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Cloning and characterization of novel human SLC4A8 gene products encoding Na+-driven Cl-/HCO3(-) exchanger variants NDCBE-A, -C, and -D.

ABSTRACT: The reported sequences of the human and mouse Na+-driven Cl-/HCO3(-) exchangers (NDCBEs) differ greatly in their extreme cytosolic COOH termini (Ct). In human NDCBE (NDCBE-B), a 17-amino acid (aa) sequence replaces 66 aa at the equivalent position in mouse NDCBE (NDCBE-A). We performed 5'- and 3'-rapid amplification of cDNA ends (RACE) on human brain cDNA, followed by PCR of full-length cDNAs to determine whether the human SLC4A8 gene was capable of producing the mouselike Ct sequence. Our study confirmed the presence in human cDNA of mouse NDCBE-like transcripts (human NDCBE-A) and also disclosed the existence of three further novel NDCBE transcripts that we have called NDCBE-C, NDCBE-D, and NDCBE-D'. The novel NDCBE-C/D/D' transcripts initiate at a novel "exon 0" positioned approximately 35 kb upstream of the first exon of NDCBE-A/B. NDCBE-C/D/D' protein products are predicted to be truncated by 54 aa in the cytosolic NH(2) terminus (Nt) compared with NDCBE-A/B. Our data, combined with a new in silico analysis of partial transcripts reported by others in the region of the human SLC4A8 gene, increase the known extent of the SLC4A8 gene by 49 kb, to 124 kb. A functional comparison of NDCBE-A/B/C/D expressed in Xenopus oocytes demonstrates that the Nt variation does not affect the basal functional expression of NDCBE, but those with the shorter Ct have a 25-50% reduced functional expression compared with those with the longer Ct. By comparison with an artificially truncated NDCBE that contains neither 17-aa nor 66-aa Ct cassette, we determined that the functional difference is unrelated to the 66-aa cassette of NDCBE-A/C, but is instead due to an inhibitory effect of the 17-aa cassette of NDCBE-B/D.

SUBMITTER: Parker MD

PROVIDER: S-EPMC2519961 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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Cloning and characterization of novel human SLC4A8 gene products encoding Na+-driven Cl-/HCO3(-) exchanger variants NDCBE-A, -C, and -D.

Parker Mark D MD Bouyer Patrice P Daly Christopher M CM Boron Walter F WF

Physiological genomics 20080624 3

The reported sequences of the human and mouse Na+-driven Cl-/HCO3(-) exchangers (NDCBEs) differ greatly in their extreme cytosolic COOH termini (Ct). In human NDCBE (NDCBE-B), a 17-amino acid (aa) sequence replaces 66 aa at the equivalent position in mouse NDCBE (NDCBE-A). We performed 5'- and 3'-rapid amplification of cDNA ends (RACE) on human brain cDNA, followed by PCR of full-length cDNAs to determine whether the human SLC4A8 gene was capable of producing the mouselike Ct sequence. Our study ...[more]

PMID: 18577713

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Project description:We have cloned the squid neuronal Na+-Ca2+ exchanger, NCX-SQ1, expressed it in Xenopus oocytes, and characterized its regulatory and ion transport properties in giant excised membrane patches. The squid exchanger shows 58% identity with the canine Na+-Ca2+ exchanger (NCX1.1). Regions determined to be of functional importance in NCX1 are well conserved. Unique among exchanger sequences to date, NCX-SQ1 has a potential protein kinase C phosphorylation site (threonine 184) between transmembrane segments 3 and 4 and a tyrosine kinase site in the Ca2+ binding region (tyrosine 462). There is a deletion of 47 amino acids in the large intracellular loop of NCX-SQ1 in comparison with NCX1. Similar to NCX1, expression of NCX-SQ1 in Xenopus oocytes induced cytoplasmic Na+-dependent 45Ca2+ uptake; the uptake was inhibited by injection of Ca2+ chelators. In giant excised membrane patches, the NCX-SQ1 outward exchange current showed Na+-dependent inactivation, secondary activation by cytoplasmic Ca2+, and activation by chymotrypsin. The NCX-SQ1 exchange current was strongly stimulated by both ATP and the ATP-thioester, ATP gamma S, in the presence of F- (0.2 mM) and vanadate (50 microM), and both effects reversed on application of a phosphatidylinositol-4',5'-bisphosphate antibody. NCX1 current was stimulated by ATP, but not by ATP gamma S. Like NCX1 current, NCX-SQ1 current was strongly stimulated by phosphatidylinositol-4',5'-bisphosphate liposomes. In contrast to results in squid axon, NCX-SQ1 was not stimulated by phosphoarginine (5-10 mM). After chymotrypsin treatment, both the outward and inward NCX-SQ1 exchange currents were more strongly voltage dependent than NCX1 currents. Ion concentration jump experiments were performed to estimate the relative electrogenicity of Na+ and Ca2+ transport reactions. Outward current transients associated with Na+ extrusion were much smaller for NCX-SQ1 than NCX1, and inward current transients associated with Ca2+ extrusion were much larger. For NCX-SQ1, charge movements of Ca2+ transport could be defined in voltage jump experiments with a low cytoplasmic Ca2+ (2 microM) in the presence of high extracellular Ca2+ (4 mM). The rates of charge movements showed "U"-shaped dependence on voltage, and the slopes of both charge-voltage and rate-voltage relations (1,600 s-1 at 0 mV) indicated an apparent valency of -0.6 charges for the underlying reaction. Evidently, more negative charge moves into the membrane field in NCX-SQ1 than in NCX1 when ions are occluded into binding sites.

Dataset Information

Cloning and characterization of novel human SLC4A8 gene products encoding Na+-driven Cl-/HCO3(-) exchanger variants NDCBE-A, -C, and -D.

Publications

Cloning and characterization of novel human SLC4A8 gene products encoding Na+-driven Cl-/HCO3(-) exchanger variants NDCBE-A, -C, and -D.

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