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1 Institute of Physiology, University of Zuerich, Zuerich, Switzerland
2 Department of Physiology, Emory University School of Medicine, Atlanta, GA, USA
3 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA
* To whom correspondence should be addressed. E-mail: leilav{at}physiol.unizh.ch.
We extracted RNA from the giant fiber lobe (GFL) of the squid Loligo pealei and performed PCR using degenerate primers that were based on highly conserved regions of Na+-coupled HCO-3 transporters. This approach yielded a novel, 290-bp sequence related to the bicarbonate-transporter superfamily. Using a Loligo opalescens library, we extended the initial fragment in the 3' and 5' directions by a combination of library screening and PCR, and obtained the full-length clone (1198 amino acids) by PCR from Loligo pealei GFL. The amino-acid sequence is 46% identical to mammalian electrogenic and electroneutral Na/HCO3 cotransporters and 33% identical to the anion exchanger AE1. Northern-blot analysis showed strong signals in Loligo pealei GFL, optic lobe and heart, and weaker signals in gill and stellate ganglion. To assess function, we injected in vitro-transcribed cRNA into Xenopus oocytes and subsequently used icroelectrodes to monitor intracellular pH (pHi) and membrane voltage (Vm). Superfusing such oocytes with 5% CO2/33 mM HCO-3 caused a CO2-induced fall in pHi, followed by a slow recovery. The absence of a rapid HCO- 3-induced hyperpolarization indicates that the pHi-recovery mechanism is electroneutral. Ion substitutions showed that Na+ and Cl- are required on opposite sides of the membrane. Transport was blocked by 50 µM 5,5'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). The characteristics of our novel clone fit those of a Na+-driven Cl-HCO3 exchanger (NDCBE).
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