Extracellular HCO3/- dependence of electrogenic Na/HCO3 cotransporters cloned from salamander and rat kidney

Irina I. Grichtchenko, Michael F Romero, Walter F. Boron

Research output: Contribution to journalArticle

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Abstract

We studied the extracellular [HCO3/-] dependence of two renal clones of the electrogenic Na/HCO3 cotransporter (NBG) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (ΔV(m)) elicited by the NBC cloned from the kidney of the salamander Arabystoma 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 (ΔI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCO3/-/ CO2 (0.33-99 mM HCO3/-, pH(o) 7.5) elicited an immediate, DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid)sensitive and Na+- dependent hyperpolarization (or outward current). In ΔV3(m) experiments, the apparent K(m) for HCO3/- of akNBC (10.6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent K(m) for HCO3/- of rkNBC was less (6.5 mM). Because it has been reported that SO3/(=)/HSO3/- stimulates Na/ HCO3 cotransport in renal membrane vesicles (a result that supports the existence of a CO3/(=) binding site with which SO3/(=) interacts), we examined the effect of SO3/(=)/HSO3/- on rkNBC. In voltage-clamp studies, we found that neither 33 mM SO4/(=) nor 33 mM SO3/(=)/HSO3/- substantially affects the apparent K(m) for HCO3/-. We also used microelectrodes to monitor intracellular pH (pH(i)) while exposing rkNBC-expressing oocytes to 3.3 mM HCO3/-/0.5% CO2. We found that SO3/(=)/HSO3/- did not significantly affect the DIDS-sensitive component of the pH(i) recover), from the initial CO2-induced acidification. We also monitored the rkNBC current while simultaneously varying [CO2](o), pH(o), and [CO3/(=)](o) at a fixed [HCO3/-](o) of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [CO3/(=)](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 SO3/(=), HSO3/-, or CO3/(=).

Original languageEnglish (US)
Pages (from-to)533-545
Number of pages13
JournalJournal of General Physiology
Volume115
Issue number5
DOIs
StatePublished - May 2000
Externally publishedYes

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Urodela
Kidney
Oocytes
Microelectrodes
Xenopus
Acids
Membrane Potentials
Electrodes
Clone Cells
Binding Sites
Membranes

Keywords

  • Carbonate
  • Extracellular pH
  • Intracellular pH
  • Sulfite
  • Xenopus oocytes

ASJC Scopus subject areas

  • Physiology

Cite this

Extracellular HCO3/- dependence of electrogenic Na/HCO3 cotransporters cloned from salamander and rat kidney. / Grichtchenko, Irina I.; Romero, Michael F; Boron, Walter F.

In: Journal of General Physiology, Vol. 115, No. 5, 05.2000, p. 533-545.

Research output: Contribution to journalArticle

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abstract = "We studied the extracellular [HCO3/-] dependence of two renal clones of the electrogenic Na/HCO3 cotransporter (NBG) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (ΔV(m)) elicited by the NBC cloned from the kidney of the salamander Arabystoma 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 (ΔI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCO3/-/ CO2 (0.33-99 mM HCO3/-, pH(o) 7.5) elicited an immediate, DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid)sensitive and Na+- dependent hyperpolarization (or outward current). In ΔV3(m) experiments, the apparent K(m) for HCO3/- of akNBC (10.6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent K(m) for HCO3/- of rkNBC was less (6.5 mM). Because it has been reported that SO3/(=)/HSO3/- stimulates Na/ HCO3 cotransport in renal membrane vesicles (a result that supports the existence of a CO3/(=) binding site with which SO3/(=) interacts), we examined the effect of SO3/(=)/HSO3/- on rkNBC. In voltage-clamp studies, we found that neither 33 mM SO4/(=) nor 33 mM SO3/(=)/HSO3/- substantially affects the apparent K(m) for HCO3/-. We also used microelectrodes to monitor intracellular pH (pH(i)) while exposing rkNBC-expressing oocytes to 3.3 mM HCO3/-/0.5{\%} CO2. We found that SO3/(=)/HSO3/- did not significantly affect the DIDS-sensitive component of the pH(i) recover), from the initial CO2-induced acidification. We also monitored the rkNBC current while simultaneously varying [CO2](o), pH(o), and [CO3/(=)](o) at a fixed [HCO3/-](o) of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [CO3/(=)](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 SO3/(=), HSO3/-, or CO3/(=).",
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T1 - Extracellular HCO3/- dependence of electrogenic Na/HCO3 cotransporters cloned from salamander and rat kidney

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AU - Romero, Michael F

AU - Boron, Walter F.

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N2 - We studied the extracellular [HCO3/-] dependence of two renal clones of the electrogenic Na/HCO3 cotransporter (NBG) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (ΔV(m)) elicited by the NBC cloned from the kidney of the salamander Arabystoma 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 (ΔI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCO3/-/ CO2 (0.33-99 mM HCO3/-, pH(o) 7.5) elicited an immediate, DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid)sensitive and Na+- dependent hyperpolarization (or outward current). In ΔV3(m) experiments, the apparent K(m) for HCO3/- of akNBC (10.6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent K(m) for HCO3/- of rkNBC was less (6.5 mM). Because it has been reported that SO3/(=)/HSO3/- stimulates Na/ HCO3 cotransport in renal membrane vesicles (a result that supports the existence of a CO3/(=) binding site with which SO3/(=) interacts), we examined the effect of SO3/(=)/HSO3/- on rkNBC. In voltage-clamp studies, we found that neither 33 mM SO4/(=) nor 33 mM SO3/(=)/HSO3/- substantially affects the apparent K(m) for HCO3/-. We also used microelectrodes to monitor intracellular pH (pH(i)) while exposing rkNBC-expressing oocytes to 3.3 mM HCO3/-/0.5% CO2. We found that SO3/(=)/HSO3/- did not significantly affect the DIDS-sensitive component of the pH(i) recover), from the initial CO2-induced acidification. We also monitored the rkNBC current while simultaneously varying [CO2](o), pH(o), and [CO3/(=)](o) at a fixed [HCO3/-](o) of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [CO3/(=)](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 SO3/(=), HSO3/-, or CO3/(=).

AB - We studied the extracellular [HCO3/-] dependence of two renal clones of the electrogenic Na/HCO3 cotransporter (NBG) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (ΔV(m)) elicited by the NBC cloned from the kidney of the salamander Arabystoma 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 (ΔI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCO3/-/ CO2 (0.33-99 mM HCO3/-, pH(o) 7.5) elicited an immediate, DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid)sensitive and Na+- dependent hyperpolarization (or outward current). In ΔV3(m) experiments, the apparent K(m) for HCO3/- of akNBC (10.6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent K(m) for HCO3/- of rkNBC was less (6.5 mM). Because it has been reported that SO3/(=)/HSO3/- stimulates Na/ HCO3 cotransport in renal membrane vesicles (a result that supports the existence of a CO3/(=) binding site with which SO3/(=) interacts), we examined the effect of SO3/(=)/HSO3/- on rkNBC. In voltage-clamp studies, we found that neither 33 mM SO4/(=) nor 33 mM SO3/(=)/HSO3/- substantially affects the apparent K(m) for HCO3/-. We also used microelectrodes to monitor intracellular pH (pH(i)) while exposing rkNBC-expressing oocytes to 3.3 mM HCO3/-/0.5% CO2. We found that SO3/(=)/HSO3/- did not significantly affect the DIDS-sensitive component of the pH(i) recover), from the initial CO2-induced acidification. We also monitored the rkNBC current while simultaneously varying [CO2](o), pH(o), and [CO3/(=)](o) at a fixed [HCO3/-](o) of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [CO3/(=)](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 SO3/(=), HSO3/-, or CO3/(=).

KW - Carbonate

KW - Extracellular pH

KW - Intracellular pH

KW - Sulfite

KW - Xenopus oocytes

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