Effects of external pH on binding of external sulfate, 4.4-dinitro-stilbene-2,2'-disulfonate (DNDS), and chloride to the band 3 anion exchange protein.

A model in which two positively-charged titratable sites enhance the affinity for anionic substrates can explain the increase in external iodide dissociation constant (K(O)(I)) with increasing pH(O) (Liu, S. J., F.-Y. Law, and P.A. Knauf. 1996.f Gen.Physiol. 107:271-291). If sulfate binds to the same external site as I-, this model predicts that the SO(4)= dissociation constant (K(O)(S)) should also increase. The data at pH 0 8.5 to 10 fit this prediction, and the pK for the titration is not significantly different from that (pKc) for the low-pK group that affects K(O)(1). The dissociation constant for the apparently competitive inhibitor, DNDS (4,4-dinitrostilbene-2,2'- disulfonate), also increases greatly as pH(O) increases. Particularly at high pH(O), a noncompetitive inhibition by DNDS is also evident. Increasing pH(O) from 7.2 to 11.2 increases the competitive dissociation constant by 700-fold, but the noncompetitive is only increased 20-fold. The pK values for these effects are similar to pKc for K(O)(1), as expected if DNDS binds near the external transport site, but it seems likely that additional titratable groups also affect DNDS binding. The apparent affinity for external Cl- is also affected by pH(O), in a manner similar to that observed for I-. Pretreatment with the amino-selective reagent, bis-sulfosuccinimidyl suberate (BSSS), decreases the apparent Cl- affinity at pH 8.5, but two titrations are still evident, the first (lower) of which decreases the apparent C- affinity, and the second of which surprisingly increases it. Thus, the BSSS-reactive amino groups (probably Lys-539 and Lys-851) do not seem to be involved in the titrations that affect Cl- affinity. In general, the data support the concept that a positively charged amino group (or groups), together with a guanidino group, plays an important role in the binding of substrates and inhibitors at or near the external transport site.


INTRODUCTION
In the preceding paper (Liu et al., 1996), we have shown that the I-dissociation constant (Ko I) for the E o form of band 3 (the conformation with the transport site facing outward) increases dramatically with increasing external pH (pHo). The data can be most simply interpreted in terms of a model in which a positively charged amino acid residue with a pK of 9.5 -0.2 (probably lysine) contributes to binding of external substrates (Liu et al., 1996). The data are also compatible with a model similar to that proposed by Bjerrum (1992), with two titratable sites, one with a pK value of 9.3 -+ 0.3 and another with a pK > 11. The lower pK residue is probably a lysine, while the higher pK site is likely arginine. Bjerrum also proposed an alternate model in which both sites are arginines.
If titratable positive charges contribute to the free energy of binding of monovalent anions to the external transport site, then these charges should also have an effect on the binding of divalent anions or competitive inhibitors to the same site. The affinities of these latter agents should be affected by external pH in a manner which is predictable from the pK values determined from the I-affinity data.
In this paper, we test these predictions by examining the pHo dependence of SO4 =, CI-, and DNDS affinity. To test the hypothesis that certain amino groups are involved in external substrate binding, we also examine the effects of an amino-selective reagent, bis-sulfosuccinimidyl suberate (BSSS), on the apparent external C1-affinity. The results of these tests in general support the titratable substrate binding site model (Bjerrum, 1992;Liu et al., 1996), but do not rule out the possiblity that other titratable sites may play a role in the binding of at least some of these anions.

Cell Preparation and Flux Measurement
Red blood cells were obtained from freshly drawn human blood and washed three times with 150 KH (150 mM KC1, 24 mM sucrose and 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.2 at 0~ The C1-exchange fuxes (J) were determined by measuring 36C1 efflux, as described in the accompanying paper (Liu et al., 1996).

Sulfate and DNDS (4, 4 '-dinitrostilbene-2, 2'-disulfonate) Inhibition
s04 = is a substrate for band 3 but is transported at a much lower rate than C1-(~20,000 times slower at 8~ and pH 6.3 [Ku et al., 1979] ). It acts as a competitive inhibitor of CI-exchange (Milanick and Gunn, 1982). Therefore the apparent external SO4-dissociation constant, Ko s, for the conformation (Eo) of band 3 with unloaded transport site facing outward was determined by the method of Milanick and Gunn (1982) from the x value of the intersection point of two Dixon plots for SO4 = inhibition with different [Clo], as described in detail for I-in the preceding paper (Liu et al., 1996). This same method was also used to determine Ke for the nontransported inhibitor DNDS (ICN Pharmaceuticals, Plainview, NY).

Measurement of Transport Parameters for External Chloride
The unidirectional C1-effiux of control cells with ~150 mM [Cli] was measured in media with different [Clo], with sucrose replacing CI-, as described in the preceding paper. The data were fitted to the Michaelis-Menten equation by nonlinear least squares to calculateJ m~ the flux with maximal (saturating) [Clo], and Ka/2o, the [C1 o] that gives half-maximal flux.

BSSS (Bis-sulfosuccinimidyl suberate) Treatment
BSSS (Pierce Chemical Co., Rockford, IL) was freshly dissolved in 150 KH at pH 7.4 at room temperature. Cells were washed once with 150 KH (pH 7.4 at room temperature), and then incubated with 2.7 mM BSSS in 150 KH at ~50% hematocrit at pH 7.25 and 37~ for 1 h. The reaction was stopped by adding 40 ml of 50 mM glycine in 150 KH (pH 7.2 at 0~ to the reaction medium. Then the BSSS treated cells were washed three times with 150 KH (pH 7.2 at 0~ containing 0.2% BSA (BSA, Sigma Chemical Co.). After each wash, the resuspended cells were left at room temperature for 10 min to allow the pHi to reach equilibrium.

Data Analysis
Enzfitter (Elsevier Biosoft), a nonlinear regression data analysis program for the IBM PC, was used to fit the data for C1-flux, J, as a function of inhibitor concentration, [Inh] The dissociation constants for SO4 =, DNDS, and CI at various pHo values were fitted to a model in which the affinity change is related to titration of one or two sites, as described by Eqs. 1 and 2 of the preceding paper (Liu et al., 1996). Because the variations in the dissociation constants in general increase with the size of the constants, the data were weighted according to the inverse of their values, and nonlinear least squares estimates of the parameters were obtained by using Origin (MicroCal Software, Northampton, MA).

Effects of pHo on Anions Other Than Iodide
The titratable substrate binding site (SBS) model postulates that the electrostatic field of positive charges near the external transport site contributes significandy to the binding affinity for anionic substrates, such as I- (Liu et al., 1996). According to this model, titration of the positive charges should also affect the binding of other substrates or competitive inhibitors to the external transport site. Because of the different charges or locations of these other ions, however, the effects of pHo may show quantitative differences, although the pK values for the titrations should not be significantly different.
The dissociation constant for binding, /Q, is related to the free energy of binding by the equation: In general, AG is a complicated function of the interactions between the ion and the binding site. In the present context, however, we are primarily concerned with the effect of addition or removal of a positive charge near the binding site. For a single positive charge, the electrical contribution to the free energy (in kilocalories per mole) of an anion located at a distance r from the center of the titratable positive charge is equal to the electrostatic work and is given by (Gabler, 1978): where n is the charge of the anion (e.g., one for a univalent anion) and D is the dielectric constant. If we write Eq. 1 for the condition when the residue is protonated (subscript +) and when it is deprotonated (subscript 0), the dissociation constant for the protonated form, K+, and for the deprotonated form, K0, have the following relationship, provided that the entire free energy difference between the two forms is given by AGd: If a different anion, designated by *, is bound near the titratable charge, we can write equations for this ion similar to Eqs. 2 and 3. When these equations are solved simultaneously, we obtain: That is, the ratio of dissociation constants in the two states for one anion is proportional to the same ratio for the other anion (*), raised to a power that is directly proportional to the ratio of the charges of the anions (n/n*) and inversely proportional to the ratio of the distance between each anion and the positive charge ( r/ r*).

Effects of pH o on Sulfate Affinity
According to Eq. 4, if a divalent anion such as SO4 = is bound to the external transport site, and if the distance between the center of the SO4 = and the titratable positive charge is less than twice the distance between the charge and bound I-, the doubling of n (for SO4 =) relative to n* (for I-) in Eq. 4 predicts that the ratio of neutral and alkaline form dissociation constants for SO4 = should be larger than that for I-. Thus, qualitatively pHo would be expected to have a larger effect on the dissociation constant for SO4 =, Ko s, than on that for I-, Ko I. Because of the complex nature of the equation describing the titration (see Liu et al., 1996), however, the apparent values of Ko I and Ko s at each pHo will not follow Eq. 4. As mentioned in the preceding paper (Liu et al., 1996), larger values of K 0 relative to K+ will cause the inflection point for the pHo titration to shift to higher pHo values. At pHo < 10.5, therefore, the predicted results for divalent and monovalent ions are quite similar (e.g., compare solid triangles for I-in Fig. 3 with dashed line predicted for a divalent ion at the same position as I-). Despite this apparent pK shift, numeri-cal fits to the one-site or two-site SBS model should give pK values for SO4 = similar to those seen for I-.
To test the SBS model, therefore, we measured/co s at various pH o values. Like Ko I, Ko s was determined from the negadve of the x value of the intersection point of Dixon plots (1/flux vs [SO4o]) at different [Clo] (Milanick and Gunn, 1986;Fr6hlich and Gunn, 1986;Knauf and Brahm, 1989). Fig. 1 shows that the Dixon plots for SO4 = inhibition were nearly perfecdy linear, indicating a single site of inhibition, as required for the determination of Ko s. Table I shows/co s as a function of pH o. Ko s increases markedly as pHo is raised from 8.5 to 10, which is qualitatively consistent with the pH o dependence of KJ. This increase in Ko s is not related to the titratable site with a pK of 5.03, described by Milanick and Gunn (1982), which causes a ninefold increase in SO4 = affinity when it is protonated. Because of this site, the apparent Ko s increases with an apparent pK of 5.99. By pH 8.5, however, the lowest pHo shown in Table I, this titration should be over 99% complete. Thus, the titratable site characterized by Milanick and Gunn (1982) has no effect on Ko s in the pH o range examined here.
As shown in Table I, when/co I and Ko s at various pHo are divided by their values at pHo 8.5, the ratio for SO4 = shows a dependence on pHo that is similar to but somewhat stronger than that seen for I-. This is fairly consistent with the SBS model, suggesting that the reduction of local positive charge by titration may explain the decrease in both SO4 = and I-binding affinity with pHo. The effect of pH o on Ko s, however, is somewhat larger than predicted for a divalent ion at the same location as I-(in which case the ratios for SO4 = and I-should be nearly identical at pHo ~< 10), suggesting that SO4 = may interact with an additional titratable charge. The idea that SO4 = and I-are not located in precisely the same place when bound to the external transport site is supported by evidence that SO4 = and CI-interact differently with an inhibitor, NAP-taurine (N-[4-azido-2-nitrophenyl]-2-aminoethylsulfonate) (Fr6hlich and Gunn, 1987;Knauf et al., 1978). If the favorable electrostatic free energy resulting from interaction of this positive charge with anions were the dominant factor in determining the total binding free energy, and if I-and SO4 = were bound at exactly the same position, one would expect divalent anions such as SO4 = to have a higher apparent affinity for the external transport site than do monovalent anions such as I-(Eqs. 1 and 2). The fact that the dissociation constants are higher for SO4 = than for I- (Table  I), indicating a lower affinity for the doubly charged ion, demonstrates that factors other than the electrostatic interaction make important contributions to the binding free energy.

pK for Dependence of Ko s on pHo
If the seven determinations of Ko s between pHo 8.5 and 10 (Table I) are fitted to a single-site titration curve, the pK value is 8.7 (+-1.0). A fit to a two-site model, with the pKfor the higher pK residue (D) set to 11.35 (Bjerrum, 1992), also gives a pK for the lower pK (C) residue of 8.7 -+ 1.0. Because of the limited data range, these pK's are not very accurately determined, but they are not far from the pK values (9.5 + 0.2 or 9.3 -+ 0.3) determined by fitting similar models to the data for the pHo dependence of Ko I (Liu et al., 1996). The pK value is very close to that (8.7) determined by Bjerrum (1992) from measurements of CI-fluxes under conditions of low ionic strength. The simplest interpretation of the similar pK values would be that SO4 =, I-, and C1interact with the same titratable positive charge.
Although the data fit well with the SBS model, this is not the only model which could explain the increase of SO4 = dissociation constants with pHo. A model in which positive fixed charges act to increase the local concentration of anions near the transport site, without themselves participating in substrate binding, predicts qualitatively similar results. In this case, the ratio of local anion concentration to that in the bulk medium is governed by the electrical potential between the local region and the bulk medium. The equilibrium distribution ratio of divalent anions (SO4 =) between the local and bulk regions, given by the Nernst equation, will thus be the square of the corresponding distribution for monovalent anions (I-) at each value of pHo. Table  I shows that this relationship does not hold for pH o 10 as compared to pHo 8.5, providing evidence against this simple version of the fixed charge model. As discussed  (1996). *The average value of the parameters in the column at each pH o. tSignificanfly different from (Kol/KotpH85) 2, determined by an unpaired t test (P < 0.05), but not significantly different from/~,l/Kolpn8. 5 (P = 0.11).
in the previous paper, however, precise calculations of the expected results for variations of such fixed charge models are impossible at present, and the model in general involves complex interactions of C1-and SO4 = ions in the local region which would probably lead to nonlinearities in the Dixon plots. Because this model is more complex and less predictive than the SBS model, and because it does not seem to fit the data as well, it seems that the alternative SBS model provides a better explanation of the pHo effects.
pH Dependence of DNDS Inhibition DNDS, a stilbene derivative with two negative charges, inhibits anion transport by reversibly binding to band 3. At neutral pHo DNDS seems to act primarily as a competitive (transport site) inhibitor (Fr6hlich, 1982 Frrhlich (1982), using an optical absorbance technique to measure DNDS binding. Because Fr6hlich's data were obtained by using citrate to replace CI-and to maintain constant ionic strength, whereas the experiments in Table II had varying ionic strength and no citrate, it seems likely, as discussed in the preceding paper (Liu et al., 1996) in connection with Ko I, that the effects of ionic strength on K~ ~176 are small, or else that effects of ionic strength on the dissociation constant and on the pK values for the positive sites tend to cancel out in this pHo range. K~ ~176 increases with pHo, indicating that the inhibitory potency decreases at higher pHo. This is consistent with the dramatic reduction in the inhibition of C1-exchange by DNDS at pHo values >10 observed by Wieth and Bjerrum (1983).
If DNDS binds to the transport site, the increase in K~ ~176 with increasing pHo could be partially or completely the result of the same titration which causes the 297 LIu ET AL.  Bjerrum (1992) has also reported unpublished data showing that DNDS affinity is affected at lower pH o values than is C1-affinity. At higher pH o values, /~DNDS increases to such an extent that it is difficult to present on a linear scale, and is better seen on a logarithmic plot such as Fig. 3 B. By pHo 11.2, there is a ~700-fold increase in K, DNDs, compared to only a N40-fold increase in Ko I.

Competitive and Noncompetitive Effects of DNDS
According to the ping-pong mechanism, the distribution of band 3 among its various forms is a function of [Cli] and [Clo]. In general, the reciprocal of the IDa0 for an inhibitor such as DNDS is the average of the reciprocals of the dissociation constants of DNDS for each form of band 3 (see Fig. 4 A), weighted according to the fraction of band 3 in each form in the absence of inhibitor (Knauf et al., 1992): where/~ is the total concentration of band 3 and Kf, Kg, and Kh are dissociation constants of inhibitor for the ECI o,/~, and ECI i conformations respectively (Fig. 4 A). Thus, the IDs0 will vary with [Clo], as the distribution of band 3 among the various conformations changes. If DNDS is an external competitive inhibitor, it can bind only to the E o conformation of band 3. That is,/~, Kg,  (Fr6hlich et al., 1983;Fr6hlich and Gunn, 1986). For example, when [Clo] = K1/2o (the concentration of external CI-that gives halfmaximal flux), Eo = E,/2. The results in Table II show strong effects of [Clo] on the DNDS ID50 at pHo 7.2, as expected. In the low C1-(0.5-1 mM) media, the IDs0 for DNDS is ~25 times  Table II, for/~c and/~" are from Table II1, and for Ko l are from Liu et al. (1996). The dashed line represents predictions based on a model in which a doubly charged anion is located at the same distance from the titratable sites as I-, calculated using the two fitratable site model in which the completely deprotonated form has negligible affinity for anions, with the pK values given in the preceding paper (Liu et al., 1996).
lower than the ID50 in 149 mM CI-medium, suggesting that DNDS binds preferentially to the E o conformation, which is consistent with the idea that DNDS is a competitive inhibitor (Fr6hlich, 1982). At higher pHo, the IDs0 for DNDS does not increase as much with increasing [Clo]. In part this is due to the increase in Kl/2o, which makes C1-a less effective competitor, but it also suggests that DNDS can bind to other conformations of band 3 as well as to Eo. The possibility that DNDS displays some noncompetitive inhibition at high pHo (and even at lower pHo values) was tested as follows: if one assumes (Fig. 4 B)   with dissociation constant Ke c, can also bind noncompetitively to all forms of band 3 with the same dissociation constant K. n, and that the competitive and noncompetitive binding of DNDS to E o are mutually exclusive, a linear Dixon plot will be obtained as in Fig. 2 and 1/IDs0 will be given by (see Appendix):  Fig. 3, where K, ~ and /~n, divided by their values at pH 7.2, are plotted for comparison with similarly normalized values of /~nm)s and Ko I. Like /~bNDS, K c begins to increase between pHo 8 and 10 ( Fig. 3 A), and by pH o 11.2 reaches a value ~1,000 times larger than its value at pHo 7.2 (Fig. 3 B). In contrast, K~ n does not increase until pHo is raised to 9.9, as Standard errors for/~c and K~ n were < 30% of the mean, except for one /(n value at pH 10.9. Average /~DNDSvalues for each pH o were calculated from Table II; standard errors for pH o > 9 were < 38% of the mean. shown in Fig. 3 B, and the maximum change by pHo 11.2 is only ~20-fold (Fig. 3 A). The different titration profiles for Kec and Ken fit well with the concept that DNDS binds at different sites in the band 3 molecule for competitive and noncompetitive inhibition. Because Ken (~5 IxM at pH o 7.2) is much higher than Ke~ (Table III), at low concentrations DNDS acts primarily as a competitive inhibitor and the noncompetitive component does not play an important role. However, when DNDS is used at relatively high concentrations, for example, to protect the substrate binding site from chemical modification, the experiments must be interpreted cautiously, because under these conditions DNDS binds to at least one other site (the noncompetitive binding site) besides the transport site. Two recent brief reports provide further evidence that there is a noncompetitive component of DNDS inhibition (Knauf et al., 1993;Aranibar et al., 1994).

pH Dependence of Competitive and Noncompetitive DNDS Binding
If the dissociation constant for DNDS binding to the Eo form of band 3, Ke ~ is fitted to a single titratable site model, the pK value is 8.3 -+ 0.5. The large standard error probably results from the sharp increase in /~DNDS with pHo, the scatter in the high pHo data, and the failure to observe a plateau at high pHo. (In fact, the calculated value for the DNDS dissociation constant of the deprotonated form approaches infinity.) A fit to a twosite model, with pKo fixed at 11.35 (Bjerrum, 1992) gives an apparent pKc of 8.7 + 0.3. For Ker the one-site model gives a pK c of 8.3 + 0.6 and the two-site model gives 9.0 + 0.2. All of these values fall near the range (8.7-9.5) obtained from Bjerrum's (1992) model and from fits to Ko ~ data in the preceding paper (Liu et al., 1996). Thus, the data are consistent with the concept that the same titratable site(s) affect DNDS and Ibinding.
As another test of the hypothesis that DNDS and Iare affected by the same titratable sites, we calculated the expected pHo dependence of the dissociation constant for a doubly charged anion that is bound at precisely the same location as I-. This was done by taking the best-fit parameters for the pHo dependence of Ibinding, based on a model in which titration of the high-pK site (D) abolishes anion binding affinity (Bjerrum, 1992;Liu et al., 1996), and squaring the value of the dissociation constant with the C residue deprotonated (corresponding to K+I), as predicted by Eq. 4. The prediction, shown by the dashed lines in Fig. 3, A and B, falls well below the observed KeDNDS and Kec values over the entire pHo range, suggesting that additional titratable positive charges besides those that affect I-binding influence the competitive binding of DNDS. Evidence that DNDS binds in a region with several positive charges (Passow, 1986) makes this possibility more likely. Moreover, site-directed mutations of Lys-558 and Lys 869 of mouse band 3 (corresponding to Lys-539 and Lys-851 of human band 3) have much larger effects on DNDS affinity than on C1-affinity , consistent with the idea that additional charge interactions are involved in DNDS binding.
The lower-affinity noncompetitive component of DNDS inhibition, Ken, fits better to a one or two-titratable site model, with pK's of 9.4 _+ 0.5 in either case, although the affinities for the protonated and deprotonated forms are very poorly defined. This is not significantly different from the pK value obtained from the Ko I data (Liu et al., 1996) or from Bjerrum's model, so the data do not disprove the hypothesis that the same (probably Lys) site may affect both binding of transported substrates and the noncompetitive binding of DNDS. This might seem an unlikely situation, but it is easier to envision if the amino group is actually located several A from the transport site (see below). On the other hand, a separate group with a similar pK may be responsible for the pH dependence of Ke n.

pHo Dependence of External Cl-Half Saturation K1/2o and Maximum Flux, jmo
The data in Table II for the effect of pH o on K1/2o are plotted in Fig. 5 (solid circles). It is apparent that Kl/2o increases dramatically at pH values > 9/According to the ping-pong model for anion exchange (see Appendix and Fr6hlich and Gunn, 1986), K1/2o is given by: 1 The values of K~/2o at pH 7-8.5 are somewhat lower than those previously reported by Gunn and Fr6hlich (1979) at pH 7.8. This may be due to the fact that citrate was not used in these experiments to maintain ionic strength. Thus, as discussed in the preceding paper (Liu et al., 1996), competitive effects of citrate were avoided, but the conslants are "apparent" in the sense that they apply strictly only to conditions where the ionic strength is allowed to vary with [CIo]. (7) where K o and/~-are the dissociation constants for binding of el-to the outward-facing or inward-facing transport sites, respectively, and where k is the rate constant for conversion from the inward-facing ECli form to the outward-facing ECIo form, while k' is the rate-constant for the reverse conformational change (see Fig. 4). Because [Cli] is high and constant, the term/~-/[Cli] is unlikely to strongly influence K1/2o. An increase in K1/2o could, however, reflect an increase in Ko with pH o, or could be related to either an increase in k or a decrease in k', any one of which would increase the numerator in Eq. 7.
The maximum CI-exchange flux, with constant [Cli] and with saturating [Clo], is given by: jmo is plotted as a function ofpHo in Fig. 5 (open circles). Like K1/2o, jmo also increases with increasing pHo, but in this case the increase is smaller and occurs only at pHo around 11. If Eq. 7 is divided by Eq. 8, one is left with: because the identical denominators of Eq. 7 and 8 cancel out. (This is the reason why, in a ping-pong model [Gunn and Fr6hlich, 1979], the ratio K1/2o/J m~ is independent of [eli].) When the ratio Ki/zo/J m~ is plotted against pH o (Fig. 6, solid circles), there is a large increase with increasing pHo. This increase must be related either to an increase in Ko or to a decrease in k' or both, because E,, the total amount of band 3, remains constant.  with pH o, we multiplied K1/zo/J m~ at each pHo by the calculated ratio of k' to k' at pH 8.5 (k'r), determined as described in Fig. 7 of Liu et al. (1996). This gives Ko/(k'rEt) which, since k'r and/~ are constants, should reflect the effects of pHo on K o. The solid line through the open circles is a least-squares best-fit (using Origin) to a two titratable site model, with each point weighted as its inverse, including both our data and Bjerrum's (1992) data, as shown in Fig. 7.
In the preceding paper (Liu et al., 1996), we calculated the pHo dependence of k', relative to its value at pH 8.5, k'r, based on the assumption that external pH has the same effects on el-binding as on I-binding. If we multiply equation 6 by k'/k'r, we obtain: Because all of the terms on the right-hand side are conslants except Ko, a plot of this function (Fig. 6, open circles) should indicate the effects of pHo on do.
These observations complement and are consistent with Bjerrum's (1992) report thatJ m~ increases slightly as pHo is raised >11, that K1/2o increases dramatically with pHo, and that K1/zo/J m~ tends to increase 10-fold for a 1-U change in pH at high pHo. In fact, when Bjerrum's data for Kl/zo/J m~ are multiplied by the k'/k'r data from the preceding paper (Liu et al., 1996), the resulting Ko/k'rEt values (Fig. 7, solid circles) fit well with those in Fig. 6 (open circles in Fig. 7).
When all of the Ko/k'rE t data are fitted to a two titratable site model (assuming no CI-binding to the completely deprotonated form), keeping the dissociation constant for the neutral form at the measured value of 0.02, we obtain pKc = 8.8 +-0.6 and pKn = 12.1 --0.5, in reasonable agreement with the pK values (9.3 -+ 0.3 and 12.7 -+ 0.3) determined by fitting the Ko x data to the same model (Liu et al., 1996). The Ko/k'rEt value for the alkaline form, with one residue deprotonated, is very poorly determined but is ~1.8 + 1.3. The mean value  Table I of the preceding paper (Liu et al., 1996). The right axis shows values of Ko/(k'~EO from Fig. 6 (open circles) and calculated from Fig. 13 of Bjerrum (1992) (solid circles), using the conversion factor 0.38 pmol/cm -2 s -1 = 1 mmol kg -1 min -l (Gunn and Frthlich, 1989). The solid line represents a best fit to a two titratable site model assuming negligible binding to the completely deprotonated form. The Ko/(k'~E 0 value for the protonated form is fixed at 0.02 mM s kg mmo1-1, the value for the alkaline form with the C residue deprotonated is 1.8 _+ 1.3 mM s kg mmo1-1, the pKc value is 8.8 -+ 0.6, and the pIgv value is 12.1 _+ 0.5. The axes for /<,/(k'~t) and Ko I are scaled so that the values match at pHo 7.2.
corresponds to a 90-fold increase in K o, not significantly different from the 70-fold increase seen for Ko I. Fig. 7 also shows a direct comparison of the Ko/ (k'~Et) data with the Ko I data. When the scales are adjusted for the different magnitude of each parameter at pHo 7.2, it appears that the major difference is that some of the KJ values, particularly at pH o 11.6 and 12.4, fall somewhat below the Ko/(k'~Et) values. Because Ko should be the only pH-dependent variable in Ko/(k'~Et), this seems to violate the assumption that pH o has parallel effects on CI-and I-dissociation constants. Most of the apparent discrepancy, however, seems to be related to scatter in the Ko ~ values; for example, the fit of the Ko I data to the CI-data is much better around pHo 12 than at pHo 12.4. Also, as mentioned above, fits to the data indicate no significant difference in the effect of deprotonation of the low-pK C residue on Ko/(k'~) as compared to Ko I, so the assumption that I-and C1-affinities are affected similarly by pHo is at least approximately valid.
Even if the data in Fig. 7 are taken at face value, the additional increase in Ko/(k'/Et) relative to that for Ko l would be only about twofold. If we make the corresponding assumption that the alkaline form of band 3 (with the C group deprotonated) exhibits twice as large an increase in Ko (for C1-) as in Ko I, Eq. T2.6 in the preceding paper (Liu et al., 1996) must be replaced by: T b ----K,,,+IK I = 2T = 2Ko.++/K~++ where Tb represents the ratio of C1-to I-affinities for the alkaline state and the other terms have the meanings defined in the preceding paper. Insertion of this equation yields the final result that the term (k'+/k'++) in Eq. 4 of the preceding paper is replaced by (k'+/ k'++)/2. Thus, the fit to the Dixon plot slope data gives half of (k'+/k'++), rather than (k'+/k'++). In general, the value obtained from the fit is equal to T/Tb (k'+/ k'++). Thus, the true value of (k'+/k'++) would be 0.28 instead of 0.14. Such a change would not substantially affect the conclusions drawn in the preceding paper.
To see whether or not particular externally-accessible lysines are involved in external anion binding, we used BSSS, a nonpenetrating cross linking reagent (Starts and Kakkad, 1983) which probably reacts with the same Lys residues with which H2DIDS reacts (Jennings et al., 1985;Okubo et al., 1994). BSSS has the advantage that it does not introduce new charged groups into band 3, as do the disulfonic stilbenes, and that upon reaction with an amino group, the pK is changed so that the positive charge is absent at neutral pH. Also, since BSSS does not completely inhibit anion exchange, the modified transport system may be studied. In fact, although BSSS inhibits Cli-Bro exchange by at least 90% at pHo > 8, Cli-Br o exchange of BSSS treated cells could be activated by lowering external pH (Jennings et al., 1985). In BSSS treated cells the half-saturation constant for extracellular Br-was higher than the corresponding K1/2 for control cells, indicating that reaction with BSSS decreases substrate affinity (Jennings et al., 1985). C1-exchange was inhibited by :"90% at pHo 7.2 in BSSS treated cells (Knauf and Spinelli, 1995), but the remaining flux could still be measured easily. Therefore, we examined how BSSS treatment affects the pH dependence of substrate binding to band 3. Because Cli-Io exchange is stimulated by BSSS (Jennings et al., 1985), we were unable to measure KJ in BSSS treated cells, since the Dixon plot intersection technique requires that Cli/CIo exchange be much faster than Cli/I o exchange (Knauf and Brahm, 1989;Liu et al., 1996). We chose instead to measure the effects of pHo on the apparent half-saturation constant for external CI-, Kl/2o, and on the apparent maximum flux, jmo, in BSSStreated cells with a constant pHi (7.2) and constant [Cli] (:--150 mM), as described above for control cells.
j .... (solid circles in Fig. 8) is much lower in BSSStreated cells than in control cells (compare solid circles in Fig. 8 with open circles in Fig. 5; note difference in scale), and remains fairly constant above pHo 8.7, decreasing slightly above pH 11. This suggests (Eq. 8) that k is greatly reduced in BSSS-treated cells. The increase inJ m~ at low pHo (6.4) is similar to the behavior that is seen for Cli/Br o exchange, where more acidic pHo acti-vates transport with a pK of N7 (Jennings et al., 1985). Fig. 8 also shows data for K~/~o and K1/2o/J m~ for BSSStreated cells as a function of pHo. Even at the lowest pH values shown, K1/2o (open squares) is much higher than in control cells (Fig. 5). This probably reflects an increase in Ko (Eq. 7), which could be explained either if neutralization of an amino group reduces the favorable electrostatic free energy for CI-binding or if BSSS sterically or allosterically hinders C1-binding. If k/k' is assumed to remain constant, the increase in K~/2 o would correspond to a 32-fold increase in Ko relative to untreated cells around pH o 8.5-8.7.
When K1/2o//J m~ is plotted (open circles in Fig. 8), it is clear that the value is much greater than for the control (solid circles in Fig. 6; note difference in scale) at all pH values. At pH 8.7, for example, with BSSS Kl/~o/J m~ is ~'-,2,400 times larger than for the control. This could be explained by the combined effects of a 32-fold increase in Ko, as discussed above, and a 75-fold decrease in k' (Eq. 9).
The increase in both K1/2o and K1/2o/J m~ as pH o is increased from 8.7 to 10 could be due to either a further increase in K o or a decrease in k' (Eq. 7 and 9). In either case, the data indicate that a low-pK titration is preserved in BSSS-treated cells, suggesting that the C residue does not react with BSSS. If we fit the K~/2o/J m~ data to a two titratable site model, with pK c fixed at 8.8 and pKD at 11.35 (Bjerrum's (1992) parameters for relatively low ionic strength media), the fit is not too good for the left limb of the bell-shaped curve (dashed line in Fig. 9). An even poorer fit (not shown) is obtained if the value of 9.3 from the preceding paper (Liu et al., 1996) is used for pKc. IfpKc is treated as a variable, the best-fit value is 7.5 + 0.2, and a rather good fit is ob- rained (solid line in Fig. 9). The different value for pKc does not rule out the possibility that the C residue remains titratable after BSSS treatment, because BSSS might alter the environment of the residue and hence might lower its apparent pK. Surprisingly, both K1/2o and Ka/zo/J m~ in BSSStreated cells decrease markedly at pHo > 10 (Fig. 8). The simplest explanation for this (Eqs. 7 and 9) is that Ko decreases, but an increase in k' cannot be ruled out. At pHo > 11, K1/2o for BSSS-treated cells is actually lower than for control cells (compare open squares in Fig. 8 with solid circles in Fig. 5), implying that under these conditions the BSSS-modified band 3 has a higher apparent affinity for C1-than does unmodified band 3. As shown in Fig. 9, this behavior is compatible with a two-site model in which the second titration lowers the apparent dissociation constant for CI-, rather than raising it as in control cells.
The ability of the cross-linker BSSS to prevent the decrease in apparent C1-affinity that normally occurs at high pH might indicate that the increasing negative charge on band 3 causes electrostatic repulsion that "opens up" the band 3 structure and perturbs the external anion binding site. The BSSS intramolecular cross-link would act to stabilize the normal structure and prevent this effect. As the effects of pHo are completely reversible (Wieth and Bjerrum, 1982), any changes in structure which take place must also be reversible. On the other hand, it is possible that the decrease in K1/2o and in K1/2o/ jmo is caused in whole or in part by an increase in k', that would decrease the number of outward-facing sites and thereby decrease the apparent K1/2o ( (Bjerrum, 1992), with Kl/2o/J m~ of 30 + 2, 980 _+ 230, and 44 _+ 3 mM kg s mmo1-1 for the doubly protonated, singly protonated, and unprotonated forms. Solid line depicts best fit with pKD fixed at 11.35 (Bjerrum, 1992)

Identity of the Charged Residues
Structural models of band 3 (Tanner et al., 1988;Lux et al., 1989;Wood, 1992) indicate that there are many Lys and Arg residues in the transmembrane segments or in the loops adjacent to them which could function as the charges that bind external anions. At least one of these, Lys-430, seems unlikely because it is the site of reaction of eosin-5-maleimide, which is a noncompetitive inhibitor and which therefore must not bind to the external transport site (Knauf et al., 1993;Liu and Knauf, 1993;Liu et al., 1995). However, as discussed in the preceding paper (Liu et al., 1996) the amino group that increases substrate affinity could be > 4.4 A from the anion binding site (assuming a dielectric constant of 30 in the region near the transport site), so it might be possible for an inhibitor to react with the amino group and still not block the transport site. Lys-851, which reacts with a transported substrate, pyridoxal phosphate (Kawano et al., 1988) and with a competitive inhibitor, HzDIDS (Okubo et al., 1994), would seem to be a good candidate. The fact that transport can proceed even if the corresponding Lys in the mouse band 3 is altered by site-directed mutation  fits with the idea that this charge increases anion affinity, but is not essential for transport. Jennings et al. (1985), however, have interpreted the effects of BSSS on reductive methylation of this Lys as indicating that BSSS reacts with "Lys-b," now known to be Lys-851 (Okubo et al., 1994). The observation of a low-pK titration in BSSS-treated cells would, therefore, suggest that Lys-851 is not the titratable Lys. Passow (1986) has also argued that the HzDIDS-reactive lysines are not directly involved in substrate binding. There are, however, several other candidate lysines in band 3, as well as several arginines that might be the high-pK residue. Much further work with chemical probes and site-directed mutagenesis will be necessary to identify the functionally-important residues and to determine their precise role in transport.

Determination of K~ n and K~ c for DNDSfrom//)5o and [Cllo
The assumption here is that DNDS inhibits anion transport by binding competitively to the Eo form of band 3 with a dissociation constant of/~c and noncompetitively to Eo,/~, EClo, and ECli with the same dissociation constant, K~ n ( Fig. 4 B) (AI.1) Subscripts i and 0 represent the band 3 conformations with the transport site facing the cytoplasmic or extracellular side of the membrane, respectively. DNDS binds to Eo to form ED o (in competition with substrates) or to form Eo D as a noncompetitive inhibitor (Fig. 4 B)  For a ping-pong model of C1-exchange such as that depicted in Fig. 4, with no inhibitors present, if the ion binding and dissociation steps are rapid compared to the ion translocation steps, the unidirectional effiux is given by (see Frthlich and Gunn, 1986; where Et is the total amount of band 3 and the other parameters are as defined in Fig. 4 with K1/2o and then setting the rightmost term in (A2.1) equal to the other three terms in the right-hand parenthesis, because under these conditions the denominator will be twice the value when [Clo] is maximal, meaning that the flux will be half of the maximal flux (since the numerator is constant): This is text Eq. 7.