The anion conductance of the glutamate transporter EAAC1 depends on the direction of glutamate transport

The steady-state and pre-steady-state kinetics of glutamate transport by the neuronal glutamate transporter EAAC1 were determined under conditions of outward glutamate transport and compared to those found for the inward transport mode. In both transport modes, the glutamate-induced current is composed of two components, the coupled transport current and the uncoupled anion current, and inhibited by a specific non-transportable inhibitor. Furthermore, the glutamate-independent leak current is observed in both transport modes. Upon a glutamate concentration jump outward transport currents show a distinct transient phase that deactivates within 15 ms. The results demonstrate that the general properties of EAAC1 are symmetric, but the rates of substrate transport and anion flux are asymmetric with respect to the orientation of the substrate binding site in the membrane. Therefore, the EAAC1 anion conductance differs from normal ligand-gated ion channels in that it can be activated by glutamate and Na(+) from both sides of the membrane.     

Single channel characteristics of a high conductance anion channel in "sarcoballs"

Previously undescribed high conductance single anion channels from frog skeletal muscle sarcoplasmic reticulum (SR) were studied in native membrane using the "sarcoball" technique (Stein and Palade, 1988). Excised inside-out patches recorded in symmetrical 200 mM TrisCl show the conductance of the channel''s predominant state was 505 +/- 25 pS (n = 35). From reversal potentials, the Pcl/PK ratio was 45. The slope conductance vs. Cl- ion concentration curve saturates at 617 pS, with K0.5 estimated at 77 mM. The steady-state open probability (Po) vs. holding potential relationship produces a bell-shaped curve, with Po values reaching a maximum near 1.0 at 0 mV, and falling off to 0.05 at +/- 25 mV. Kinetic analysis of the voltage dependence reveals that while open time constants are decreased somewhat by increases in potential, the largest effect is an increase in long closed times. Despite the channel''s high conductance, it maintains a moderate selectivity for smaller anions, but will not pass larger anions such as gluconate, as determined by reversal-potential shifts. At least two substates different from the main open level are distinguishable. These properties are unlike those described for mitochondrial voltage- dependent anion channels or skeletal muscle surface membrane Cl channels and since SR Ca channels are present in equally high density in sarcoball patches, we propose these sarcoball anion channels originate from the SR. Preliminary experiments recording currents from frog SR anion channels fused into liposomes indicate that either biochemical isolation and/or alterations in lipid environment greatly decrease the channel''s voltage sensitivity. These results help underline the potential significance of using sarcoballs to study SR channels. The steep voltage sensitivity of the sarcoball anion channel suggests that it could be more actively involved in the regulation of Ca2+ transport by the SR.     

On the relationship between anion binding and chloride conductance in the CFTR anion channel

Mutations at many sites within the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel pore region result in changes in chloride conductance. Although chloride binding in the pore – as well as interactions between concurrently bound chloride ions – are thought to be important facets of the chloride permeation mechanism, little is known about the relationship between anion binding and chloride conductance. The present work presents a comprehensive investigation of a number of anion binding properties in different pore mutants with differential effects on chloride conductance. When multiple pore mutants are compared, conductance appears best correlated with the ability of anions to bind to the pore when it is already occupied by chloride ions. In contrast, conductance was not correlated with biophysical measures of anion:anion interactions inside the pore. Although these findings suggest anion binding is required for high conductance, mutations that strengthened anion binding had very little effect on conductance, especially at high chloride concentrations, suggesting that the wild-type CFTR pore is already close to saturated with chloride ions. These results are used to support a revised model of chloride permeation in CFTR in which the overall chloride occupancy of multiple loosely-defined chloride binding sites results in high chloride conductance through the pore.     

Distinct conformational states mediate the transport and anion channel properties of the glutamate transporter EAAT-1

Glutamate transport by the excitatory amino acid transporters (EAATs) is coupled to the co-transport of 3 Na(+), 1 H(+), and the counter-transport of 1 K(+) ion. In addition to coupled ion fluxes, glutamate and Na(+) binding to the transporter activates a thermodynamically uncoupled anion conductance through the transporter. In this study, we have distinguished between these two conductance states of the EAAT-1 transporter using a [2-(trimethylammonium)ethyl]methanethiosulfonate-modified V452C mutant transporter. Glutamate binds to the modified mutant transporter and activates the uncoupled anion conductance but is not transported. The selective alteration of the transport function without altering the anion channel function of the V452C mutant transporter suggests that the two functions are generated by distinct conformational states of the transporter.     

Characterization of a large conductance non-selective anion channel in B lymphocytes

Studies on ion channel currents in freshly isolated murine B lymphocytes with the patch clamp technique revealed the presence of a non-selective anion channel of large conductance in inside-out (i/o) patches. This channel is characterized here according to its unitary conductance, ion selectivity, regulatory factors, distribution and kinetic behaviour. With a unitary conductance of 348 +/- 4.4 pS in a normal physiological ion gradient, it exhibited an indiscriminate selectivity to cations (Na+ and K+). Selectivity to chloride over sodium was established by substitution of high concentrations of NaCl (450 mM) in the bath (i/o patches), resulting in a selectivity ratio (PCl/PNa) of 33. Selectivity to chloride over potassium was confirmed in a similar manner by substitution of TEA-Cl for KCl, yielding a selectivity ratio (PCl/Pk) greater than 80. Conductance of aspartate through the channel demonstrated the non-selective nature of this anion channel. Voltage proved to be a regulatory factor but other influences on channel activity were also present, including the configuration of the patch (channel is inactive in cell attached patches), and the enhancement of activity at negative membrane voltages by previous pulsing. Intracellular levels of calcium (i/o patches) did not appear to control channel conductances or regulate kinetic activity. Kinetic behaviour of this channel was complex, with periods of bursting and flickering activity interspersed with prolonged closed/open intervals. Multiple subconductance states were also present. The complex properties and behaviour of this channel suggest a possible role in signal transduction in B cell activation.     

Characterization of the anion transport channel protein in human erythrocytes. Induced circular dichroism of inhibitors bound to the anion transport channel

The induced circular dichroism (CD) of erythrocyte ghosts with anion-transport inhibitors has been studied. A ghost-EITC (eosin 5-isothiocyanate) system shows an induced CD spectrum at the wavelength region corresponding to the absorption bands of EITC. Also a ghost-EMI (eosin 5-maleimide) system shows induced CD, but has bands of opposite sign to the EITC system. From the change of the CD intensity, the number of EITC molecules bound to one erythrocyte was estimated to be about 1.4 X 10(6), being close to the number of band 3 copies per ghost. The CD spectra of EITC and EMI systems show that a configurational structure of the moiety anchoring the EMI molecule is the reverse to that of EITC. The preferred conformation of bound EITC may be twisted in a right-handed sense. From the signs of the induced CD bands in ghost-stilbene disulfonate systems, the chirality of twisted stilbene derivatives seems to be a left-handed sense, as is the case for the EMI derivative. The CD spectra of EITC in the presence of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate) shows that the binding site of EITC may not be identical with that of DIDS. The results observed in this study reflect the ternary arrangement of the functional amino groups in anion recognition sites.     

The discovery of slowness: low-capacity transport and slow anion channel gating by the glutamate transporter EAAT5

Excitatory amino acid transporters (EAATs) control the glutamate concentration in the synaptic cleft by glial and neuronal glutamate uptake. Uphill glutamate transport is achieved by the co-/countertransport of Na⁺ and other ions down their concentration gradients. Glutamate transporters also display an anion conductance that is activated by the binding of Na⁺ and glutamate but is not thermodynamically coupled to the transport process. Of the five known glutamate transporter subtypes, the retina-specific subtype EAAT5 has the largest conductance relative to glutamate uptake activity. Our results suggest that EAAT5 behaves as a slow-gated anion channel with little glutamate transport activity. At steady state, EAAT5 was activated by glutamate, with a K_m= 61 ± 11 μM. Binding of Na⁺ to the empty transporter is associated with a K_m = 229 ± 37 mM, and binding to the glutamate-bound form is associated with a K_m = 76 ± 40 mM. Using laser-pulse photolysis of caged glutamate, we determined the pre-steady-state kinetics of the glutamate-induced anion current of EAAT5. This was characterized by two exponential components with time constants of 30 ± 1 ms and 200 ± 15 ms, which is an order of magnitude slower than those observed in other glutamate transporters. A voltage-jump analysis of the anion currents indicates that the slow activation behavior is caused by two slow, rate-limiting steps in the transport cycle, Na⁺ binding to the empty transporter, and translocation of the fully loaded transporter. We propose a kinetic transport scheme that includes these two slow steps and can account for the experimentally observed data. Overall, our results suggest that EAAT5 may not act as a classical high-capacity glutamate transporter in the retina; rather, it may function as a slow-gated glutamate receptor and/or glutamate buffering system.     

Thiamine triphosphate activates an anion channel of large unit conductance in neuroblastoma cells

In neuroblastoma cells, the intracellular thiamine triphosphate (TTP) concentration was found to be about 0.5 m, which is several times above the amount of cultured neurons or glial cells. In inside-out patches, addition of TTP (1 or 10) m to the bath activated an anion channel of large unit conductance (350–400 pS) in symmetrical 150 mm NaCl solution. The activation occurred after a delay of about 4 min and was not reversed when TTP was washed out. A possible explanation is that the channel has been irreversibly phosphorylated by TTP. The channel open probability (P _o) shows a bell-shaped behavior as a function of pipette potential (V _p). P _o is maximal for –25 mV<V _p<10 mV and steeply decreases outside this potential range. From reversal potentials, permeability ratios of P_Cl/ P_Na = 20 and P_Cl/P_gluconate = 3 were estimated. ATP (5 mm) at the cytoplasmic side of the channel decreased the mean single channel conductance by about 50%, but thiamine derivatives did not affect unit conductance; 4,4 -diisothiocyanostilbene-2,2-disulfonic acid (0.1 mm) increased the flickering of the channel between the open and closed state, finally leading to its closure. Addition of oxythiamine (1 mm), a thiamine antimetabolite, to the pipette filling solution potentiates the time-dependent inactivation of the channel at V _p=–20 mV but had the opposite effect at +30 mV. This finding corresponds to a shift of P _o towards more negative resting membrane potentials. These observations agree with our previous results showing a modulation of chloride permeability by thiamine derivatives in membrane vesicles from rat brain.We would like to thank the National Funds for Scientific Research (Belgium) for financially supporting the stay of L.B. in Konstanz. We wish to thank A. Ngezahayo, F. Mendez and Dr. P. Wins for helpful discussions. This work was in part supported by a research grant from the Fonds special pour la Recherche à l'Université de Liege to L.B., the SFB 156 of the DFG and a grant of the Hermann and Lilly Schilling Stiftung to H.-A.K. Neuroblastoma, PC-12 and glioma cell lines were a gift from Prof. G. Moonen (Department of Human Physiology, University of Liège).     

Active recovery attenuates the fall in sweat rate but not cutaneous vascular conductance after supine exercise.

The purpose of this study was to identify whether baroreceptor unloading was responsible for less efficient heat loss responses (i.e., skin blood flow and sweat rate) previously reported during inactive compared with active recovery after upright cycle exercise (Carter R III, Wilson TE, Watenpaugh DE, Smith ML, and Crandall CG. J Appl Physiol 93: 1918-1929, 2002). Eight healthy adults performed two 15-min bouts of supine cycle exercise followed by inactive or active (no-load pedaling) supine recovery. Core temperature (T(core)), mean skin temperature (T(sk)), heart rate, mean arterial blood pressure (MAP), thoracic impedance, central venous pressure (n = 4), cutaneous vascular conductance (CVC; laser-Doppler flux/MAP expressed as percentage of maximal vasodilation), and sweat rate were measured throughout exercise and during 5 min of recovery. Exercise bouts were similar in power output, heart rate, T(core), and T(sk). Baroreceptor loading and thermal status were similar during trials because MAP (90 +/- 4, 88 +/- 4 mmHg), thoracic impedance (29 +/- 1, 28 +/- 2 Omega), central venous pressure (5 +/- 1, 4 +/- 1 mmHg), T(core) (37.5 +/- 0.1, 37.5 +/- 0.1 degrees C), and T(sk) (34.1 +/- 0.3, 34.2 +/- 0.2 degrees C) were not significantly different at 3 min of recovery between active and inactive recoveries, respectively; all P > 0.05. At 3 min of recovery, chest CVC was not significantly different between active (25 +/- 6% of maximum) and inactive (28 +/- 6% of maximum; P > 0.05) recovery. In contrast, at this time point, chest sweat rate was higher during active (0.45 +/- 0.16 mg.cm(-2).min(-1)) compared with inactive (0.34 +/- 0.19 mg.cm(-2).min(-1); P < 0.05) recovery. After exercise CVC and sweat rate are differentially controlled, with CVC being primarily influenced by baroreceptor loading status while sweat rate is influenced by other factors.     

Inner mitochondrial membrane anion channel is present in brown adipocytes but is not identical with the uncoupling protein

Summary Vesicles of inner mitochondrial membrane, mitoplasts, from rat brown adipose tissue were prepared by osmotic swelling and studied using the patch-clamp technique. Current events of a 107.8±8.7 pS (n=16, 21°C) channel were recorded in the mitoplast-attached mode. This channel was selective for anions and its kinetics resembled those of channels previously found in liver and heart mitochondria of mouse and ox. In whole-mitoplast mode each of five purine nucleotides (20 m) blocked the channel. This is the first demonstration of pharmacological blockade of this type of channel. Although a similar anion channel in mouse and ox mitochondria was suggested to be the uncoupling protein (UCP) associated with nonshivering thermogenesis, we present several arguments against this possibility. Thus we describe a high-conductance, purine-nucleotide-binding, anion selective mitochondrial channel, that is not the UCP.     

Neuronal influences are necessary to produce mitochondrial co‐localization with glutamate transporters in astrocytes

Recent evidence suggests that the predominant astrocyte glutamate transporter, GLT‐1/ Excitatory Amino Acid Transporter 2 (EAAT2) is associated with mitochondria. We used primary cultures of mouse astrocytes to assess co‐localization of GLT‐1 with mitochondria, and tested whether the interaction was dependent on neurons, actin polymerization or the kinesin adaptor, TRAK2. Mouse primary astrocytes were transfected with constructs expressing V5‐tagged GLT‐1, pDsRed1‐Mito with and without dominant negative TRAK2. Astrocytes were visualized using confocal microscopy and co‐localization was quantified using Volocity software. Image analysis of confocal z‐stacks revealed no co‐localization between mitochondria and GLT‐1 in pure astrocyte cultures. Co‐culture of astrocytes with primary mouse cortical neurons revealed more mitochondria in processes and a positive correlation between mitochondria and GLT‐1. This co‐localization was not further enhanced after neuronal depolarization induced by 1 h treatment with 15 mM K⁺. In pure astrocytes, a rho kinase inhibitor, Y27632 caused the distribution of mitochondria to astrocyte processes without enhancing GLT‐1/mitochondrial co‐localization, however, in co‐cultures, Y27632 abolished mitochondrial:GLT‐1 co‐localization. Disrupting potential mitochondrial: kinesin interactions using dominant negative TRAK2 did not alter GLT‐1 distribution or GLT‐1: mitochondrial co‐localization. We conclude that the association between GLT‐1 and mitochondria is modest, is driven by synaptic activity and dependent on polymerized actin filaments.

     

Evidence for functional interaction of plasma membrane electron transport, voltage-dependent anion channel and volume-regulated anion channel in frog aorta

Frog aortic tissue exhibits plasma membrane electron transport (PMET) owing to its ability to reduce ferricyanide even in the presence of mitochondrial poisons, such as cyanide and azide. Exposure to hypotonic solution (108 mOsmol/kg H2O) enhanced the reduction of ferricyanide in excised aortic tissue of frog. Increment in ferricyanide reductase activity was also brought about by the presence of homocysteine (100 microM dissolved in isotonic frog Ringer solution), a redox active compound and a potent modulator of PMET. Two plasma-membrane-bound channels, the volume-regulated anion channel (VRAC) and the voltage-dependent anion channel (VDAC), are involved in the response to hypotonic stress. The presence of VRAC and VDAC antagonists-tamoxifen, glibenclamide, fluoxetine and verapamil, and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), respectively-inhibited this enhanced activity brought about by either hypotonic stress or homocysteine. The blockers do not affect the ferricyanide reductase activity under isotonic conditions. Taken together, these findings indicate a functional interaction of the three plasma membrane proteins, namely, ferricyanide reductase (PMET), VDAC and VRAC.     

The nature of amino acid 482 of human ABCG2 affects substrate transport and ATP hydrolysis but not substrate binding

Several members of the ATP-binding cassette (ABC) transporter superfamily, including P-glycoprotein and the half-transporter ABCG2, can confer multidrug resistance to cancer cells in culture by functioning as ATP-dependent efflux pumps. ABCG2 variants harboring a mutation at arginine 482 have been cloned from several drug-resistant cell lines, and these variants differ in their substrate transport phenotype. In this study, we changed the wild-type arginine 482 in human ABCG2 to each one of the 19 other standard amino acids and expressed each one transiently in HeLa cells. Using the 5D3 antibody that recognizes a cell surface epitope of ABCG2, we observed that all the mutants were expressed at the cell surface. However, the mutant ABCG2 proteins differed markedly in transport activity. All of the variants were capable of transporting one or more of the substrates used in this study, with the exception of the R482K mutant, which is completely devoid of transport ability. Six of the mutants (R482G, R482H, R482K, R482P, R482T, and R482Y) and the wild-type protein (R482wt) were selected for studies of basal and stimulated ATPase activity and photoaffinity labeling with the substrate analog [125I]iodoarylazidoprazosin. Whereas these seven ABCG2 variants differed markedly in ATPase activity, all were able to specifically bind the substrate analog [125I]iodoarylazidoprazosin. These data suggest that residue 482 plays an important role in substrate transport and ATP turnover, but that the nature of this amino acid may not be important for substrate recognition and binding.     

Molecular determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel pore

Ionic selectivity in many cation channels is achieved over a short region of the pore known as the selectivity filter, the molecular determinants of which have been identified in Ca(2+), Na(+), and K(+) channels. However, a filter controlling selectivity among different anions has not previously been identified in any Cl(-) channel. In fact, because Cl(-) channels are only weakly selective among small anions, and because their selectivity has proved so resistant to site-directed mutagenesis, the very existence of a discrete anion selectivity filter has been called into question. Here we show that mutation of a putative pore-lining phenylalanine residue, F337, in the sixth membrane-spanning region of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel, dramatically alters the relative permeabilities of different anions in the channel. Specifically, mutations that reduce the size of the amino acid side chain present at this position virtually abolish the relationship between anion permeability and hydration energy, a relationship that characterizes the anion selectivity not only of wild-type CFTR, but of most classes of Cl(-) channels. These results suggest that the pore of CFTR may indeed contain a specialized region, analogous to the selectivity filter of cation channels, at which discrimination between different permeant anions takes place. Because F337 is adjacent to another amino acid residue, T338, which also affects anion selectivity in CFTR, we suggest that selectivity is predominantly determined over a physically discrete region of the pore located near these important residues.     

Decreased expression of vesicular GABA transporter, but not vesicular glutamate, acetylcholine and monoamine transporters in rat brain following focal ischemia

In nerve terminals, vesicular transporters pack neurotransmitters into synaptic vesicles, which is an essential prerequisite for transmitter release. To date, three distinct families of vesicular transporters have been identified which are specific for (a) excitatory amino acids (glutamate and aspartate), (b) inhibitory amino acids (GABA and glycine) and (c) acetylcholine and monoamines. The present study evaluated the effect of transient focal cerebral ischemia on the expression of these vesicular transporters in adult rat brain. Ischemia was induced by a 1 h transient middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats. At various reperfusion periods (3-72 h), mRNA levels of the vesicular transporters were estimated in the contralateral and the ipsilateral cerebral cortex by real-time PCR analysis. Following transient focal ischemia, mRNA expression of the vesicular GABA transporter (VGAT) decreased significantly by 3 h of reperfusion and remained at a significantly lower level than sham until at least 72 h of reperfusion. Western blotting showed a significant decrease in the VGAT immunoreactive protein levels in the ipsilateral cortex of rats subjected to focal ischemia and 24 h reperfusion. Immunohistochemistry demonstrated many VGAT immunopositive puncta in the contralateral cortex, which were significantly decreased in the ipsilateral cortex at 24 h reperfusion. Focal ischemia had no effect on the mRNA levels of the vesicular transporters specific for glutamate/aspartate, acetylcholine and monoamines at either 6 h or 24 h of reperfusion.     

Sex differences in lizard escape decisions vary with latitude,but not sexual dimorphism

Sexual selection is a powerful evolutionary mechanism that has shaped the physiology, behaviour and morphology of the sexes to the extent that it can reduce viability while promoting traits that enhance reproductive success. Predation is one of the underlying mechanisms accounting for viability costs of sexual displays. Therefore, we should expect that individuals of the two sexes adjust their anti-predator behaviour in response to changes in predation risk. We conducted a meta-analysis of 28 studies (42 species) of sex differences in risk-taking behaviour in lizards and tested whether these differences could be explained by sexual dichromatism, by sexual size dimorphism or by latitude. Latitude was the best predictor of the interspecific heterogeneity in sex-specific behaviour. Males did not change their escape behaviour with latitude, whereas females had increasingly reduced wariness at higher latitudes. We hypothesize that this sex difference in risk-taking behaviour is linked to sex-specific environmental constraints that more strongly affect the reproductive effort of females than males. This novel latitudinal effect on sex-specific anti-predator behaviour has important implications for responses to climate change and for the relative roles of natural and sexual selection in different species.     

The oxidation of glutamine and glutamate in relation to anion transport in enterocyte mitochondria.

The oxidation of L-glutamate and L-glutamine by enterocyte mitochondria was supported by malate. The stimulation of the rate of oxidation of the two amino acids by small amounts of added malate was 93% and 76% respectively. This could not be accounted for by the oxidation of the small amounts of malate added. Amino-oxyacetate added initially inhibited malate-supported oxidation of L-glutamate by 81% and that of L-glutamine by 38%. The inhibition of L-glutamate oxidation was partially reversed by L-glutamine. The dicarboxylate-carrier inhibitor 2-phenylsuccinate inhibited the malate-supported oxidation of both amino acids, but appeared to be slightly stimulatory to L-glutamine oxidation when added initially. The inhibition of L-glutamate oxidation was reversed by L-glutamine. The mitochondrial uncoupler FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) inhibited malate-supported oxidation of L-glutamate by 78% when added initially. The oxidation of L-glutamine was completely inhibited. However, the uncoupler stimulated the oxidation of both amino acids when added finally. Pyruvate inhibited aspartate synthesis when either of these amino acids was the main substrate, alanine being synthesized. There was no effect on O2 uptake. Mitochondria did not swell in KCl solution, but swelled rapidly in water. Mitochondrial swelling in potassium phosphate and potassium acetate solutions was activated by valinomycin and to a lesser extent by the further addition of FCCP. With potassium malate, swelling was mainly activated by phosphate. The swelling of enterocyte mitochondria in potassium glutamate was slow. In glutamine solution, mitochondrial swelling was greater and appeared to be enhanced by the initial presence of small amounts of phosphate.     

Neuronal survival depends on EGFR signaling in cortical but not midbrain astrocytes

Mice lacking epidermal growth factor receptor (EGFR) develop a neurodegeneration of unknown etiology affecting exclusively the frontal cortex and olfactory bulbs. Here, we show that EGFR signaling controls cortical degeneration by regulating cortical astrocyte apoptosis. Whereas EGFR(-/-) midbrain astrocytes are unaffected, mutant cortical astrocytes display increased apoptosis mediated by an Akt-caspase-dependent mechanism and are unable to support neuronal survival. The expression of many neurotrophic factors is unaltered in EGFR(-/-) cortical astrocytes suggesting that neuronal loss occurs as a consequence of increased astrocyte apoptosis rather than impaired secretion of trophic factors. Neuron-specific expression of activated Ras can compensate for the deficiency of EGFR(-/-) cortical astrocytes and prevent neuronal death. These results identify two functionally distinct astrocyte populations, which differentially depend on EGFR signaling for their survival and also for their ability to support neuronal survival. These spatial differences in astrocyte composition provide a mechanism for the region-specific neurodegeneration in EGFR(-/-) mice.     

Disease-associated mutations in the extracytoplasmic loops of cystic fibrosis transmembrane conductance regulator do not impede biosynthetic processing but impair chloride channel stability

Consistent with its function as a chloride channel regulated entirely from the cytoplasmic side of the plasma membrane, the cystic fibrosis transmembrane conductance regulator (CFTR) glycoprotein exposes little of its mass on the exterior surface of cells. The first and fourth extracytoplasmic loops (ELs) contain approximately 15 and 30 residues, respectively; the other four ELs are extremely short. To examine the influence of missense mutants in ELs detected in patients with cystic fibrosis, we have expressed them in mammalian (baby hamster kidney (BHK21)) cells and assessed their biosynthetic processing and chloride channel activity. In contrast to previous findings that 18 of 30 disease-associated missense mutations in cytoplasmic loops caused retention of the nascent polypeptides in the endoplasmic reticulum, all the EL mutants studied matured and were transported to the cell surface. This pronounced asymmetry is consistent with the notion that endoplasmic reticulum quality control of nascent CFTR is exerted primarily on the cytoplasmic side of the membrane. Although this set of EL mutations has little effect on CFTR maturation, most of them seriously compromise its chloride channel activity. Substitutions at six different positions in EL1 and single positions in EL2 and EL4 all destabilized the open state, some of them severely, indicating that the ELs contribute to the stability of the CFTR ion pore.