Expression of the rat renal electhogenic Na/HCO3 cothansporter (rNBC)

M. F. Romero, M. A. Hediger, P. Fong, W. F. Boron

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Abstract

We have recently expression-cloned the salamander renal electrogenic Na/HCO3 cotransporter (aNBC) (Bornera et al, FASEB J. 10:A89,1996] and by homology the first mammalian form, named rNBC for rat Na Bicarbonate Cotransporter (rNBC) [Romero et al. JASN 7:1259, 1996]. NBC is the major basolateral membrane route for HCO3- reabsorption in the renal proximal tubule, and assists in Na+ reabsorption, rNBC is a 3.6 kb cDNA, encoding a 1035 amino acid protein (-116KD) and is 86% identical to aNBC. Probing a rat tissue northern with rNBC shows transcripts of 7.5kb in kidney, brain, liver, and heart. Using microelectrodes to measure membrane potential (Vm) and intracellular pH (pHi), or to voltage clamp, we examined the transport characteristics of rNBC. Our assay was to apply CO2/HCO3-(pH 7.5), to wait for pHi to stabilize after the CO2-induced pHi acidification, and then to remove Na+. Subcloning rNBC into a Xenopus expression vector increased rNBC expression by ∼50-fold. With the new construct, bath addition of 1.5% CO2/10 mM HCO3- causes the rNBC-oocytes to hyperpolarize (ΔVm=-63±5mV, n=12; Na+ and HCO3- influx), whereas, native or water-injected controls depolarize (ΔVm∼+20mV). In voltage clamp experiments (Vm=-60mV) CO2/HCO3- elicits a peak Δ outward current (∼500nA). With subsequent Na+ removal (choline replacement in CO2/HCO3-) rNBC-oocytes depolarize (ΔVm= +61±4mV, n=27) and ↓ pHi, or show a peak Δ inward current (∼500nA, Na+ and HCO3- eftlux as in the proximal tubule). Bath Na+ removal in native and control oocytes causes a hyperpolarization (ΔVm ∼ -5mV) and pHi does not change. To demonstrate HCO3--dependence, we acidified rNBC-oocytes with butyrate rather than CO2/HCO3+. Butyrate addition slightly hyperpolarizes oocytes (ΔVm=-8±2mV, n=5), moreover, Na+ removal (in butyrate) depolarizes (ΔVm = -5±1mV, n=9) rNBC-oocytes. Additionally, replacing Na+ by Li+ in the presence of CO2/HCO3- mimics the effects of Na+ removal depolarization (ΔVm=+42±3mV, n=3) and ↓ pHi. Cl- removal (replacement by gluconate) does not effect the activity of rNBC. Voltage clamp studies reveal an outward rectification at hypetpolarized Vm in CO2/HCO3- and an inward rectification of rNBC-cotransporter current with Na+ removal. Finally, the functional activity of rNBC is blocked by 200 μM DIDS. Comparing the aNSC and rNBC sequences to the GenBank database revealed they are unique. Thus, rNBC is the first mammalian member of the Na+-linked HCO3- transporters Therefore, rNBC like aNBC is a transporter that is (i) electrogenic and voltage dependent, (ii) HCO3- dependent, (iiii) Na+ not Li+ dependent, (iv) Cl- independent, and (v) blocked by DIDS.

Original languageEnglish (US)
Pages (from-to)A25
JournalFASEB Journal
Volume11
Issue number3
StatePublished - Dec 1 1997

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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    Romero, M. F., Hediger, M. A., Fong, P., & Boron, W. F. (1997). Expression of the rat renal electhogenic Na/HCO3 cothansporter (rNBC). FASEB Journal, 11(3), A25.