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J Gen Physiol
1997 Mar 01;1093:345-60. doi: 10.1085/jgp.109.3.345.
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Electrogenic sulfate/chloride exchange in Xenopus oocytes mediated by murine AE1 E699Q.
Chernova MN
,
Jiang L
,
Crest M
,
Hand M
,
Vandorpe DH
,
Strange K
,
Alper SL
.
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Functional evaluation of chemically modified human erythrocytes has led to the proposal that amino acid residue E681 of the band 3 anion exchanger AE1 lies on the anion translocation pathway and is a proton carrier required for H+/SO4(2-) cotransport. We have tested in Xenopus oocytes the functional consequences of mutations in the corresponding residue E699 of mouse AE1. Most mutations tested abolished AE1-mediated Cl- influx and efflux. Only the E699Q mutation increased stilbene disulfonate-sensitive efflux and influx of SO4(2-). E699Q-mediated Cl- influx was activated by elevation of intracellular SO4(2-), but E699Q-mediated Cl- efflux was undetectable. The DNDS (4,4'-dinitrostilbene-2,2'-disulfonic acid) sensitivity of E699Q-mediated SO4(2-) efflux was indistinguishable from that of wt AE1-mediated Cl- efflux. The extracellular anion selectivity of E699Q-mediated SO4(2-) efflux was similar to that of wt AE1-mediated Cl- efflux. The stoichiometry of E699Q-mediated exchange of extracellular Cl- with intracellular SO4(2-) was 1:1. Whereas SO4(2-) injection into oocytes expressing wt AE1 produced little change in membrane potential or resistance, injection of SO4(2-), but not of Cl- or gluconate, into oocytes expression E699Q depolarized the membrane by 17 mV and decreased membrane resistance by 66%. Replacement of bath Cl- with isethionate caused a 28-mV hyperpolarization in SO4(2-)-loaded oocytes expressing E699Q, but had no effect on oocytes expressing wt AE1. Extracellular Cl(-)-dependent depolarization of SO4(2-)-preloaded oocytes was blocked by DNDS. AE1 E699Q-mediated inward current measured in the presence of extracellular Cl- was of magnitude sufficient to account for measured 35SO4(2-) efflux. Thus, AE1 E699Q-mediated SO4(2-)/Cl- exchange operated largely, if not exclusively, as an electrogenic, asymmetric, 1:1 anion exchange. The data confirm the proposal that E699 resides on or contributes to the integrity of the anion translocation pathway of AE1. A single amino acid change in the sequence of AE1 converted electroneutral to electrogenic anion exchange without alteration of SO4(2-)/Cl- exchange stoichiometry.
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9089441
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Figure 2. SO42− efflux by mutant AE1 polypeptides. (A) Extracellular SO42− supported 35SO42− efflux from AE1 E699Q-expressing oocytes (lower two traces) but not from oocytes expressing wt AE1 or AE1 mutants with T, K, G, or R in place of E699 (upper cluster of 8 traces). Extracellular condition shown above x axis. DIDS was added in the continued presence of SO42−. Representative of 3 similar experiments performed 4–5 d after cRNA injection. Each trace represents a single oocyte. (B) SO42−/Cl− exchange mediated by AE1 E699Q was 70% faster than SO42−/SO42− exchange. DIDS inhibited efflux in the continued presence of Cl−. The two uppermost traces show efflux from water-injected oocytes. The six traces below show efflux from AE1 E699Q-expressing oocytes. (C) Bar graph of the data in B, (n) for each condition above bars. *P < 0.02.
Figure 3. Biosynthesis and cell surface expression of AE1 E699Q and wt AE1. 72 h after cRNA injection, metabolically labelled oocytes expressing wt AE1 (lanes 1 and 2), AE1 E699Q (lanes 3 and 4), or no cRNA (lane 5, water-injected) were incubated for 3 h in the absence (lanes 1, 3, and 5) or presence (lanes 2 and 4) of 5 mg/ml chymotrypsin as described in methods. Triton X100 extracts of the oocytes were subjected to immunoprecipitation with anti-AE1 antibody, SDS-PAGE, and autoradiography. Arrows indicate holoprotein and the 60-kD NH2-terminal chymotryptic fragment of AE1 (p60). Representative of four similar experiments.
Figure 4. DNDS inhibits anion exchange by wt AE1 and by E699Q AE1 with similar potency. (A) Effect of increasing concentrations of DNDS on AE1 E699Q-mediated 35SO42− efflux into Cl− medium. Blockade was reversible. Each trace represents an individual oocyte. Upper trace is water-injected oocyte. (B) DNDS inhibition dose-response curves of wt AE1-mediated 36Cl− efflux and of AE1 E699Q-mediated 35SO42− efflux. Each of 5 oocytes was exposed for 9 or 12 min to incrementally increasing DNDS concentrations.
Figure 5. (A) Extracellular halide dependence of AE1 E699Q-mediated SO42− efflux. Each line represents a single oocyte. (B) Extracellular anion dependence of AE1 E699Q-mediated 35SO42− efflux. Summary of three similar experiments, with number oocytes evaluated in each condition noted above columns. Relative efflux into iodide medium lacking gluconate salts of Mg2+, Ca2+, and K+ was further reduced to 0.09 ± 0.03 (n = 5), but omission of these gluconate salts did not change values for other anions (not shown).
Figure 6. Experimental format for measurement of stoichiometry of influx vs. efflux. (A) 36Cl− influx into acutely SO42−-loaded or native oocytes injected 13 d earlier with water or with AE1 E699Q cRNA (*P < 0.01). Representative of five similar, paired experiments. AE1 E699Q-mediated 36Cl− influx was measurable in native oocytes from 8 of 10 frogs tested. Influx was measurable in SO42−-loaded oocytes from 9 of 10 frogs tested. (B) Oocytes from the frog of panel A injected 13 d earlier with water (upper trace) or with cRNA (seven lower traces) were acutely injected with 35SO42− to a final intracellular [SO42−] of ∼14 mM, then subjected to the efflux assay. Note that fractional efflux into Cl− medium was less than observed in Fig. 2 B, in which intracellular [SO42−] was ∼1 mM.
Figure 7. Intracellular anion dependence of electrogenic anion exchange. (A) Oocytes injected 72 h previously with cRNA encoding AE1 E699Q were acutely injected (arrow) with 50 nl of 87 mM K salts of SO42−, Cl−, or gluconate as indicated while membrane potential and resistance were recorded. Regular spaced vertical displacements in potential are in response to 10-nA current pulses. (B) An oocyte injected 72 h previously with cRNA encoding wt AE1 was injected with 50 nl 87 mM K2SO4. (C) A water-injected oocyte was injected with 50 nl 87 mM K2SO4.
Figure 8. Membrane potential changes in Na2SO4-loaded oocytes in response to varied extracellular conditions. (A) Membrane potential of a Na2SO4-loaded E699Q-expressing oocyte during two sequential transitions from extracellular Cl− to isethio-nate. (B) Membrane potential of a Na2SO4-loaded E699Q-expressing oocyte in extracellular Cl− medium during introduction and removal of DNDS. (C) Membrane potential of a Na2SO4-loaded wt AE1-expressing oocyte during transition from extracellular Cl− to isethionate. (D) Membrane potential of a Na2SO4-loaded oocyte, previously injected with water, during transition from extracellular Cl− to isethionate. Gaps in the voltage records represent application of stepped voltage protocols (see Fig. 9).
Figure 9. AE1 E699Q-associated currents. (A) Mean I-V curves and representative current traces from Na2SO4-loaded oocytes in extracellular Cl− medium expressing wt AE1 (filled squares, n = 20 oocytes from 6 frogs) and AE1 E699Q (filled circles, n = 54 oocytes from 8 frogs). *P < 0.04; NS, P > 0.05. (B) Comparison of E699Q-mediated currents in Na2SO4-loaded oocytes in the presence of extracellular Cl− (filled circles), extracellular isethionate (open squares, n = 44 oocytes from 7 frogs), and extracellular sulfate (open triangles, n = 5 oocytes from 1 frog). *P < 0.002. (C) Comparison of E699Q-mediated currents in Na2SO4-loaded oocytes in extracellular chloride in the absence (filled circles) and presence of 200 μM DNDS (open diamonds, n = 24 oocytes from 4 frogs) *P < 0.03. Voltage was stepped from a holding potential of −50 mV to test potentials between −80 and +60 mV in 20-mV increments. At potentials ≥+80 mV, endogenous outward currents were activated in all groups of oocytes. Data recorded in extracellular chloride from AE1 E699Q-expressing oocytes is replicated in all three panels.
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