Store-operated Ca2+ influx is mediated by store002Doperated Ca2+ channels (SOCs) and is a central component of receptor-evoked Ca2+ signals1. The Orai channels mediate SOCs2–4 and STIM1 is the ER-resident Ca2+ sensor that gates the channels5, 6. How STIM1 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(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. Orai1 activated by SOAR recapitulates all the entire kinetic properties of Orai1 activated by STIM1. Mutations of STIM1 within SOAR prevent activation of Orai1 without preventing 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 C-terminus α-helicial region of Orai is required for activation by SOAR. Deleting most of Orai1 N terminus impaired Orai1 activation by STIM1, but (Δ1–73)Orai1 interacts with and is fully activated by SOAR. Accordingly, the characteristic inward rectification of Orai is mediated by an interaction between the polybasic STIM1(672–685) and a proline-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.

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.

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 (NP(o)) 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.

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.

Transepithelial Cl− and HCO3− 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 ). HCO3− is the biological buffer that maintains acid-base balance, thereby preventing metabolic and respiratory acidosis ( 48 ). HCO3− also buffers the pH of the mucosal layers that line all epithelia, protecting them from injury ( 2 ). Being a chaotropic ion, HCO3− is essential for solubilization of ions and macromolecules such as mucins and digestive enzymes in secreted fluids. Most epithelia have a Cl−/HCO3 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 HCO3− transporters, the SLC26 family ( 73 , 78 ). This review will highlight structural features, the functional diversity, and several regulatory aspects of the SLC26 transporters.

SLC26A9 is a member of the SLC26 family of anion transporters, which is expressed at high levels in airway and gastric surface epithelial cells.

Unexpectedly, deletion of slc26a6 in mice and measurement of fluid and HCO 3 À secretion into sealed intralobular pancreatic ducts revealed that deletion of slc26a6 enhanced spontaneous and decreased stimulated secretion. Remarkably, inhibition of CFTR activity with CFTR inh -172, knock-down of CFTR by siRNA and measurement of CFTR current in WT and slc26a6 À/À 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 HCO 3 À secretion in vivo.

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a singlemolecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5′-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotidebased polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time singlemolecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.single-molecule sequencing | nanopore | DNA sequencing by synthesis | polymer-tagged nucleotides | chip array T he importance of DNA sequencing has increased dramatically from its inception four decades ago. It is recognized as a crucial technology for most areas of biology and medicine and as the underpinning for the new paradigm of personalized and precision medicine. Information on individuals' genomes and epigenomes can help reveal their propensity for disease, clinical prognosis, and response to therapeutics, but routine application of genome sequencing in medicine will require comprehensive data delivered in a timely and cost-effective manner (1). Although 35 years of technological advances have improved sequence throughput and have reduced costs exponentially, genome analysis still takes several days and thousands of dollars to complete (1, 2). To realize the potential of personalized medicine fully, the speed and cost of sequencing must be brought down another order of magnitude while increasing sequencing accuracy and read length. Singlemolecule approaches are thought to be essential to meet these requirements and offer the additional benefit of eliminating amplification bias (3, 4). Although optical methods for singlemolecule sequencing have been achieved and commercialize...