminobutyric acid type A receptor (GABA(A) receptor) can be concatenated to yield a functional receptor. This concatenated receptor alpha(1)-beta(2)-alpha(1)-gamma(2)-beta(2) has the advantage of a known subunit arrangement. Most of its functional properties are not significantly different from a receptor formed by individual subunits. Extent of expression amounted to about 40% of that of non-concatenated receptors in Xenopus oocytes, after injection of oocytes with comparable amounts of cRNA coding for concatenated and non-concatenated receptors. The ability to express receptors consisting of five subunits enables detailed studies of GABA(A) receptor subtype selective compounds.

Divalent metal-ion transporter DMT1 mediates both H+ -coupled Fe2+ transport and uncoupled fluxes

The H(+) -coupled divalent metal-ion transporter DMT1 serves as both the primary entry point for iron into the body (intestinal brush-border uptake) and the route by which transferrin-associated iron is mobilized from endosomes to cytosol in erythroid precursors and other cells. Elucidating the molecular mechanisms of DMT1 will therefore increase our understanding of iron metabolism and the etiology of iron overload disorders. We expressed wild type and mutant DMT1 in Xenopus oocytes and monitored metal-ion uptake, currents and intracellular pH. DMT1 was activated in the presence of an inwardly directed H(+) electrochemical gradient. At low extracellular pH (pH(o)), H(+) binding preceded binding of Fe(2+) and its simultaneous translocation. However, DMT1 did not behave like a typical ion-coupled transporter at higher pH(o), and at pH(o) 7.4 we observed Fe(2+) transport that was not associated with H(+) influx. His(272) --> Ala substitution uncoupled the Fe(2+) and H(+) fluxes. At low pH(o), H272A mediated H(+) uniport that was inhibited by Fe(2+). Meanwhile H272A-mediated Fe(2+) transport was independent of pH(o). Our data indicate (i) that H(+) coupling in DMT1 serves to increase affinity for Fe(2+) and provide a thermodynamic driving force for Fe(2+) transport and (ii) that His-272 is critical in transducing the effects of H(+) coupling. Notably, our data also indicate that DMT1 can mediate facilitative Fe(2+) transport in the absence of a H(+) gradient. Since plasma membrane expression of DMT1 is upregulated in liver of hemochromatosis patients, this H(+) -uncoupled facilitative Fe(2+) transport via DMT1 can account for the uptake of nontransferrin-bound plasma iron characteristic of iron overload disorders.

Late-onset manifestation of antenatal Bartter syndrome as a result of residual function of the mutated renal Na+-K+-2Cl- co-transporter

Genetic defects of the Na+-K+-2Cl- (NKCC2) sodium potassium chloride co-transporter result in severe, prenatal-onset renal salt wasting accompanied by polyhydramnios, prematurity, and life-threatening hypovolemia of the neonate (antenatal Bartter syndrome or hyperprostaglandin E syndrome). Herein are described two brothers who presented with hyperuricemia, mild metabolic alkalosis, low serum potassium levels, and bilateral medullary nephrocalcinosis at the ages of 13 and 15 yr. Impaired function of sodium chloride reabsorption along the thick ascending limb of Henle's loop was deduced from a reduced increase in diuresis and urinary chloride excretion upon application of furosemide. Molecular genetic analysis revealed that the brothers were compound heterozygotes for mutations in the SLC12A1 gene coding for the NKCC2 co-transporter. Functional analysis of the mutated rat NKCC2 protein by tracer-flux assays after heterologous expression in Xenopus oocytes revealed significant residual transport activity of the NKCC2 p.F177Y mutant construct in contrast to no activity of the NKCC2-D918fs frameshift mutant construct. However, coexpression of the two mutants was not significantly different from that of NKCC2-F177Y alone or wild type. Membrane expression of NKCC2-F177Y as determined by luminometric surface quantification was not significantly different from wild-type protein, pointing to an intrinsic partial transport defect caused by the p.F177Y mutation. The partial function of NKCC2-F177Y, which is not negatively affected by NKCC2-D918fs, therefore explains a mild and late-onset phenotype and for the first time establishes a mild phenotype-associated SLC12A1 gene mutation.

Functional properties of multiple isoforms of human divalent metal-ion transporter 1 (DMT1)

DMT1 (divalent metal-ion transporter 1) is a widely expressed metal-ion transporter that is vital for intestinal iron absorption and iron utilization by most cell types throughout the body, including erythroid precursors. Mutations in DMT1 cause severe microcytic anaemia in animal models. Four DMT1 isoforms that differ in their N- and C-termini arise from mRNA transcripts that vary both at their 5'-ends (starting in exon 1A or exon 1B) and at their 3'-ends giving rise to mRNAs containing (+) or lacking (-) the 3'-IRE (iron-responsive element) and resulting in altered C-terminal coding sequences. To determine whether these variations result in functional differences between isoforms, we explored the functional properties of each isoform using the voltage clamp and radiotracer assays in cRNA-injected Xenopus oocytes. 1A/IRE+-DMT1 mediated Fe2+-evoked currents that were saturable (K(0.5)(Fe) approximately 1-2 microM), temperature-dependent (Q10 approximately 2), H+-dependent (K(0.5)(H) approximately 1 muM) and voltage-dependent. 1A/IRE+-DMT1 exhibited the provisional substrate profile (ranked on currents) Cd2+, Co2+, Fe2+, Mn2+>Ni2+, V3+>>Pb2+. Zn2+ also evoked large currents; however, the zinc-evoked current was accounted for by H+ and Cl- conductances and was not associated with significant Zn2+ transport. 1B/IRE+-DMT1 exhibited the same substrate profile, Fe2+ affinity and dependence on the H+ electrochemical gradient. Each isoform mediated 55Fe2+ uptake and Fe2+-evoked currents at low extracellular pH. Whereas iron transport activity varied markedly between the four isoforms, the activity for each correlated with the density of anti-DMT1 immunostaining in the plasma membrane, and the turnover rate of the Fe2+ transport cycle did not differ between isoforms. Therefore all four isoforms of human DMT1 function as metal-ion transporters of equivalent efficiency. Our results reveal that the N- and C-terminal sequence variations among the DMT1 isoforms do not alter DMT1 functional properties. We therefore propose that these variations serve as tissue-specific signals or cues to direct DMT1 to the appropriate subcellular compartments (e.g. in erythroid cells) or the plasma membrane (e.g. in intestine).

Novel chloride channel mutations leading to mild myotonia among Chinese

We describe two Chinese families with a mild form of the myotonia congenita due to novel chloride channel (ClCN1) mutations. In one case, heterozygous I553F and H555N mutations were found. The patient shared the I553F mutation with his healthy father, and his mother had a history of mild myotonia when she was younger. In another family, autosomal dominant myotonia congenita was due to a L844F change. The physiological effects of the mutations were examined by using the two-electrode voltage-clamp technique after expression of the channels in Xenopus oocytes. All mutations drastically shifted the voltage required for half-maximal activation, more under conditions mimicking the homozygous situation, than under conditions mimicking the heterozygous situation. The larger effect was seen in the compound heterozygous situation combining the I553F and the H555N mutations. Our data suggest that myotonia congenita caused by CLCN1 mutations in Chinese have similar variable features to those found in the West.

Gain-of-function haplotype in the epithelial calcium channel TRPV6 is a risk factor for renal calcium stone formation

The rate-limiting step of dietary calcium absorption in the intestine requires the brush border calcium entry channel TRPV6. The TRPV6 gene was completely sequenced in 170 renal calcium stone patients. The frequency of an ancestral TRPV6 haplotype consisting of three non-synonymous polymorphisms (C157R, M378V, M681T) was significantly higher (P = 0.039) in calcium stone formers (8.4%; derived = 502, ancestral = 46) compared to non-stone-forming individuals (5.4%; derived = 645, ancestral = 37). Mineral metabolism was investigated on four different calcium regimens: (i) free-choice diet, (ii) low calcium diet, (iii) fasting and (iv) after a 1 g oral calcium load. When patients homozygous for the derived haplotype were compared with heterozygous patients, no differences were found with respect to the plasma concentrations of 1,25-vitamin D, PTH and calcium, and the urinary excretion of calcium. In one stone-forming patient, the ancestral haplotype was found to be homozygous. This patient had absorptive hypercalciuria. We therefore expressed the ancestral protein (157R+378V+681T) in Xenopus oocytes and found a significantly enhanced calcium permeability when tested by a (45)Ca(2+) uptake assay (7.11 +/- 1.93 versus 3.61 +/- 1.01 pmol/min/oocyte for ancestral versus derived haplotype, P < 0.01). These results suggest that the ancestral gain-of-function haplotype in TRPV6 plays a role in calcium stone formation in certain forms of absorptive hypercalciuria.