Characterization of human SLC4A10 as an electroneutral Na/HCO3 cotransporter (NBCn2) with Cl- self-exchange activity

Abstract

The SLC4A10 gene product, commonly known as NCBE, is highly expressed in rodent brain and has been characterized by others as a Na(+)-driven Cl-HCO(3) exchanger. However, some of the earlier data are not consistent with Na(+)-driven Cl-HCO(3) exchange activity. In the present study, northern blot analysis showed that, also in humans, NCBE transcripts are predominantly expressed in brain. In some human NCBE transcripts, splice cassettes A and/or B, originally reported in rats and mice, are spliced out. In brain cDNA, we found evidence of a unique partial splice of cassette B that is predicted to produce an NCBE protein with a novel C terminus containing a protein kinase C phosphorylation site. We used pH-sensitive microelectrodes to study the molecular physiology of human NCBE expressed in Xenopus oocytes. In agreement with others we found that NCBE mediates the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid-sensitive, Na(+)-dependent transport of HCO(3)(-). For the first time, we demonstrated that this transport process is electroneutral. Using Cl(-)-sensitive microelectrodes positioned at the oocyte surface, we found that, unlike both human and squid Na(+)-driven Cl-HCO(3) exchangers, human NCBE does not normally couple the net influx of HCO(3)(-) to a net efflux of Cl(-). Moreover we found that that the (36)Cl efflux from NCBE-expressing oocytes, interpreted by others to be coupled to the influx of Na(+) and HCO(3)(-), actually represents a CO(2)/HCO(3)(-)-stimulated Cl(-) self-exchange not coupled to either Na(+) or net HCO(3)(-) transport. We propose to rename NCBE as the second electroneutral Na/HCO(3) cotransporter, NBCn2.

FIGURE 1.

Human splice variants of NCBE. The horizontal bars are scale representations of aligned NCBE protein variants. The putative boundaries of the soluble N terminus (Nt), transmembrane domain (TMD), and soluble C terminus (Ct) are marked along the top of the figure. The numbered, vertical bars mark positions of individual transmembrane spans. The total length in amino acids of each NCBE is given on the right-hand side of the diagram. Human variants NCBE-A (GenBank protein accession number BAB18301) and NCBE-B (reported in the present study, GenBank protein accession number AAQ83632) are shown. The sites of protein variations introduced by the presence or absence of RNA splice cassettes are labeled “A” and “B.” The brace identifies the position of the cDNA hybridization sequence used to probe the northern blots presented in Fig. 2.

FIGURE 2.

Northern blots of human NCBE. Multiple tissue northern blots (Clontech) were probed with a ³²P-labeled cDNA designed to hybridize with the region of NCBE indicated in Fig. 1. These blots were of human cRNA from various organs (A) and human cRNA from various brain regions (B).

FIGURE 3.

pH_i recovery mediated by NCBE. Representative traces from experiments in which we simultaneously recorded pH_i and V_m are shown. A, NCBE-expressing oocytes. B, H₂O-injected oocytes. In both cases, the oocytes were acidified in our solution (segment ab). pH_i recovery from the acid load (bc) was observed in oocytes expressing NCBE. The application of to NCBE-expressing oocytes did not elicit and rapid changes in V_m (arrow in lower panel).

FIGURE 4.

dependence of pH_i recovery mediated by NCBE. Shown are representative traces from a series of experiments in which we simultaneously recorded pH_i and V_m in NCBE-expressing oocytes that we acidified either in our solution (segment ab) or in our butyrate solution (segment de). pH_i recovery from the acid load was only observed when cells were acidified in the presence of extracellular (bc versus ef). The unusually large depolarization in segment a′c′ of this experiment was typical for this particular set of experiments but not representative of NCBE expressing oocytes as a whole (see Fig. 3 and Figs. 5, 6, 7, 8) and likely reflects an unusually tight membrane (as evidenced by the relatively negative resting V_m) in the presence of the usual, small, endogenous acid-stimulated current.

FIGURE 5.

Na⁺ dependence of NCBE-mediated transport (Part I). Shown is a representative trace from a series of experiments in which we simultaneously recorded pH_i and V_m in NCBE-expressing oocytes that we acidified in a solution (segment ab) in which NMDG⁺ fully replaced Na⁺. pH_i recovery from the acid load did not occur in this solution (bc) or in a Li⁺-containing solution (cd). pH_i recovery was only permitted when Na⁺ was restored to the bath (de). pH_i recovery ceased again upon subsequent removal of bath Na⁺ (ef). The application of to NCBE-expressing oocytes did not elicit and rapid changes in V_m (arrow in lower panel).

FIGURE 6.

Na⁺ dependence of NCBE-mediated transport (Part II). Shown are representative traces from a series of experiments in which we simultaneously recorded pH_i and V_m in NCBE-expressing oocytes that we acidified in -containing solutions (segments ab). A, NCBE-expressing oocyte. B, H₂O-injected oocyte. In the presence of Na⁺, pH_i recovery from the acid load occurred in oocytes expressing NCBE (segment bc in A; 12 ± 1 × 10^–5 pH units/s, n = 46) but not H₂O-injected oocytes (segment bc in B; 4 ± 1 × 10^–5 pH units/s, n = 24). In both panels, removal of bath Na⁺ caused pH_i to fall slowly (cd) as summarized in the text.

FIGURE 7.

Extracellular Cl^– independence of NCBE-mediated transport. A representative trace from a series of experiments in which we simultaneously recorded pH_i and V_m in NCBE-expressing oocytes that we twice acidified in solutions, once in the presence and once in the absence of bath Cl^–. In half of the experiments and in the trace shown here, the oocyte was acidified initially in a Cl^–-containing solution (segment ab) and subsequently in a Cl^–-free solution (segment a′b′). In the other half of the experiments, we reversed the order of the two pulses (not shown); oocytes were acidified initially in a Cl^–-free solution and subsequently in a Cl^–-containing solution. The mean pH_i recovery rate from the acid load was identical in both solutions (bc versus b′c′).

FIGURE 8.

DIDS blockade of NCBE-mediated transport. Shown are representative traces from a series of experiments in which we simultaneously recorded pH_i and V_m. A, NCBE-expressing oocytes. B, H₂O-injected oocytes. In both cases, we acidified the cells in a -containing solution (segments ab). pH_i recovery from the acid load occurred in oocytes expressing NCBE (segment bc in A; 17 ± 2 × 10^–5 pH units/s, n = 14) but not H₂O-injected oocytes (segment bc₁ in B; 1 ± 1 × 10^–5 pH units/s, n = 7). Addition of 200 μm DIDS (Sigma) to the bath caused the pH_i of both populations of oocytes to fall slowly (cd). In many oocytes, we observed a brief alkalinization upon DIDS application, such as that shown in B, segment c₁c.

FIGURE 9.

Perturbations of surface [Cl^–] in response to the application of . Shown are representative traces from a series of experiments in which we simultaneously monitored [Cl^–]atthe oocyte surface ([Cl^–]_S) as well as pH_i and V_m as we exposed cells to a solution containing (instant of solution change marked with arrows in panels C–F). Representative [Cl^–]_S measurements are shown for oocytes heterologously expressing AE1 (A, upper panel), human NDCBE-EGFP (B), squid NDCBE (sqNDCBE) (C), NBCn1-EGFP (D), nothing (i.e. H₂O-injected oocytes) (E), and NCBE-EGFP (hNDCBE) (F). Before each experiment, the [Cl^–]_S electrode was calibrated once in the absence of (represented in the inset, Calibration A; this calibration was applied to the portion of the [Cl^–]_S trace gathered in the absence of (black bars)), and once in the presence of (represented in the inset, Calibration B; this calibration was applied to the portion of the [Cl^–]_S trace gathered in the presence of (gray bars)). Average [Cl^–]_S and pH_i data for all populations of oocytes are provided in Fig. 10. A representative pH_i trace is provided for a cell expressing AE1 (A, lower panel).

FIGURE 10.

Average maximal changes in [Cl^–]_S and average rates of pH_i recovery in oocytes exposed to . Shown are mean data from experiments such as those presented in Fig. 9 in which we simultaneously monitored [Cl^–]_S (A) and pH_i (B) of oocytes during exposure to solution. Black bars are data gathered from oocytes that had been preincubated with 200 μm DIDS for 1 h prior to assay. Gray bars are data gathered from oocytes that were exposed to solution in the absence of bath Na⁺. Values are means ± S.E. with number of oocytes in parentheses. hNDCBE, human NDCBE; sqNDCBE, squid NDCBE.

FIGURE 11.

Efflux of ³⁶Cl from oocytes. A, NDCBE-EGFP. B, NCBE-EGFP. C, oocytes injected with H₂O. The values on the ordinate represent the fractional efflux of ³⁶Cl (mean ± S.E.) measured over one of two 30-min collection periods. In each panel, the gray“A” bar represents the normalized value for the first collection period in ND96 solution. The white bars represent data from a second collection period (bars B–H), normalized to the value in the corresponding first collection period. The horizontal dashed line represents the average estimated normalized background (i.e.“zero” efflux) value for the second period. We calculated that, at the beginning of the first efflux period (i.e. before gray bar), NDCBE-expressing oocytes contained 553 ± 30 cpm (n = 66), NCBE-expressing oocytes contained 774 ± 30 cpm (n = 34), and H₂O-injected oocytes contained 325 ± 48 cpm (n = 27). For a given panel, these initial cpm values did not differ significantly among oocytes subsequently assigned to groups B–J (one-way ANOVA, p > 0.10 in all cases). The fractional loss of ³⁶Cl during the first 30-min efflux period in ND96 (i.e. during gray bar) was 13 ± 1% (n = 66) for NDCBE-expressing oocytes, 9 ± 1% (n = 79) for NCBE-expressing oocytes, and 33 ± 3% (n = 38) for H₂O-injected oocytes. For a given panel, these first period fractional losses did not differ significantly among oocytes subsequently assigned to groups B–J (one-way ANOVA, p > 0.08 in all cases). Bars marked with p values are significantly different from all unmarked bars within that panel (one-way ANOVA with Student-Newman-Keuls posthoc analysis). In B, bars F and G are not significantly different from each other (p = 0.247). *, in B, bar H is significantly different from bars F (p = 0.0004), G (p = 0.0002), and I (p = 0.028) but not bar J (p = 0.05) or bars B–E (p > 0.07). Unmarked bars are not significantly different from each other (p > 0.71).