Crystal structure and mechanism of a calcium-gated potassium channel

Ion channels exhibit two essential biophysical properties; that is, selective ion conduction, and the ability to gate-open in response to an appropriate stimulus. Two general categories of ion channel gating are defined by the initiating stimulus: ligand binding (neurotransmitter- or second-messenger-gated channels) or membrane voltage (voltage-gated channels). Here we present the structural basis of ligand gating in a K(+) channel that opens in response to intracellular Ca(2+). We have cloned, expressed, analysed electrical properties, and determined the crystal structure of a K(+) channel (MthK) from Methanobacterium thermoautotrophicum in the Ca(2+)-bound, opened state. Eight RCK domains (regulators of K(+) conductance) form a gating ring at the intracellular membrane surface. The gating ring uses the free energy of Ca(2+) binding in a simple manner to perform mechanical work to open the pore.     

The open pore conformation of potassium channels

Living cells regulate the activity of their ion channels through a process known as gating. To open the pore, protein conformational changes must occur within a channel's membrane-spanning ion pathway. KcsA and MthK, closed and opened K(+) channels, respectively, reveal how such gating transitions occur. Pore-lining 'inner' helices contain a 'gating hinge' that bends by approximately 30 degrees. In a straight conformation four inner helices form a bundle, closing the pore near its intracellular surface. In a bent configuration the inner helices splay open creating a wide (12 A) entryway. Amino-acid sequence conservation suggests a common structural basis for gating in a wide range of K(+) channels, both ligand- and voltage-gated. The open conformation favours high conduction by compressing the membrane field to the selectivity filter, and also permits large organic cations and inactivation peptides to enter the pore from the intracellular solution.     

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 A, 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 alpha-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.     

X-ray structure of a voltage-dependent K+ channel

Voltage-dependent K+ channels are members of the family of voltage-dependent cation (K+, Na+ and Ca2+) channels that open and allow ion conduction in response to changes in cell membrane voltage. This form of gating underlies the generation of nerve and muscle action potentials, among other processes. Here we present the structure of KvAP, a voltage-dependent K+ channel from Aeropyrum pernix. We have determined a crystal structure of the full-length channel at a resolution of 3.2 A, and of the isolated voltage-sensor domain at 1.9 A, both in complex with monoclonal Fab fragments. The channel contains a central ion-conduction pore surrounded by voltage sensors, which form what we call 'voltage-sensor paddles'-hydrophobic, cationic, helix-turn-helix structures on the channel's outer perimeter. Flexible hinges suggest that the voltage-sensor paddles move in response to membrane voltage changes, carrying their positive charge across the membrane.     

Determining the architectures of macromolecular assemblies

To understand the workings of a living cell, we need to know the architectures of its macromolecular assemblies. Here we show how proteomic data can be used to determine such structures. The process involves the collection of sufficient and diverse high-quality data, translation of these data into spatial restraints, and an optimization that uses the restraints to generate an ensemble of structures consistent with the data. Analysis of the ensemble produces a detailed architectural map of the assembly. We developed our approach on a challenging model system, the nuclear pore complex (NPC). The NPC acts as a dynamic barrier, controlling access to and from the nucleus, and in yeast is a 50 MDa assembly of 456 proteins. The resulting structure, presented in an accompanying paper, reveals the configuration of the proteins in the NPC, providing insights into its evolution and architectural principles. The present approach should be applicable to many other macromolecular assemblies.     

The molecular architecture of the nuclear pore complex

Nuclear pore complexes (NPCs) are proteinaceous assemblies of approximately 50 MDa that selectively transport cargoes across the nuclear envelope. To determine the molecular architecture of the yeast NPC, we collected a diverse set of biophysical and proteomic data, and developed a method for using these data to localize the NPC's 456 constituent proteins (see the accompanying paper). Our structure reveals that half of the NPC is made up of a core scaffold, which is structurally analogous to vesicle-coating complexes. This scaffold forms an interlaced network that coats the entire curved surface of the nuclear envelope membrane within which the NPC is embedded. The selective barrier for transport is formed by large numbers of proteins with disordered regions that line the inner face of the scaffold. The NPC consists of only a few structural modules that resemble each other in terms of the configuration of their homologous constituents, the most striking of these being a 16-fold repetition of 'columns'. These findings provide clues to the evolutionary origins of the NPC.     

Essential role for collectrin in renal amino acid transport

Angiotensin -converting enzyme 2 (ACE2) is a regulator of the renin angiotensin system involved in acute lung failure, cardiovascular functions and severe acute respiratory syndrome (SARS) infections in mammals. A gene encoding a homologue to ACE2, termed collectrin (Tmem27), has been identified in immediate proximity to the ace2 locus. The in vivo function of collectrin was unclear. Here we report that targeted disruption of collectrin in mice results in a severe defect in renal amino acid uptake owing to downregulation of apical amino acid transporters in the kidney. Collectrin associates with multiple apical transporters and defines a novel group of renal amino acid transporters. Expression of collectrin in Xenopus oocytes and Madin-Darby canine kidney (MDCK) cells enhances amino acid transport by the transporter B(0)AT1. These data identify collectrin as a key regulator of renal amino acid uptake.     

Uptake of labelled DNA by isolated perfused organs

Résumé L'absorption et la dégradation du DNA ont été étudiées dans le foie perfusé in vitro. Alors que les DNA très polymérisés peuvent être absorbés par l'organe et y être retenus (en partie tout au moins), les DNA dégradés sont hydrolysés en composés acido solubles. Les DNA très polymérisés ne sont pas dégradés dans le sang ni dans les organes étudiés.