Nikolay Shcheynikov

A molecular mechanism for aberrant CFTR-dependent HCO(3)(-) transport in cystic fibrosis.

Aberrant HCO3− transport is a hallmark of cystic fibrosis (CF) and is associated with aberrant Cl−‐dependent HCO3− transport by the cystic fibrosis transmembrane conductance regulator (CFTR). We show here that HCO3− current by CFTR cannot account for CFTR‐activated HCO3− transport and that CFTR does not activate AE1–AE4. In contrast, CFTR markedly activates Cl− and OH−/HCO3− transport by members of the SLC26 family DRA, SLC26A6 and pendrin. Most notably, the SLC26s are electrogenic transporters with isoform‐specific stoichiometries. DRA activity occurred at a Cl−/HCO3− ratio ≥2. SLC26A6 activity is voltage regulated and occurred at HCO3−/Cl− ≥2. The physiological significance of these findings is demonstrated by interaction of CFTR and DRA in the mouse pancreas and an altered activation of DRA by the R117H and G551D mutants of CFTR. These findings provide a molecular mechanism for epithelial HCO3− transport (one SLC26 transporter—electrogenic transport; two SLC26 transporters with opposite stoichiometry in the same membrane domain—electroneutral transport), the CF‐associated aberrant HCO3− transport, and reveal a new function of CFTR with clinical implications for CF and congenital chloride diarrhea.

A molecular mechanism for aberrantCFTR-dependent HCO3– transport in cystic fibrosis

Aberrant HCO3− transport is a hallmark of cystic fibrosis (CF) and is associated with aberrant Cl−‐dependent HCO3− transport by the cystic fibrosis transmembrane conductance regulator (CFTR). We show here that HCO3− current by CFTR cannot account for CFTR‐activated HCO3− transport and that CFTR does not activate AE1–AE4. In contrast, CFTR markedly activates Cl− and OH−/HCO3− transport by members of the SLC26 family DRA, SLC26A6 and pendrin. Most notably, the SLC26s are electrogenic transporters with isoform‐specific stoichiometries. DRA activity occurred at a Cl−/HCO3− ratio ≥2. SLC26A6 activity is voltage regulated and occurred at HCO3−/Cl− ≥2. The physiological significance of these findings is demonstrated by interaction of CFTR and DRA in the mouse pancreas and an altered activation of DRA by the R117H and G551D mutants of CFTR. These findings provide a molecular mechanism for epithelial HCO3− transport (one SLC26 transporter—electrogenic transport; two SLC26 transporters with opposite stoichiometry in the same membrane domain—electroneutral transport), the CF‐associated aberrant HCO3− transport, and reveal a new function of CFTR with clinical implications for CF and congenital chloride diarrhea.

The Solute Carrier 26 Family of Proteins in Epithelial Ion Transport

Transepithelial Cl(-) and HCO(3)(-) transport is critically important for the function of all epithelia and, when altered or ablated, leads to a number of diseases, including cystic fibrosis, congenital chloride diarrhea, deafness, and hypotension (78, 111, 119, 126). HCO(3)(-) is the biological buffer that maintains acid-base balance, thereby preventing metabolic and respiratory acidosis (48). HCO(3)(-) also buffers the pH of the mucosal layers that line all epithelia, protecting them from injury (2). Being a chaotropic ion, HCO(3)(-) is essential for solubilization of ions and macromolecules such as mucins and digestive enzymes in secreted fluids. Most epithelia have a Cl(-)/HCO(3) exchange activity in the luminal membrane. The molecular nature of this activity remained a mystery for many years until the discovery of SLC26A3 and the realization that it is a member of a new family of Cl(-) and HCO(3)(-) transporters, the SLC26 family (73, 78). This review will highlight structural features, the functional diversity, and several regulatory aspects of the SLC26 transporters.

Coupling Modes and Stoichiometry of Cl−/HCO3− Exchange by slc26a3 and slc26a6

The SLC26 transporters are a family of mostly luminal Cl− and HCO3 − transporters. The transport mechanism and the Cl−/HCO3 − stoichiometry are not known for any member of the family. To address these questions, we simultaneously measured the HCO3 − and Cl− fluxes and the current or membrane potential of slc26a3 and slc26a6 expressed in Xenopus laevis oocytes and the current of the transporters expressed in human embryonic kidney 293 cells. slc26a3 mediates a coupled 2Cl−/1HCO3 − exchanger. The membrane potential modulated the apparent affinity for extracellular Cl− of Cl−/HCO3 − exchange by slc26a3. Interestingly, the replacement of Cl− with NO3 − or SCN− uncoupled the transport, with large NO3 − and SCN− currents and low HCO3 − transport. An apparent uncoupled current was also developed during the incubation of slc26a3-expressing oocytes in HCO3 −-buffered Cl−-free media. These findings were used to develop a turnover cycle for Cl− and HCO3 − transport by slc26a3. Cl− and HCO3 − flux measurements revealed that slc26a6 mediates a 1Cl−/2HCO3 − exchange. Accordingly, holding the membrane potential at 40 and −100 mV accelerated and inhibited, respectively, Cl−-mediated HCO3 − influx, and holding the membrane potential at −100 mV increased HCO3 −-mediated Cl− influx. These findings indicate that slc26a6 functions as a coupled 1Cl−/2HCO3 − exchanger. The significance of isoform-specific Cl− and HCO3 − transport stoichiometry by slc26a3 and slc26a6 is discussed in the context of diseases of epithelial Cl− absorption and HCO3 − secretion.

TRP-ML1 Is a Lysosomal Monovalent Cation Channel That Undergoes Proteolytic Cleavage

Mutations in the gene MCOLN1 coding for the TRP (transient receptor potential) family ion channel TRP-ML1 lead to the lipid storage disorder mucolipidosis type IV (MLIV). The function and role of TRP-ML1 are not well understood. We report here that TRP-ML1 is a lysosomal monovalent cation channel. Both native and recombinant TRP-ML1 are cleaved resulting in two products. Recombinant TRP-ML1 is detected as the full-length form and as short N- and C-terminal forms, whereas in native cells mainly the cleaved N and C termini are detected. The N- and C-terminal fragments of TRP-ML1 were co-immunoprecipitated from cell lysates and co-eluted from a Ni2+ column. TRP-ML1 undergoes proteolytic cleavage that is inhibited by inhibitors of cathepsin B (CatB) and is altered when TRP-ML1 is expressed in CatB-/- cells. N-terminal sequencing of purified C-terminal fragment of TRP-ML1 expressed in Sf9 cells indicates a cleavage site at Arg200 ↓ Pro201. Consequently, the conserved R200H mutation changed the cleavage pattern of TRP-ML1. The cleavage inhibited TRP-ML1 channel activity. This work provides the first example of inactivation by cleavage of a TRP channel. The significance of the cleavage to the function of TRP-ML1 is under investigation.

Slc26a6 regulates CFTR activity in vivo to determine pancreatic duct HCO3− secretion: relevance to cystic fibrosis

Fluid and HCO3− secretion are vital functions of the pancreatic duct and other secretory epithelia. CFTR and Cl−/HCO3− exchange activity at the luminal membrane are required for these functions. The molecular identity of the Cl−/HCO3− exchangers and their relationship with CFTR in determining fluid and HCO3− secretion are not known. We show here that the Cl−/HCO3− exchanger slc26a6 controls CFTR activity and ductal fluid and HCO3− secretion. Unexpectedly, deletion of slc26a6 in mice and measurement of fluid and HCO3− secretion into sealed intralobular pancreatic ducts revealed that deletion of slc26a6 enhanced spontaneous and decreased stimulated secretion. Remarkably, inhibition of CFTR activity with CFTRinh‐172, knock‐down of CFTR by siRNA and measurement of CFTR current in WT and slc26a6−/− duct cells revealed that deletion of slc26a6 resulted in dis‐regulation of CFTR activity by removal of tonic inhibition of CFTR by slc26a6. These findings reveal the intricate regulation of CFTR activity by slc26a6 in both the resting and stimulated states and the essential role of slc26a6 in pancreatic HCO3− secretion in vivo.

The Slc26a4 transporter functions as an electroneutral Cl-/I-/HCO3-exchanger : role of Slc26a4 and Slc26a6 in I-and HCO3-secretion and in regulation of CFTR in the parotid duct

Transcellular Cl− and HCO3− transport is a vital function of secretory epithelia and exit across the luminal membrane is mediated by members of the SLC26 transporters in conjunction with cystic fibrosis transmembrane conductance regulator (CFTR) channel. Typically, secretory epithelia express several SLC26 transporters in the same tissue; however, how their specific function is determined in vivo is not known. In the present work we used the parotid gland duct which expressed Slc26a4 and Slc26a6 and the model systems of Slc26a4−/− and Slc26a6−/− mice to study the role and regulation of these SLC26 transporters. We examined the transport modes of SLC26A4 expressed in Xenopus oocytes and report that SLC26A4 functions as a coupled, electroneutral I−/Cl−, I−/HCO3− and Cl−/HCO3− exchanger with 1: 1 stoichiometry, with I− as the preferred anion. In the duct, Slc26a4 is expressed in the luminal membrane and mainly mediates I− secretion with minimal role in luminal HCO3− transport. By contrast, Slc26a6 mediates luminal Cl−/HCO3− exchange activity with minimal role in I− secretion. Furthermore, silencing of CFTR altered Cl−/HCO3− exchange by Slc26a6, but had no effect on I− secretion by Slc26a4. Accordingly, deletion of Slc26a6, but not deletion of Slc26a4, results in dysregulation of CFTR. These findings provide the first evidence for a selective role of the SLC26 transporters expressed in the same tissue in epithelial anion transport and suggest that transport specificity is achieved by both the properties of the transporters and the composition of the complexes they form.

SLC26A7 Is a Cl– Channel Regulated by Intracellular pH

Members of the SLC26 transporter family play an essential role in several epithelial functions, as revealed by diseases associated with mutations in members of the family. Several members were shown to function as Cl– and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} transporters that likely play an important role in epithelial Cl– absorption and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} secretion. However, the mechanism of most transporters is not well understood. SLC26A7 is a member of the SLC26 transporter family reported to be expressed in the basolateral membrane of the cortical collecting duct and parietal cells and functions as a coupled \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Cl}^{-}{/}\mathrm{HCO}_{3}^{-}\) \end{document} exchanger. In the present work we examined the transport properties of SLC26A7 to determine its transport characteristics and electrogenicity. We found that when expressed in Xenopus oocytes or HEK293 cells SLC26A7 functions as a pHi-regulated Cl– channel with minimal \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{OH}^{-}{/}\mathrm{HCO}_{3}^{-}\) \end{document} permeability. Expression of SLC26A7 in oocytes or HEK293 cells generated a Cl– current with linear I/V and an instantaneous current that was voltage- and time-independent. Based on measurement of reversal potential the selectivity of SLC26A7 is \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NO}_{3}^{-}{\gg}\mathrm{Cl}^{-}=\mathrm{Br}^{-}=\mathrm{I}^{-}{>}\mathrm{SO}_{4}^{2-}=\mathrm{Glu}^{-}\) \end{document}, although I– partially inhibited the current. Incubating the cells with \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} or butyrate acidified the cytosol and increased the selectivity of SLC26A7 for Cl–. Measurement of membrane potential and pHi showed minimal OH– and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} transport by SLC26A7 when the cells were incubated in Cl–-containing or Cl–-free media. The activity of SLC26A7 was inhibited by all inhibitors of anion transporters tested, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, diphenylamine-2-carboxylic acid, and glybenclamide. These findings reveal that SLC26A7 functions as a unique Cl– channel that is regulated by intracellular H+.

IRBIT coordinates epithelial fluid and HCO3– secretion by stimulating the transporters pNBC1 and CFTR in the murine pancreatic duct

Fluid and HCO3- secretion are vital functions of secretory epithelia. In most epithelia, this entails HCO3- entry at the basolateral membrane, mediated by the Na+-HCO3- cotransporter, pNBC1, and exit at the luminal membrane, mediated by a CFTR-SLC26 transporters complex. Here we report that the protein IRBIT (inositol-1,4,5-trisphosphate [IP3] receptors binding protein released with IP3), a previously identified activator of pNBC1, activates both the basolateral pNBC1 and the luminal CFTR to coordinate fluid and HCO3- secretion by the pancreatic duct. We used video microscopy and ion selective microelectrodes to measure fluid secretion and Cl- and HCO3- concentrations in cultured murine sealed intralobular pancreatic ducts. Short interference RNA-mediated knockdown of IRBIT markedly inhibited ductal pNBC1 and CFTR activities, luminal Cl- absorption and HCO3- secretion, and the associated fluid secretion. Single-channel measurements suggested that IRBIT regulated CFTR by reducing channel mean close time. Furthermore, expression of IRBIT constructs in HEK cells revealed that activation of pNBC1 required only the IRBIT PEST domain, while activation of CFTR required multiple IRBIT domains, suggesting that IRBIT activates these transporters by different mechanisms. These findings define IRBIT as a key coordinator of epithelial fluid and HCO3- secretion and may have implications to all CFTR-expressing epithelia and to cystic fibrosis.

Dynamic control of cystic fibrosis transmembrane conductance regulator Cl(-)/HCO3(-) selectivity by external Cl(-).

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} secretion is a vital activity in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing epithelia. However, the role of CFTR in this activity is not well understood. Simultaneous measurements of membrane potential and pHi and/or current in CFTRexpressing Xenopus oocytes revealed dynamic control of CFTR \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Cl}^{-}{/}\mathrm{HCO}_{3}^{-}\) \end{document} permeability ratio, which is regulated by external Cl– (Cl–o). Thus, reducing external Cl– from 110 to 0–10 mm resulted in the expected increase in membrane potential, but with no corresponding OH– or \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} influx. Approximately 3–4 min after reducing \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(Cl_{o}^{-}\) \end{document} to 0 mm, an abrupt switch in membrane potential occurs that coincided with an increased rates of OH– and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} influx. The switch in membrane permeability to \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{OH}^{-}{/}\mathrm{HCO}_{3}^{-}\) \end{document} can also be recorded as a leftward shift in the reversal potential. Furthermore, an increased rate of OH– influx in response to elevating pHo to 9.0 was observed only after the switch in membrane potential. The time to switch increased to 11 min at \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(Cl_{o}^{-}\) \end{document} of 5 mm. Conversely, re-addition of external Cl– after the switch in membrane potential did not stop \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} influx, which continued for about 3.9 min after Cl– addition. Importantly, addition of external Cl– to cells incubated in Cl–-free medium never resulted in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} efflux. Voltage and current clamp experiments showed that the delayed \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} transport is electrogenic. These results indicate that CFTR exists in two conformations, a Cl– only and a Cl– and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{OH}^{-}{/}\mathrm{HCO}_{3}^{-}\) \end{document} permeable state. The switch between the states is controlled by external Cl–. Accordingly, a different tryptic pattern of CFTR was found upon digestion in Cl–-containing and Cl–-free media. The physiological significance of these finding is discussed in the context of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} secretion by tissues such as the pancreas and salivary glands.