Reversible and irreversible interactions of DIDS with the human electrogenic Na/HCO3 cotransporter NBCe1-A: role of lysines in the KKMIK motif of TM5.
Others have shown that H(2)DIDS reversibly and covalently binds to the first lysine (K) in the SKLIK motif at the extracellular end of transmembrane segment 5 of the Cl-HCO(3) exchanger AE1. Here we mutated K558, K559, and/or K562 in the homologous KKMIK motif of human NBCe1-A. We expressed constructs in Xenopus oocytes, and used a two-electrode voltage clamp to test the sensitivity of the NBC current (-160 to +20 mV) to DIDS. A 30-s DIDS exposure decreased the current at 0 mV, and a subsequent albumin wash returned the current to the initial value (less any irreversible DIDS inhibition), permitting the determination of a complete dose-response curve on a single oocyte. For all constructs, the reversible DIDS inhibition of the NBC current decreased at more negative voltages. The apparent inhibitory constant for reversible DIDS binding increased in the sequence RRMIR < KKMIK (wt, approximately 40 microM) < NKMIK congruent with NKMIN congruent with KKMIN < KNMIN congruent with KNMIK < NNMIK < NNMIN ( approximately 400 microM) < DDMID < EEMIE ( approximately 800 microM). Thus the second K is the most important for reversible DIDS blockade. Nevertheless, these mutations had relatively little effect on slope conductance in the absence of DIDS. For KKMIK, RRMIR, NKMIK, KKMIN, KNMIK, and NNMIN, the rates of irreversible inhibition by DIDS roughly parallel the apparent affinities for reversible DIDS binding. The rate was extremely low for DDMID. The fitted maximal inhibitions were 80-91% for the first five constructs, and 66% for NNMIN. Thus DIDS probably reversibly binds before irreversibly reacting with NBCe1-A. Finally, tenidap blocks not only KKMIK, but also NNMIN and EEMIE.
Lu
Jing
J
Department Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520-8026, USA. jing.lu@yale.edu
Boron
Walter F
WF
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000378:metabolism* / Q000494:pharmacology; D020816:Amino Acid Motifs; D000818:Animals; D004305:Dose-Response Relationship, Drug; D049452:Green Fluorescent Proteins; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D008564:Membrane Potentials / Q000187:drug effects; D008845:Microinjections; D008954:Models, Biological; D009154:Mutation; D009865:Oocytes; D000078183:Oxindoles; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D007211:Indoles; D000078183:Oxindoles; D011993:Recombinant Fusion Proteins; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C062866:tenidap; D008239:Lysine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid