Grichtchenko II et al. (2000), Extracellular HCO(3)(-) dependence of electroge...

XB-ART-11143

J Gen Physiol 2000 May 01;1155:533-46.

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Extracellular HCO(3)(-) dependence of electrogenic Na/HCO(3) cotransporters cloned from salamander and rat kidney.

Grichtchenko II , Romero MF , Boron WF .

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We studied the extracellular [HCOabstract (3) (-)] dependence of two renal clones of the electrogenic Na/HCO(3) cotransporter (NBC) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (DeltaV(m)) elicited by the NBC cloned from the kidney of the salamander Ambystoma tigrinum (akNBC) and by the NBC cloned from the kidney of rat (rkNBC). We used a two-electrode voltage clamp to measure the change in current (DeltaI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCOabstract (3 )(-)/CO(2) (0.33-99 mM HCOabstract (3)(-), pH(o) 7.5) elicited an immediate, DIDS (4, 4-diisothiocyanatostilbene-2,2-disulfonic acid)-sensitive and Na(+)-dependent hyperpolarization (or outward current). In DeltaV(m) experiments, the apparent K(m ) for HCOabstract (3)(-) of akNBC (10. 6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent K(m) for HCOabstract (3)(-) of rkNBC was less (6.5 mM). Because it has been reported that SOabstract (3)(=)/HSO abstract (3)(-) stimulates Na/HCO(3 ) cotransport in renal membrane vesicles (a result that supports the existence of a COabstract (3)(=) binding site with which SOabstract (3)(=) interacts), we examined the effect of SOabstract (3)(=)/HSO abstract (3)(-) on rkNBC. In voltage-clamp studies, we found that neither 33 mM SOabstract (4)(=) nor 33 mM SOabstract (3) (=)/HSOabstract (3)(-) substantially affects the apparent K(m) for HCO abstract (3)(-). We also used microelectrodes to monitor intracellular pH (pH(i)) while exposing rkNBC-expressing oocytes to 3.3 mM HCOabstract (3 )(-)/0.5% CO(2). We found that SO abstract (3)(=)/HSOabstract (3 )(-) did not significantly affect the DIDS-sensitive component of the pH(i) recovery from the initial CO(2 )-induced acidification. We also monitored the rkNBC current while simultaneously varying [CO(2)](o), pH(o), and [COabstract (3)(=)](o) at a fixed [HCOabstract (3)(-)](o) of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [COabstract (3)(=)](o ). However, a pH titration curve nicely fitted the data expressed as current versus pH(o). Thus, rkNBC expressed in Xenopus oocytes does not appear to interact with SOabstract (3 )(=), HSOabstract (3)(-), or COabstract (3)(=).

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Species referenced: Xenopus laevis
Genes referenced: pkm sult2a1

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	Figure 1. Membrane potential and pHi of Xenopus laevis oocytes during a superfusion of 33 HCO3â/5% CO2 solution. (A) Water-injected oocyte. The CO2/HCO3 â solution is Solution 2 in Table . Typical of six experiments. (B) Oocyte expressing rkNBC. Typical of nine experiments. pHo 7.5, 22Â°C.
	Figure 2. Membrane potential of akNBC-expressing Xenopus laevis oocytes during superfusion of solutions with different levels of HCO3 â/CO2. In our assay, we bracketed each test pulse with a pulse of the standard (std) CO2/HCO3 â solution (33 mM HCO3â/5% CO 2, Solution 2 in Table ). We normalized the ÎVm under test conditions to the mean ÎVm for the bracketing std pulses. The HEPES-buffered solution was Solution 1 in Table . Typical of nine experiments.
	Figure 4. Dependence of HCO3â-evoked currents on the expression rkNBC and the presence of Na+. (A) H2 O-injected, control oocyte. (B) Oocyte expressing rkNBC. (C) Effect of removing Na+ in an oocyte expressing rkNBC. In each case, we pulsed the oocyte with a pH 7.5 solution containing 99 mM HCO3 â/15% CO2. Vhold = â60 mV, 22Â°C.
	Figure 3. [HCO3â]o dependence of akNBC and rkNBC, based on ÎVm data. The solid curve represents the result of a nonlinear least-squares curve fit of to the akNBC data (âª) similar to those shown in Fig. 2 . The broken curve represents the result of a similar fit to the rkNBC data (â¡). Each symbol represents the mean of six to nine data points, obtained in separate experiments. The vertical bars represent SEMs; the bars are omitted when they are smaller than the size of the symbol. The kinetic parameters are summarized in the first two lines of Table .
	Figure 6. [HCO3â]o dependence of rkNBC current. The solid curve represents the result of a nonlinear least-squares curve fit of to the data (â¢) similar to those shown in Fig. 5. Each symbol represents the mean of five to eight data points obtained in separate experiments. The vertical bar represents the SEM; the bars are omitted when they are smaller than the size of the symbol. The kinetic parameters are summarized in the last line of Table .
	Figure 5. Membrane current of rkNBC-expressing Xenopus laevis oocyte during superfusion of solutions with different levels of HCO3 â/CO2. The protocol for changing the extracellular solutions was the same as in Fig. 2 . The standard (std) solution contained 33 mM HCO3 â/5% CO2 (Table , Solution 2). Typical of eight experiments. Vhold = â60 mV, 22Â°C.
	Figure 7. Effect of SO4= and SO3=/HSO 3â on the current carried by rkNBC. The oocyte was exposed five times to a solution containing 33 mM HCO3 â/5% CO2. For the first, third, and fifth pulses, we switched from a HEPES solution (Table , Solution 1) to a solution containing 33 mM HCO3â/5% CO2 solution (Table , Solution 2). For the second HCO3 â/CO2 pulse, we switched from a HEPES solution containing 33 mM SO4= (Table , Solution 3) to a 33 mM HCO3â/5% CO2 that also contained 33 mM SO4= (Table , Solution 4). For the fourth HCO3â /CO2 pulse, we switched from a HEPES-containing 33 mM SO3 =/HSO3â (Table , Solution 5) to a 33-mM HCO3â/5% CO2 solution that also contained 33 mM SO3=/HSO3 â (Table , Solution 6). Typical of six experiments. Vhold = â60 mV, 22Â°C.
	Figure 8. Effect of SO4= and SO3=/HSO 3â on the [HCO3â] o dependence of the current carried by rkNBC. (A) Experiments conducted in 33 mM SO4=. The experimental protocol was the same as in Fig. 5, except that all solutions contained 33 mM SO4=. Typical of eight experiments. Vhold = â60 mV, 22Â°C, pH 7.5. (B) Experiments conducted in 26.4 mM SO3=/6.6 mM HSO 3â. The protocol was the same as in A. Typical of 10 experiments. (C) Effect of SO4= and SO3 =/HSO3â on [HCO3 â]o dependency of rkNBC. One of the solid curves is the same as that in Fig. 6, and represents the fit of to the data obtained in the absence of SO4= and SO3=/HSO3 â (â). The other two solid curves represent the fits of to the data obtained in SO4= (âµ), as in A, and the data obtained in SO3=/HSO 3â (â´), as in B. Each symbol represents the mean of 6â17 data points, obtained in separate experiments. The bars representing SEM are omitted because they are smaller than the size of the symbol.
	Figure 9. Effect of SO3=/HSO3â on the DIDS-sensitive recovery of pHi from a CO2 -induced acid load. (A) Absence of SO3=/HSO 3â. During the indicated time, the solution bathing an oocyte expressing rkNBC was switched from standard HEPES ( Table , Solution 1) to a solution containing 3.3 mM HCO3 â/0.5% CO2. During the pHi recovery from the CO2-induced acid load, we blocked rkNBC by applying 1 mM DIDS. (B) Presence of 26.4 mM SO3=/6.6 mM HSO 3â. The protocol was the same as in A, except that all solutions contained 26.4 mM SO3=/6.6 mM HSO 3â.
	Figure 10. Effect of varying [CO3=]o and pHo on the rkNBC current. (A) Relative rkNBC current as a function of [CO3=]o. â€¢ represent data obtained in the presence of Ca2+, and â—‹, with Mg2+ replacing Ca2+. The dashed curve is the result of a nonlinear least-squares fit of the data by a normalized Michaelis-Menten equation. The best-fit value for Km(CO3=) was 6.1 Â± 1.5 Î¼Îœ, and for Imax, 1.09. The solid curve represents the best fit of the data by a normalized Michaelis-Menten equation plus a linear component. The best-fit value for K m(CO3=) was 4.5 Â± 0.6 Î¼Îœ, for Imax was 1.05, and for Î± was 0.000122 Î¼Îœâˆ’1. (B) Relative rkNBC current as a function of pHo. The solid curve is the result of a nonlinear least-squares fit of the data by a normalized pH titration curve ( Boron and Knakal 1992). The best-fit value for pK was 7.50 Â± 0.05. The number of determinations is given in parentheses. The vertical bars indicate SEM values; they are omitted where the length of the bar is smaller than the size of the symbol.
	Figure 11. Predicted effects of SO3= and/or HSO3 â on the pHi changes mediated by NBC. AâG refer to a general scheme in which NBC transports one Na+ , one CO3=, and one HCO3 â. (A) Neither SO3= nor HSO3 â interact with cotransporter. The entering CO 3= can neutralize two H+, and the entering HCO3â can neutralize an additional H +, for a total of three H+ neutralized. (B) SO3 = replaces CO3=. To the extent that SO3= replaces CO3=, only 1.24 H+ are neutralized, and thus the expected rate of pHi increase will be 41% of that in A. (C) HSO3â replaces HCO3â. Only two H+ are neutralized, and thus the expected rate of pHi increase will be only 67% of that in A. (D) SO3= and HSO 3â replace, respectively, CO3= and HCO3â. Only total 0.24 H+ ions are neutralized, and thus the expected rate of pHi increase will be only 8% of that in A. (E) SO3= acts as a competitive inhibitor of CO3=. (F) HSO3 â acts as a competitive inhibitor of HCO3 â. (G) SO3= and HSO3 â both are competitive inhibitors. Aâ²âGâ² refer to a general scheme in which NBC transports one Na+ and three HCO3â. (Aâ²) Neither SO3= nor HSO3â interact with cotransporter. A total of three H+ are neutralized. (Bâ²) HSO3â replaces one HCO 3â. Only two H+ are neutralized, and thus the expected rate of pHi increase will be 67% of that in Aâ². (Câ²) Two HSO3â replace two HCO3â. Only one H+ is neutralized, and thus the expected rate of pHi increase will be only 33% of that in Aâ². (Dâ²) Three HSO3 â replace three HCO3â. No H+ ions are neutralized, and thus the expected rate of pH i increase will be 0% of that in Aâ². In Eâ²âGâ², HSO3â acts as a competitive inhibitor at one, two, and three HCO3â -binding sites, respectively. Figure 11a.
	Figure 11b.

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