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1.  Gating of CFTR by the STAS domain of SLC26 transporters 
Nature cell biology  2004;6(4):343-350.
Chloride absorption and bicarbonate secretion are vital functions of epithelia1–6, 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 CFTR7, which activates electrogenic chloride-bicarbonate exchange by SLC26T8. 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.
doi:10.1038/ncb1115
PMCID: PMC3943213  PMID: 15048129
2.  Distinct gating mechanisms revealed by the structures of a multi-ligand gated K+ channel 
eLife  2012;1:e00184.
The gating ring-forming RCK domain regulates channel gating in response to various cellular chemical stimuli in eukaryotic Slo channel families and the majority of ligand-gated prokaryotic K+ channels and transporters. Here we present structural and functional studies of a dual RCK-containing, multi-ligand gated K+ channel from Geobacter sulfurreducens, named GsuK. We demonstrate that ADP and NAD+ activate the GsuK channel, whereas Ca2+ serves as an allosteric inhibitor. Multiple crystal structures elucidate the structural basis of multi-ligand gating in GsuK, and also reveal a unique ion conduction pore with segmented inner helices. Structural comparison leads us to propose a novel pore opening mechanics that is distinct from other K+ channels.
DOI: http://dx.doi.org/10.7554/eLife.00184.001
eLife digest
Most cells are surrounded by a semipermeable membrane, and although this membrane allows very few molecules to pass through it, cells can use transmembrane proteins to overcome this barrier. Some of these proteins import glucose, amino acids and other nutrients into the cell, while others transport ions into or out of the cell. Ion transport across the cell membrane is essential for a wide variety of biological processes, including signal transduction and the generation of electrical impulses in nerve cells.
The pores that allow ions to travel through the cell membrane are known as ion channels, and most channels allow only one type of ion—usually sodium, calcium or potassium (K+) ions—to pass through them. There are many different types of ion channels and they are classified according to the type of ion they allow to pass through them, and by the gating mechanism that is used to open and close the channel. For example, ligand-gated K+ channels facilitate the passage of potassium ions and are opened and closed by ligands binding and unbinding to and from the channel.
Most K+ channels are made up of four identical subunits, and in the majority of ligand-gated K+ channels in prokaryotes, each of these subunits will have one or two ligand-binding RCK domains (where RCK stands for regulating the conductance of K+). This is also true for some K+ channels in eukaryotes. While it is known that RCK domains are responsible for regulating the transport of potassium ions across the cell membranes of diverse organisms, little is known about the structure or gating mechanisms of K+ channels that are gated by more than one ligand.
Kong et al. have studied a ligand-gated K+ channel called GsuK that has two RCK domains per subunit and is found in the bacterium G. sulfurreducens. They found that the opening process was mediated by a ligand that contains adenine, such as NAD+ or ADP, and the channel was closed by the presence of calcium ions. And by determining multiple crystal structures, Kong et al. were able to understand, from a structural point of view, how these ligands regulate this channel, and to propose a gating mechanism that is distinct from the mechanisms that are known to control other potassium channels.
DOI: http://dx.doi.org/10.7554/eLife.00184.002
doi:10.7554/eLife.00184
PMCID: PMC3510474  PMID: 23240087
Geobacter sulfurreducens; K+ channel; ligand gating; Other
3.  Crucial Points within the Pore as Determinants of K+ Channel Conductance and Gating 
Journal of molecular biology  2011;411(1):27-35.
While selective for K+ ions, K+ channels vary significantly among their rate of ion permeation. Here we probe the effect of steric hindrance and electrostatics within the ion conduction pathway on K+ permeation in the MthK K+ channel using structure-based mutagenesis combined with single channel electrophysiology and x-ray crystallography. We demonstrate that changes in side chain size and polarity at Ala88, which forms the constriction point of the open MthK pore, have profound effects on the single channel conductance as well as open probability. We also reveal that the negatively charged Glu92s at the intracellular entrance of the open pore form an electrostatic trap, which stabilizes a hydrated K+ ion and facilitates the ion permeation. This electrostatic attraction is also responsible for intracellular divalent blockage, which renders the channel inward rectified in the presence Ca2+. In light of the high structural conservation of the selectivity filter, the size and chemical environment differences within the portion of the ion conduction pathway other than the filter are likely the determinants for the conductance variations among K+ channels.
doi:10.1016/j.jmb.2011.04.058
PMCID: PMC3171199  PMID: 21554888
MthK K+ channel; steric hindrance; electrostatics; constriction point; single channel electrophysiology
4.  Peptidyl-prolyl isomerase FKBP52 controls chemotropic guidance of neuronal growth cones via regulation of TRPC1 channel opening 
Neuron  2009;64(4):471-483.
Summary
Immunophilins, including FK506-binding proteins (FKBPs), are protein chaperones with peptidyl-prolyl isomerase (PPIase) activity. Initially identified as pharmacological receptors for immunosuppressants to regulate immune responses via isomerase independent mechanisms, FKBPs are most highly expressed in the nervous system where their physiological function as isomerases remains unknown. We demonstrate that FKBP12 and FKBP52 catalyze cis/trans isomerization of regions of TRPC1 implicated in controlling channel opening. FKBP52 mediates stimulus-dependent TRPC1 gating through isomerization, which is required for chemotropic turning of neuronal growth cones to netrin-1 and myelin-associated glycoprotein and for netrin-1/DCC-dependent midline axon guidance of commissural interneurons in the developing spinal cord. By contrast, FKBP12 mediates spontaneous opening of TRPC1 through isomerization and is not required for growth cone responses to netrin-1. Our study demonstrates a novel physiological function of proline isomerases in chemotropic nerve guidance through TRPC1 gating and may have significant implication in clinical applications of immunophilin-related therapeutic drugs.
doi:10.1016/j.neuron.2009.09.025
PMCID: PMC2786904  PMID: 19945390
5.  STIM1 gates TRPC channels but not Orai1 by electrostatic interaction 
Molecular cell  2008;32(3):439-448.
Summary
The receptor-evoked Ca2+ signal includes activation of the store-operated channels (SOCs) TRPC and Orai channels. Although both are gated by STIM1, it is not known how STIM1 gates the channels and whether STIM1 gates the TRPCs and Orais by the same mechanism. Here, we report the molecular mechanism by which STIM1 gates TRPC1, which involves interaction between two conserved, negatively charged aspartates in TRPC1(639DD640) with the positively charged STIM1(684KK685) in STIM1 polybasic domain. Charge swapping and functional analysis revealed that exact orientation of the charges on TRPC1 and STIM1 are required, but all positive-negative charge combinations on TRPC1 and STIM1, except STIM1(684EE685)+TRPC1(639RR640), are functional as long as they are reciprocal, indicating that STIM1 gates TRPC1 by intermolecular electrostatic interaction. Similar gating was observe with TRPC3(697DD698). STIM1 gates Orai1 by a different mechanism since the polybasic and S/P domains of STIM1 are not required for activation of Orai1 by STIM1.
doi:10.1016/j.molcel.2008.09.020
PMCID: PMC2586614  PMID: 18995841
6.  TRPC channels as STIM1-regulated store-operated channels 
Cell calcium  2007;42(2):205-211.
Receptor-activated Ca2+ influx is mediated largely by store-operated channels (SOCs). TRPC channels mediate a significant portion of the receptor-activated Ca2+ influx. However, whether any of the TRPC channels function as a SOC remains controversial. Our understanding of the regulation of TRPC channels and their function as SOCs is being reshaped with the discovery of the role of STIM1 in the regulation of Ca2+ influx channels. The findings that STIM1 is an ER resident Ca2+ binding protein that regulates SOCs allow an expanded and molecular definition of SOCs. SOCs can be considered as channels that are regulated by STIM1 and require the clustering of STIM1 in response to depletion of the ER Ca2+ stores and its translocation towards the plasma membrane. TRPC1 and other TRPC channels fulfill these criteria. STIM1 binds to TRPC1, TRPC2, TRPC4 and TRPC5 but not to TRPC3, TRPC6 and TRPC7, and STIM1 regulates TRPC1 channel activity. Structure-function analysis reveals that the C-terminus of STIM1 contains the binding and gating function of STIM1. The ERM domain of STIM1 binds to TRPC channels and a lysine-rich region participates in the gating of SOCs and TRPC1. Knock-down of STIM1 by siRNA and prevention of its translocation to the plasma membrane inhibit the activity of native SOCs and TRPC1. These findings support the conclusion that TRPC1 is a SOC. Similar studies with other TRPC channels should further clarify their regulation by STIM1 and function as SOCs.
doi:10.1016/j.ceca.2007.03.004
PMCID: PMC2764400  PMID: 17517433
7.  SOAR and the polybasic STIM1 domains gate and regulate the Orai channels 
Nature cell biology  2009;11(3):337-343.
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.
doi:10.1038/ncb1842
PMCID: PMC2663385  PMID: 19182790
8.  STIM1 heteromultimerizes TRPC channels to determine their function as store-operated channels 
Nature cell biology  2007;9(6):636-645.
Stromal interacting molecule 1 (STIM1) is a Ca2+ sensor that conveys the Ca2+ load of the endoplasmic reticulum to store-operated channels (SOCs) at the plasma membrane. Here, we report that STIM1 binds TRPC1, TRPC4 and TRPC5 and determines their function as SOCs. Inhibition of STIM1 function inhibits activation of TRPC5 by receptor stimulation, but not by La3+, suggesting that STIM1 is obligatory for activation of TRPC channels by agonists, but STIM1 is not essential for channel function. Through a distinct mechanism, STIM1 also regulates TRPC3 and TRPC6. STIM1 does not bind TRPC3 and TRPC6, and regulates their function indirectly by mediating the heteromultimerization of TRPC3 with TRPC1 and TRPC6 with TRPC4. TRPC7 is not regulated by STIM1. We propose a new definition of SOCs, as channels that are regulated by STIM1 and require the store depletion-mediated clustering of STIM1. By this definition, all TRPC channels, except TRPC7, function as SOCs.
doi:10.1038/ncb1590
PMCID: PMC2699187  PMID: 17486119
9.  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.
doi:10.1172/JCI36983
PMCID: PMC2613461  PMID: 19033647
10.  Homer proteins in Ca2+ signaling by excitable and non-excitable cells 
Cell calcium  2007;42(4-5):363-371.
Homers are scaffolding proteins that bind Ca2+ signaling proteins in cellular microdomains. The Homers participate in targeting and localization of Ca2+ signaling proteins in signaling complexes. However, recent work showed that the Homers are not passive scaffolding proteins, but rather they regulate the activity of several proteins within the Ca2+ signaling complex in an isoform specific manner. Homer2 increases the GAP activity of RGS proteins and PLCβ that accelerate the GTPase activity of Gα subunits. Homer1 gates the activity of TRPC channels, controls the rates of their translocation and retrieval from the plasma membrane and mediates the conformational coupling between TRPC channels and IP3Rs. Homer1 stimulates the activity of the cardiac and neuronal L-type Ca2+ channels Cav1.2 and Cav1.3. Homer1 also mediates the communication between the cardiac and smooth muscle ryanodine receptor RyR2 and Cav1.2 to regulate E–C coupling. In many cases the Homers function as a buffer to reduce the intensity of Ca2+ signaling and create a negative bias that can be reversed by the immediate early gene form of Homer 1. Hence, the Homers should be viewed as the buffers of Ca2+ signaling that ensure a high spatial and temporal fidelity of the Ca2+ signaling and activation of downstream effects.
doi:10.1016/j.ceca.2007.05.007
PMCID: PMC2100435  PMID: 17618683
11.  The Mammalian Sec6/8 Complex Interacts with Ca2+ Signaling Complexes and Regulates Their Activity 
The Journal of Cell Biology  2000;150(5):1101-1112.
The localization of various Ca2+ transport and signaling proteins in secretory cells is highly restricted, resulting in polarized agonist-stimulated Ca2+ waves. In the present work, we examined the possible roles of the Sec6/8 complex or the exocyst in polarized Ca2+ signaling in pancreatic acinar cells. Immunolocalization by confocal microscopy showed that the Sec6/8 complex is excluded from tight junctions and secretory granules in these cells. The Sec6/8 complex was found in at least two cellular compartments, part of the complex showed similar, but not identical, localization with the Golgi apparatus and part of the complex associated with Ca2+ signaling proteins next to the plasma membrane at the apical pole. Accordingly, immunoprecipitation (IP) of Sec8 did not coimmunoprecipitate βCOP, Golgi 58K protein, or mannosidase II, all Golgi-resident proteins. By contrast, IP of Sec8 coimmunoprecipitates Sec6, type 3 inositol 1,4,5-trisphosphate receptors (IP3R3), and the Gβγ subunit of G proteins from pancreatic acinar cell extracts. Furthermore, the anti-Sec8 antibodies coimmunoprecipitate actin, Sec6, the plasma membrane Ca2+ pump, the G protein subunits Gαq and Gβγ, the β1 isoform of phospholipase C, and the ER resident IP3R1 from brain microsomal extracts. Antibodies against the various signaling and Ca2+ transport proteins coimmunoprecipitate Sec8 and the other signaling proteins. Dissociation of actin filaments in the immunoprecipitate had no effect on the interaction between Sec6 and Sec8, but released the actin and dissociated the interaction between the Sec6/8 complex and Ca2+ signaling proteins. Hence, the interaction between the Sec6/8 and Ca2+ signaling complexes is likely mediated by the actin cytoskeleton. The anti-Sec6 and anti-Sec8 antibodies inhibited Ca2+ signaling at a step upstream of Ca2+ release by IP3. Disruption of the actin cytoskeleton with latrunculin B in intact cells resulted in partial translocation of Sec6 and Sec8 from membranes to the cytosol and interfered with propagation of agonist-evoked Ca2+ waves. Our results suggest that the Sec6/8 complex has multiple roles in secretory cells including governing the polarized expression of Ca2+ signaling complexes and regulation of their activity.
PMCID: PMC2175249  PMID: 10973998
Sec6/8 complex; Ca2+ signaling proteins; assembly; actin cytoskeleton; Ca2+ signaling
12.  Sodium and potassium competition in potassium-selective and non-selective channels 
Nature Communications  2013;4:2721.
Potassium channels selectively conduct K+, primarily to the exclusion of Na+, despite the fact that both ions can bind within the selectivity filter. Here we perform crystallographic titration and single-channel electrophysiology to examine the competition of Na+ and K+ binding within the filter of two NaK channel mutants; one is the potassium-selective NaK2K mutant and the other is the non-selective NaK2CNG, a CNG channel pore mimic. With high-resolution structures of these engineered NaK channel constructs, we explicitly describe the changes in K+ occupancy within the filter upon Na+ competition by anomalous diffraction. Our results demonstrate that the non-selective NaK2CNG still retains a K+-selective site at equilibrium, whereas the NaK2K channel filter maintains two high-affinity K+ sites. A double-barrier mechanism is proposed to explain K+ channel selectivity at low K+ concentrations.
K+ channels are selective for K+ despite the fact that Na+ can bind and conduct through the selectivity filter. Sauer et al. show that a K+-selective NaK2K channel has two high-affinity K+-binding sites, whereas a non-selective NaK2CNG channel has one, and propose a double-barrier mechanism for ion selectivity.
doi:10.1038/ncomms3721
PMCID: PMC3831281  PMID: 24217363

Results 1-12 (12)