Electrogenic Na/HCO3 cotransporters from rat and salamander kidney have similar external HCO3 dependencies

I. I. Grichtchenko, Michael F Romero, W. F. Boron

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

The deduced amino-acid sequences of the electrogenic Na/HCO3 cotransporters cloned from Ambystoma (aNBC) and rat (rNBC) are 86% identical. Here, we studied the extracellular HCO3 dependencies of aNBC and rNBC expressed in Xenopus oocytes, injected with cRNA encoding aNBC or rNBC (in Xenopusexpression vector pTLN2). We monitored the shift in membrane potential (dVm) or outward clamp current (Iout, Vhold = -60 mV) caused by switching the external buffer from HEPES to CO2 /HCO3 at a fixed pHo of 7.5 (22°C). The major external anion was gluconate; we varied [HCO3]o from nominally 0 to 99 mM by exchanging gluconate for HCO3, maintaining a constant [CO2]/[HCO3] ratio. In water-injected oocytes, 5% CO2 /33 mM HCO3 caused a small, slow depolarization or inward current. In NBC-expressing oocytes, CO2 /HCO3 caused a large, immediate dVm of -30 to -100 mV (aNBC) or an immediate dIout of 150 - 400 nA (rNBC). The dVm and dIout were 60% inhibited by 140 uM by DIDS, and 97% by 0 Na+. In aNBC-expressing oocytes, a non-linear, least-squares curve fit of the dVm vs. [HCO3]o data by a normalized Michaelis-Menten equation yielded an apparent Km for extracellular HCO3 of 15.1 (SD=1.3) mM, and a Vmax 1.54 (SD=0.05) fold greater than the dVm caused by 33 mM HCO3. In similar dVm experiments with rNBC, the Km was 12.9 (SD=3.1) mM, and the normalized Vmax was 1.40 (SD=0.12). In rNBC oocytes, Iout was large enough for determining the HCO3 dependence: Km was 15.2 (SD=1.4) mM, and normalized Vmax was 1.59 (SD=0.04). Our data shows that external HCO3 dependence is similar for two NBCs from species in different classes (amphibian vs. mammal).

Original languageEnglish (US)
JournalFASEB Journal
Volume12
Issue number5
StatePublished - Mar 20 1998
Externally publishedYes

Fingerprint

Urodela
salamanders and newts
Oocytes
Rats
oocytes
kidneys
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid
HEPES
Kidney
Complementary RNA
Mammals
rats
Depolarization
Clamping devices
Anions
Buffers
Membranes
Amino Acids
Water
Ambystoma

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Electrogenic Na/HCO3 cotransporters from rat and salamander kidney have similar external HCO3 dependencies. / Grichtchenko, I. I.; Romero, Michael F; Boron, W. F.

In: FASEB Journal, Vol. 12, No. 5, 20.03.1998.

Research output: Contribution to journalArticle

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abstract = "The deduced amino-acid sequences of the electrogenic Na/HCO3 cotransporters cloned from Ambystoma (aNBC) and rat (rNBC) are 86{\%} identical. Here, we studied the extracellular HCO3 dependencies of aNBC and rNBC expressed in Xenopus oocytes, injected with cRNA encoding aNBC or rNBC (in Xenopusexpression vector pTLN2). We monitored the shift in membrane potential (dVm) or outward clamp current (Iout, Vhold = -60 mV) caused by switching the external buffer from HEPES to CO2 /HCO3 at a fixed pHo of 7.5 (22°C). The major external anion was gluconate; we varied [HCO3]o from nominally 0 to 99 mM by exchanging gluconate for HCO3, maintaining a constant [CO2]/[HCO3] ratio. In water-injected oocytes, 5{\%} CO2 /33 mM HCO3 caused a small, slow depolarization or inward current. In NBC-expressing oocytes, CO2 /HCO3 caused a large, immediate dVm of -30 to -100 mV (aNBC) or an immediate dIout of 150 - 400 nA (rNBC). The dVm and dIout were 60{\%} inhibited by 140 uM by DIDS, and 97{\%} by 0 Na+. In aNBC-expressing oocytes, a non-linear, least-squares curve fit of the dVm vs. [HCO3]o data by a normalized Michaelis-Menten equation yielded an apparent Km for extracellular HCO3 of 15.1 (SD=1.3) mM, and a Vmax 1.54 (SD=0.05) fold greater than the dVm caused by 33 mM HCO3. In similar dVm experiments with rNBC, the Km was 12.9 (SD=3.1) mM, and the normalized Vmax was 1.40 (SD=0.12). In rNBC oocytes, Iout was large enough for determining the HCO3 dependence: Km was 15.2 (SD=1.4) mM, and normalized Vmax was 1.59 (SD=0.04). Our data shows that external HCO3 dependence is similar for two NBCs from species in different classes (amphibian vs. mammal).",
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N2 - The deduced amino-acid sequences of the electrogenic Na/HCO3 cotransporters cloned from Ambystoma (aNBC) and rat (rNBC) are 86% identical. Here, we studied the extracellular HCO3 dependencies of aNBC and rNBC expressed in Xenopus oocytes, injected with cRNA encoding aNBC or rNBC (in Xenopusexpression vector pTLN2). We monitored the shift in membrane potential (dVm) or outward clamp current (Iout, Vhold = -60 mV) caused by switching the external buffer from HEPES to CO2 /HCO3 at a fixed pHo of 7.5 (22°C). The major external anion was gluconate; we varied [HCO3]o from nominally 0 to 99 mM by exchanging gluconate for HCO3, maintaining a constant [CO2]/[HCO3] ratio. In water-injected oocytes, 5% CO2 /33 mM HCO3 caused a small, slow depolarization or inward current. In NBC-expressing oocytes, CO2 /HCO3 caused a large, immediate dVm of -30 to -100 mV (aNBC) or an immediate dIout of 150 - 400 nA (rNBC). The dVm and dIout were 60% inhibited by 140 uM by DIDS, and 97% by 0 Na+. In aNBC-expressing oocytes, a non-linear, least-squares curve fit of the dVm vs. [HCO3]o data by a normalized Michaelis-Menten equation yielded an apparent Km for extracellular HCO3 of 15.1 (SD=1.3) mM, and a Vmax 1.54 (SD=0.05) fold greater than the dVm caused by 33 mM HCO3. In similar dVm experiments with rNBC, the Km was 12.9 (SD=3.1) mM, and the normalized Vmax was 1.40 (SD=0.12). In rNBC oocytes, Iout was large enough for determining the HCO3 dependence: Km was 15.2 (SD=1.4) mM, and normalized Vmax was 1.59 (SD=0.04). Our data shows that external HCO3 dependence is similar for two NBCs from species in different classes (amphibian vs. mammal).

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