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Background: Differentiating among HCO 3- , CO 3= , and H + movements across membranes has long seemed impossible. We now seek to discriminate unambiguously among three alternate mechanisms: the inward flux of 2 HCO 3- (mechanism 1), the inward flux of 1 CO 3= (mechanism 2), and the CO 2 /HCO 3- -stimulated outward flux of 2 H + (mechanism 3)., Methods: As a test case, we use electrophysiology and heterologous expression in Xenopus oocytes to examine SLC4 family members that appear to transport "bicarbonate" ("HCO 3- ")., Results: First, we note that cell-surface carbonic anhydrase should catalyze the forward reaction CO 2 +OH - →HCO 3- if HCO 3- is the substrate; if it is not, the reverse reaction should occur. Monitoring changes in cell-surface pH ( Δ pH S ) with or without cell-surface carbonic anhydrase, we find that the presumed Cl-"HCO 3 " exchanger AE1 (SLC4A1) does indeed transport HCO 3- (mechanism 1) as long supposed, whereas the electrogenic Na/"HCO 3 " cotransporter NBCe1 (SLC4A4) and the electroneutral Na + -driven Cl-"HCO 3 " exchanger NDCBE (SLC4A8) do not. Second, we use mathematical simulations to show that each of the three mechanisms generates unique quantities of H + at the cell surface (measured as Δ pH S ) per charge transported (measured as change in membrane current, ΔIm ). Calibrating ΔpH S /Δ Im in oocytes expressing the H + channel H V 1, we find that our NBCe1 data align closely with predictions of CO 3= transport (mechanism 2), while ruling out HCO 3- (mechanism 1) and CO 2 /HCO 3- -stimulated H + transport (mechanism 3)., Conclusions: Our surface chemistry approach makes it possible for the first time to distinguish among HCO 3- , CO 3= , and H + fluxes, thereby providing insight into molecular actions of clinically relevant acid-base transporters and carbonic-anhydrase inhibitors., (Copyright © 2022 by the American Society of Nephrology.)

In most cell types and heterologous expression systems, the electrogenic sodium-bicarbonate cotransporter NBCe1 operates with a 1Na + -2HCO 3 - stoichiometry that, given typical transmembrane electrochemical gradients, promotes Na+ and HCO 3 - influx. However, NBCe1 in the kidney mediates HCO 3 - efflux (HCO 3 - reabsorption), a direction that has been predicted to be favored only if NBCe1 operates with a 1:3 stoichiometry. The phosphorylation state of Ser982 in the cytosolic carboxy-terminal domain of NBCe1 has been reported to be a key determinant of the transporter stoichiometry, with non-phosphorylated Ser982 favoring a 1:3 stoichiometry. Conversely, phosphoproteomic data from renal cortical preparations have revealed the presence of NBCe1 peptides including phosphoserine982 (pSer982) and/or pSer985 although it was not known what proportion of NBCe1 molecules were phosphorylated. In the present study, we report the generation, characterization, and application of a novel phosphospecific antibody raised against NBCe1/pSer982 and show that, contrary to expectations, Ser982 is more prevalently phosphorylated in murine kidneys (in which NBCe1 mediates HCO 3 - efflux) than in murine colons (in which NBCe1 mediates HCO 3 - influx). Using phosphomimetic mutants of murine NBCe1 expressed in Xenopus oocytes, we found no evidence that the phosphorylation state of Ser982 or Ser985 alone influences the transport stoichiometry or conductance. Furthermore, we found that the phosphorylation of NBCe1/Ser982 is enhanced in murine kidneys following a 24 h induction of metabolic acidosis. We conclude that the phosphorylation status of Ser982 is not a key determinant of NBCe1 stoichiometry but correlates with presumed NBCe1 activity.

The SLC4 family Cl - /[Formula: see text] cotransporters (NBCe1, NBCe2, NBCn1, and NBCn2) contribute to a variety of vital physiological processes including pH regulation and epithelial fluid secretion. Accordingly, their dysfunction can have devastating effects. Disorders such as epilepsy, hemolytic anemia, glaucoma, hearing loss, osteopetrosis, and renal tubular acidosis are all genetically linked to SLC4-family gene loci. This review summarizes how studies of Slc4-modified mice have enhanced our understanding of the etiology of SLC4-linked pathologies and the interpretation of genetic linkage studies. The review also surveys the novel disease signs exhibited by Slc4-modified mice which could either be considered to presage their description in humans, or to highlight interspecific differences. Finally, novel Slc4-modified mouse models are proposed, the study of which may further our understanding of the basis and treatment of SLC4-linked disorders of [Formula: see text]-transporter dysfunction.

The SLC4 family consists of 10 genes (SLC4A1-5; SLC4A7-11). All encode integral membrane proteins with very similar hydropathy plots-consistent with 10-14 transmembrane segments. Nine SLC4 members encode proteins that transport HCO3(-) (or a related species, such as CO3(2-)) across the plasma membrane. Functionally, eight of these proteins fall into two major groups: three Cl-HCO3 exchangers (AE1-3) and five Na(+)-coupled HCO3(-) transporters (NBCe1, NBCe2, NBCn1, NBCn2, NDCBE). Two of the Na(+)-coupled transporters (NBCe1, NBCe2) are electrogenic; the other three Na(+)-coupled HCO3(-) transporters and all three AEs are electroneutral. In addition, two other SLC4 members (AE4, SLC4A9 and BTR1, SLC4A11) do not yet have a firmly established function. Most, though not all, SLC4 members are functionally inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). SLC4 proteins play important roles many modes of acid-base homeostasis: the carriage of CO2 by erythrocytes, the transport of H(+) or HCO3(-) by several epithelia, as well as the regulation of cell volume and intracellular pH., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In the basolateral membrane of proximal-tubule cells, NBCe1-A (SLC4A4, variant A), operating with an apparent Na(+):HCO(3)(-) stoichiometry of 1:3, contributes to the reclamation of HCO(3)(-) from the glomerular filtrate, thereby preventing whole body acidosis. Others have reported that NBCe1-like activity in human, rabbit, and rat renal preparations is substantially influenced by lithium, sulfite, oxalate, and harmaline. These data were taken as evidence for the presence of distinct Na(+) and CO(3)(2-) binding sites in NBCe1-A, favoring a model of 1 Na(+):1 HCO(3)(-):1 CO(3)(2-). Here, we reexamine these findings by expressing human or rabbit NBCe1-A clones in Xenopus oocytes. In oocytes, NBCe1-A exhibits a 1:2 stoichiometry and could operate in one of five thermodynamically equivalent transport modes: 1) cotransport of Na(+) + 2 HCO(3)(-), 2) cotransport of Na(+) + CO(3)(2-), 3) transport of NaCO(3)(-), 4) exchange of Na(+) + HCO(3)(-) for H(+), or 5) HCO(3)(-)-activated exchange of Na(+) for 2 H(+). In contrast to the behavior of NBCe1-like activity in renal preparations, we find that cloned NBCe1-A is only slightly stimulated by Li(+), not at all influenced by sulfite or oxalate, and only weakly inhibited by harmaline. These negative data do not uniquely support any of the five models above. In addition, we find that NBCe1-A mediates a small amount of Na(+)-independent NO(3)(-) transport and that NBCe1-A is somewhat inhibited by extracellular benzamil. We suggest that the features of NBCe1-like activity in renal preparations are influenced by yet-to-be-identified renal factors. Thus the actual ionic substrates of NBCe1 remain to be identified.

The renal electrogenic Na(+)/HCO(3)(−) cotransporter (NBCe1-A) contributes to the basolateral step of transepithelial HCO(3)(−) reabsorption in proximal tubule epithelia, contributing to the buffering of blood pH. Elsewhere in the body (e.g. muscle cells) NBCe1 variants contribute to, amongst other processes, maintenance of intracellular pH. Others have described a homozygous mutation in NBCe1 (NBCe1-A p.Ala799Val) in an individual with severe proximal renal tubular acidosis (pRTA; usually associated with defective HCO(3)(−) reabsorption in proximal tubule cells) and hypokalaemic periodic paralysis (hypoPP; usually associated with leaky cation channels in muscle cells). Using biotinylation and two-electrode voltage-clamp on Xenopus oocytes expressing NBCe1, we demonstrate that the mutant NBCe1-A (A(A799V)) exhibits a per-molecule transport defect that probably contributes towards the observed pRTA. Furthermore, we find that A(A799V) expression is associated with an unusual HCO(3)(−)-independent conductance that, if associated with mutant NBCe1 in muscle cells, could contribute towards the appearance of hypokalaemic paralysis in the affected individual. We also study three novel lab mutants of NBCe1-A: p.Ala799Ile, p.Ala799Gly and p.Ala799Ser. All three exhibit a per-molecule transport defect, but only A(A799I) exhibits an A(A799V)-like ion conductance. A(A799G) and A(A799S) exhibit unusual outward rectification in their HCO(3)(−)-dependent conductance and A(A799G) exhibits reduced sensitivity to both DIDS and tenidap. A799G is the first mutation shown to affect the apparent tenidap affinity of NBCe1. Finally we show that A(A799V) and A(A799I), which accumulate poorly in the plasma membrane of oocytes, exhibit signs of abnormal intracellular accumulation in a non-polarized renal cell-line.

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.

The red cell anion exchanger (band 3; AE1) is a multispanning membrane protein that traverses the bilayer up to 14 times and mediates the stilbene-disulfonate-sensitive, electroneutral exchange of chloride and bicarbonate. Previous studies showed that the integrity of the third extracellular loop (EC3) of the protein was not essential for stilbene-disulfonate-sensitive chloride uptake. Here we demonstrate that the chloride uptake mediated by assemblies separated at EC3 represents the physiological electroneutral Cl(-)/HCO(3)(-) activity associated with intact AE1 protein. This provides further evidence that the 1:5 and 6:14 regions of the protein form discrete folding domains and confirms that the third extracellular loop does not play a pivotal role in AE1 transport function.