Ernest B. Campbell

X-RAY STRUCTURE OF A CLC CHLORIDE CHANNEL AT 3.0 A REVEALS THE MOLECULAR BASIS OF ANION SELECTIVITY

The ClC chloride channels catalyse the selective flow of Cl- ions across cell membranes, thereby regulating electrical excitation in skeletal muscle and the flow of salt and water across epithelial barriers. Genetic defects in ClC Cl- channels underlie several familial muscle and kidney diseases. Here we present the X-ray structures of two prokaryotic ClC Cl- channels from Salmonella enterica serovar typhimurium and Escherichia coli at 3.0 and 3.5 Å, respectively. Both structures reveal two identical pores, each pore being formed by a separate subunit contained within a homodimeric membrane protein. Individual subunits are composed of two roughly repeated halves that span the membrane with opposite orientations. This antiparallel architecture defines a selectivity filter in which a Cl- ion is stabilized by electrostatic interactions with α-helix dipoles and by chemical coordination with nitrogen atoms and hydroxyl groups. These findings provide a structural basis for further understanding the function of ClC Cl- channels, and establish the physical and chemical basis of their anion selectivity.

Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment.

Voltage-dependent K+ (Kv) channels repolarize the action potential in neurons and muscle. This type of channel is gated directly by membrane voltage through protein domains known as voltage sensors, which are molecular voltmeters that read the membrane voltage and regulate the pore. Here we describe the structure of a chimaeric voltage-dependent K+ channel, which we call the ‘paddle-chimaera channel’, in which the voltage-sensor paddle has been transferred from Kv2.1 to Kv1.2. Crystallized in complex with lipids, the complete structure at 2.4 ångström resolution reveals the pore and voltage sensors embedded in a membrane-like arrangement of lipid molecules. The detailed structure, which can be compared directly to a large body of functional data, explains charge stabilization within the membrane and suggests a mechanism for voltage-sensor movements and pore gating.

Structure of a eukaryotic CLC transporter defines an intermediate state in the transport cycle.

Controlling Chloride Channels The CLC proteins are a large family of channels and transporters that transfer chloride ions across cell membranes. While structures of two prokaryotic CLCs have been determined, these do not include the cytoplasmic regulatory domains found in eukaryotic transporters, and the structures do not reveal the mechanism of Cl−/H+–coupled transport. L. Feng et al. (p. 635, published online 30 September; see the Perspective by Mindell) describe the structure of a eukaryotic CLC protein and found that the regulatory domains interacted closely with the transmembrane domain so that conformational changes are transmitted to the ion pathway. A gating glutamate in the eukaryote transporter is in a different conformation to prokaryotic structures, explaining the 2:1 stoichiometry of Cl−/H+ exchange in eukaryotes. The structure of a chloride transporter and its regulatory domain provides insight into the ion-exchange mechanism. CLC proteins transport chloride (Cl–) ions across cell membranes to control the electrical potential of muscle cells, transfer electrolytes across epithelia, and control the pH and electrolyte composition of intracellular organelles. Some members of this protein family are Cl– ion channels, whereas others are secondary active transporters that exchange Cl– ions and protons (H+) with a 2:1 stoichiometry. We have determined the structure of a eukaryotic CLC transporter at 3.5 angstrom resolution. Cytoplasmic cystathionine beta-synthase (CBS) domains are strategically positioned to regulate the ion-transport pathway, and many disease-causing mutations in human CLCs reside on the CBS-transmembrane interface. Comparison with prokaryotic CLC shows that a gating glutamate residue changes conformation and suggests a basis for 2:1 Cl–/H+ exchange and a simple mechanistic connection between CLC channels and transporters.

Structure of a pore-blocking toxin in complex with a eukaryotic voltage-dependent K+ channel

Pore-blocking toxins inhibit voltage-dependent K+ channels (Kv channels) by plugging the ion-conduction pathway. We have solved the crystal structure of paddle chimera, a Kv channel in complex with charybdotoxin (CTX), a pore-blocking toxin. The toxin binds to the extracellular pore entryway without producing discernable alteration of the selectivity filter structure and is oriented to project its Lys27 into the pore. The most extracellular K+ binding site (S1) is devoid of K+ electron-density when wild-type CTX is bound, but K+ density is present to some extent in a Lys27Met mutant. In crystals with Cs+ replacing K+, S1 electron-density is present even in the presence of Lys27, a finding compatible with the differential effects of Cs+ vs K+ on CTX affinity for the channel. Together, these results show that CTX binds to a K+ channel in a lock and key manner and interacts directly with conducting ions inside the selectivity filter. DOI: http://dx.doi.org/10.7554/eLife.00594.001

Domain-swapped chain connectivity and gated membrane access in a Fab-mediated crystal of the human TRAAK K+ channel.

TRAAK (TWIK-related arachidonic acid-stimulated K+ channel, K2P4.1) K+ ion channels are expressed predominantly in the nervous system to control cellular resting membrane potential and are regulated by mechanical and chemical properties of the lipid membrane. TRAAK channels are twofold symmetric, which precludes a direct extension of gating mechanisms that close canonical fourfold symmetric K+ channels. We present the crystal structure of human TRAAK in complex with antibody antigen-binding fragments (Fabs) at 2.75-Å resolution. In contrast to a previous structure, this structure reveals a domain-swapped chain connectivity enabled by the helical cap that exchanges two opposing outer helices 180° around the channel. An unrelated conformational change of an inner helix seals a side opening to the membrane bilayer and is associated with structural changes around the K+-selectivity filter that may have implications for mechanosensitivity and gating of TRAAK channels.

Molecular mechanism of proton transport in CLC Cl-/H+ exchange transporters

CLC proteins underlie muscle, kidney, bone, and other organ system function by catalyzing the transport of Cl- ions across cell and organellar membranes. Some CLC proteins are ion channels while others are pumps that exchange Cl- for H+. The pathway through which Cl- ions cross the membrane has been characterized, but the transport of H+ and the principle by which their movement is coupled to Cl- movement is not well understood. Here we show that H+ transport depends not only on the presence of a specific glutamate residue but also the presence of Cl- ions. H+ transport, however, can be isolated and analyzed in the absence of Cl- by mutating the glutamate to alanine and adding carboxylate-containing molecules to solution, consistent with the notion that H+ transfer is mediated through the entry of a carboxylate group into the anion pathway. Cl- ions and carboxylate interact with each other strongly. These data support a mechanism in which the glutamate carboxylate functions as a surrogate Cl- ion, but it can accept a H+ and transfer it between the external solution and the central Cl- binding site, coupled to the movement of 2 Cl- ions.

Structure of a CLC chloride ion channel by cryo-electron microscopy.

CLC proteins transport chloride (Cl−) ions across cellular membranes to regulate muscle excitability, electrolyte movement across epithelia, and acidification of intracellular organelles. Some CLC proteins are channels that conduct Cl− ions passively, whereas others are secondary active transporters that exchange two Cl− ions for one H+. The structural basis underlying these distinctive transport mechanisms is puzzling because CLC channels and transporters are expected to share the same architecture on the basis of sequence homology. Here we determined the structure of a bovine CLC channel (CLC-K) using cryo-electron microscopy. A conserved loop in the Cl− transport pathway shows a structure markedly different from that of CLC transporters. Consequently, the cytosolic constriction for Cl− passage is widened in CLC-K such that the kinetic barrier previously postulated for Cl−/H+ transporter function would be reduced. Thus, reduction of a kinetic barrier in CLC channels enables fast flow of Cl− down its electrochemical gradient.

Liquid chromatographic separation of enantiomers of β-amino acids using a Chiral stationary phase

Abstract The enantiomers of both α-substituted β-alanines and β-substituted β-alanines may be chromatographically separated using silica-bonded chiral stationary phases derived from N-acetylated α-arylalkylamines. The amino acids are chromatographed as alkyl esters of N-3,5-dinitrobenzoyl derivatives; separability factors range from 1.11 to 1.65 for nine α-substituted β-alanines and from 1.08 to 1.20 for nine β-substituted β-alanines. The enantiomers of β-aminoisobutyrate and β-leucine, chiral β-amino acids occurring in animal tissues and physiological fluids, are among those resolved. The enantiomers of R,S-β-aminoisobutyrate and several related α-alkyl-β-alanines were prepared by chromatographic resolution of diastereomeric dipeptides.

1H, 15N, 13C and 13CO assignments and secondary structure determination of basic fibroblast growth factor using 3D heteronuclear NMR spectroscopy

SummaryThe assignments of the 1H, 15N, 13CO and 13C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N-/13C-labeled FGF-2 with an isotope incorporation >95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of Cα, Cβ and Hα to the backbone amide 1H and 15N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of Cα, Cβ and Hα to the backbone amide 1H and 15N in the CBCANH and HNHA experiments. In addition, Cα and Cβ chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and Cα correlations from the carbonyl correlations observed in the HNCO and HCACO experiments and Cα correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic 1H and 13C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, Hα and Hβ protons as well as 3JHnHα coupling constants, amide exchange and 13Cα and 13Cβ secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel β-sheets (residues 30–34, 39–44, 48–53, 62–67, 71–76, 81–85, 91–94, 103–108, 113–118, 123–125 and 148–152) and a helix-like structure (residues 131–136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131–136 were defined as β-strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128–138) instead of the β-strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9–28. This is consistent with the X-ray structures of FGF-2, where the first 17–19 residues were ill defined.

Glutathione monoethyl ester: High-performance liquid chromatographic analysis and direct preparation of the free base form

Glutathione monoethyl ester (L-gamma-glutamyl-L-cysteinylglycine ethyl ester) was shown by R. N. Puri and A. Meister (1983, Proc. Natl. Acad. Sci. USA 80, 5258-5260) to be taken up by several tissues and intracellularly hydrolyzed to GSH. Since GSH itself is not significantly taken up by tissues, glutathione monoesters provide the most direct and convenient means available for increasing the intracellular GSH concentration of many tissues and cell types. In previous studies glutathione esters were prepared by HCl- or H2SO4-catalyzed esterification, and the product esters were precipitated as acidic salts by addition of ether to the reaction mixtures. In the present studies, glutathione monoethyl ester was synthesized by H2SO4-catalyzed esterification in the presence of sodium sulfate as the dehydrating agent. When no GSH remained, alcohol-washed Dowex-1 resin (hydroxide form) was added to remove sulfate and neutralize the reaction mixture. After the resin was removed by filtration, glutathione monoethyl ester crystallized in the chilled filtrate. The product was free of sulfate, GSH, and glutathione diester; its solutions in water or saline were neutral. Preparations obtained to date are nontoxic when administered to mice in doses up to at least 10 mmol/kg. Progress of the esterification reaction and purity of the product were determined quantitatively by HPLC after derivatization of the thiols with monobromobimane. Elution times of GSH, glutathione diester, and glutathione monoesters involving either the glutamyl or the glycyl carboxylate groups are reported.