Importance of several cysteine residues for the chloride conductance of trout anion exchanger 1 (tAE1).
In this study, we devised a cysteine-focused point mutation analysis of the chloride channel function of trout anion exchanger 1 (tAE1) expressed in X. laevis oocytes. Seven cysteines, belonging to the transmembrane domain of tAE1, were mutated into serines (either individually or in groups) and the effects of these mutations on the chloride conductance of injected oocytes were measured. We showed that three cysteines were essential for the functional expression of tAE1. Namely, mutations C462S, C583S and C588S reduced Cl(-) conductance by 68%, 52% and 83%, respectively, when compared to wild type tAE1. These residual conductances were still inhibited by 0.5 mM niflumic acid. Western blot experiments demonstrated that C462 was involved in protein expression onto the plasma membrane. A mutant devoid of this residue was unable to express onto the plasma membrane, especially if several other cysteines were missing: consequently, the cysteine-less mutant of tAE1 was not functional. C583 and C588 were involved in the channel function of tAE1 as shown by anion substitution experiments proving that selectivity of the mutated pore differs from the wild type one. On the contrary, they were not involved in the Cl(-)/HCO(3)(-) exchange function of tAE1, as demonstrated by intracellular pH measurements. These and several complementary mutations allow us to conclude that a mutant of tAE1 containing the sole C462 can drive a marginal Cl(-) current; however, the minimal configuration necessary to get optimal functional expression of the tAE1 chloride channel is that of a mutant containing unaffected residues C462, C583 and C588.
Martial
Sonia
S
Laboratoire de Physiologie Cellulaire et Moléculaire, UMR 6548, CNRS-Université de Nice, Bâtiment de Sciences Naturelles, Parc Valrose, Nice Cedex 2, France. sonia.martial@unice.fr
Guizouarn
Hélène
H
Gabillat
Nicole
N
Pellissier
Bernard
B
Borgese
Franck
F
D000595:Amino Acid Sequence; D000818:Animals; D027963:Chloride-Bicarbonate Antiporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D005260:Female; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014337:Trout / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D027963:Chloride-Bicarbonate Antiporters; D011994:Recombinant Proteins; D003545:Cysteine