Michael R. Dorwart

SOAR and the polybasic STIM1 domains gate and regulate Orai channels

Influx of Ca2+ through store-operated Ca2+ channels (SOCs) is a central component of receptor-evoked Ca2+ signals. Orai channels are SOCs that are gated by STIM1, a Ca2+ sensor located in the ER but how it gates and regulates the Orai channels is unknown. Here, we report the molecular basis for gating of Orais by STIM1. All Orai channels are fully activated by the conserved STIM1 amino acid fragment 344–442, which we termed SOAR (the STIM1 Orai activating region). SOAR acts in combination with STIM1 (450–485) to regulate the strength of interaction with Orai1. Activation of Orai1 by SOAR recapitulates all the kinetic properties of Orai1 activation by STIM1. However, mutations of STIM1 within SOAR prevent activation of Orai1 but not co-clustering of STIM1 and Orai1 in response to Ca2+ store depletion, indicating that STIM1–Orai1 co-clustering is not sufficient for Orai1 activation. An intact carboxy terminus α-helicial region of Orai is required for activation by SOAR. Deleting most of the Orai1 amino terminus impaired Orai1 activation by STIM1, but Orai1Δ1–73 interacted with and was fully activated by SOAR. Accordingly, the characteristic inward rectification of Orai is mediated by an interaction between the polybasic STIM1 (672–685) and a Pro-rich region in the N terminus of Orai1. Hence, the essential properties of Orai1 function can be rationalized by interactions with discrete regions of STIM1.

Gating of CFTR by the STAS domain of SLC26 transporters.

Chloride absorption and bicarbonate secretion are vital functions of epithelia, as highlighted by cystic fibrosis and diseases associated with mutations in members of the SLC26 chloride-bicarbonate exchangers. Many SLC26 transporters (SLC26T) are expressed in the luminal membrane together with CFTR, which activates electrogenic chloride-bicarbonate exchange by SLC26T. However, the ability of SLC26T to regulate CFTR and the molecular mechanism of their interaction are not known. We report here a reciprocal regulatory interaction between the SLC26T DRA, SLC26A6 and CFTR. DRA markedly activates CFTR by increasing its overall open probablity (NPo) sixfold. Activation of CFTR by DRA was facilitated by their PDZ ligands and binding of the SLC26T STAS domain to the CFTR R domain. Binding of the STAS and R domains is regulated by PKA-mediated phosphorylation of the R domain. Notably, CFTR and SLC26T co-localize in the luminal membrane and recombinant STAS domain activates CFTR in native duct cells. These findings provide a new understanding of epithelial chloride and bicarbonate transport and may have important implications for both cystic fibrosis and diseases associated with SLC26T.

Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP‐binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide‐binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF‐causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP‐ and ATP‐bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1‐transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1–NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.

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.

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.

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.

SLC26A9 is a Cl- channel regulated by the WNK kinases

SLC26A9 is a member of the SLC26 family of anion transporters, which is expressed at high levels in airway and gastric surface epithelial cells. The transport properties and regulation of SLC26A9, and thus its physiological function, are not known. Here we report that SLC26A9 is a highly selective Cl− channel with minimal OH−/HCO3− permeability that is regulated by the WNK kinases. Expression in Xenopus oocytes and simultaneous measurement of membrane potential or current, intracellular pH (pHi) and intracellular Cl− (Cl−i) revealed that expression of SLC26A9 resulted in a large Cl− current. SLC26A9 displays a selectivity sequence of I− > Br− > NO3− > Cl− > Glu−, but it conducts Br− > Cl− > I− > NO3− > Glu−, with NO3− and I− inhibiting the Cl− conductance. Similarly, expression of SLC26A9 in HEK cells resulted in a large Cl− current. Although detectable, OH− and HCO3− fluxes in oocytes expressing SLC26A9 were very small. Moreover, HCO3− had no discernable effect on the Cl− current, the reversal potential in the presence or absence of Cl−o and, importantly, HCO3− had no effect on Cl− fluxes. These findings indicate that SLC26A9 is a Cl− channel with minimal OH−/HCO3− permeability. Co‐expression of SLC26A9 with the WNK kinases WNK1, WNK3 or WNK4 inhibited SLC26A9 activity, and the inhibition was independent of WNK kinase activity. Immunolocalization in oocytes and cell surface biotinylation in HEK cells indicated that the WNK‐mediated inhibition of SLC26A9 activity is caused by reduced SLC26A9 surface expression. Expression of SLC26A9 in the airway and the response of the WNKs to homeostatic stress raise the possibility that SLC26A9 serves to mediate the response of the airway to stress.

Congenital chloride-losing diarrhea causing mutations in the stas domain result in misfolding and mistrafficking of SLC26A3

Congenital chloride-losing diarrhea (CLD) is a genetic disorder causing watery stool and dehydration. Mutations in SLC26A3 (solute carrier 26 family member 3), which functions as a coupled Cl-/\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} exchanger, cause CLD. SLC26A3 is a membrane protein predicted to contain 12 transmembrane-spanning α-helices and a C-terminal STAS (sulfate transporters and anti-sigma-factor) domain homologous to the bacterial anti-sigma-factor antagonists. The STAS domain is required for SLC26A3 Cl-/\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} exchange function and for the activation of cystic fibrosis transmembrane conductance regulator by SLC26A3. Here we investigate the molecular mechanism(s) by which four CLD-causing mutations (ΔY526/7, I544N, I675/6ins, and G702Tins) in the STAS domain lead to disease. In a heterologous mammalian expression system biochemical, immunohistochemical, and ion transport experiments suggest that the four CLD mutations cause SLC26A3 transporter misfolding and/or mistrafficking. Expression studies with the isolated STAS domain suggest that the I675/6ins and G702Tins mutations disrupt the STAS domain directly, whereas limited proteolysis experiments suggest that the ΔY526/7 and I544N mutations affect a later step in the folding and/or trafficking pathway. The data suggest that these CLD-causing mutations cause disease by at least two distinct molecular mechanisms, both ultimately leading to loss of functional protein at the plasma membrane.

S. aureus MscL Is a Pentamer In Vivo but of Variable Stoichiometries In Vitro: Implications for Detergent- Solubilized Membrane Proteins

Detergent-induced rearrangements of membrane-protein subunits explain why two MscL channel stoichiometries have been resolved by X-ray crystallography - but S. aureus MscL is truly a pentamer in vivo.