Roderick MacKinnon

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 Å, and of the isolated voltage-sensor domain at 1.9 Å, 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 channels outer perimeter. Flexible hinges suggest that the voltage-sensor paddles move in response to membrane voltage changes, carrying their positive charge across the membrane.

Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 A resolution.

Ion transport proteins must remove an ions hydration shell to coordinate the ion selectively on the basis of its size and charge. To discover how the K+ channel solves this fundamental aspect of ion conduction, we solved the structure of the KcsA K+ channel in complex with a monoclonal Fab antibody fragment at 2.0 Å resolution. Here we show how the K+ channel displaces water molecules around an ion at its extracellular entryway, and how it holds a K+ ion in a square antiprism of water molecules in a cavity near its intracellular entryway. Carbonyl oxygen atoms within the selectivity filter form a very similar square antiprism around each K+ binding site, as if to mimic the waters of hydration. The selectivity filter changes its ion coordination structure in low K+ solutions. This structural change is crucial to the operation of the selectivity filter in the cellular context, where the K+ ion concentration near the selectivity filter varies in response to channel gating.

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.

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 Ca2+. We have cloned, expressed, analysed electrical properties, and determined the crystal structure of a K+ channel (MthK) from Methanobacterium thermoautotrophicum in the Ca2+-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 Ca2+ binding in a simple manner to perform mechanical work to open the pore.

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.

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 channels 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°. 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 Å) 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.

Crystal Structures of a Complexed and Peptide-Free Membrane Protein–Binding Domain: Molecular Basis of Peptide Recognition by PDZ

Modular PDZ domains, found in many cell junction-associated proteins, mediate the clustering of membrane ion channels by binding to their C-terminus. The X-ray crystallographic structures of the third PDZ domain from the synaptic protein PSD-95 in complex with and in the absence of its peptide ligand have been determined at 1.8 angstroms and 2.3 angstroms resolution, respectively. The structures reveal that a four-residue C-terminal stretch (X-Thr/Ser-X-Val-COO(-)) engages the PDZ domain through antiparallel main chain interactions with a beta sheet of the domain. Recognition of the terminal carboxylate group of the peptide is conferred by a cradle of main chain amides provided by a Gly-Leu-Gly-Phe loop as well as by an arginine side chain. Specific side chain interactions and a prominent hydrophobic pocket explain the selective recognition of the C-terminal consensus sequence.

Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure.

Intracellular Ca2+ release and reuptake are essential for contraction and relaxation of normal heart muscle. Intracellular Ca2+ transients were recorded with aequorin during isometric contraction of myocardium from patients with end-stage heart failure. In contrast to controls, contractions and Ca2+ transients of muscles from failing hearts were markedly prolonged, and the Ca2+ transients exhibited 2 distinct components. Muscles from failing hearts showed a diminished capacity to restore low resting Ca2+ levels during diastole. These experiments provide the first direct evidence from actively contracting human myocardium that intracellular Ca2+ handling is abnormal and may cause systolic and diastolic dysfunction in heart failure.

The principle of gating charge movement in a voltage-dependent K + channel

The steep dependence of channel opening on membrane voltage allows voltage-dependent K+ channels to turn on almost like a switch. Opening is driven by the movement of gating charges that originate from arginine residues on helical S4 segments of the protein. Each S4 segment forms half of a ‘voltage-sensor paddle’ on the channels outer perimeter. Here we show that the voltage-sensor paddles are positioned inside the membrane, near the intracellular surface, when the channel is closed, and that the paddles move a large distance across the membrane from inside to outside when the channel opens. KvAP channels were reconstituted into planar lipid membranes and studied using monoclonal Fab fragments, a voltage-sensor toxin, and avidin binding to tethered biotin. Our findings lead us to conclude that the voltage-sensor paddles operate somewhat like hydrophobic cations attached to levers, enabling the membrane electric field to open and close the pore.

Mutations in the K+ channel signature sequence.

Potassium channels share a highly conserved stretch of eight amino acids, a K+ channel signature sequence. The conserved sequence falls within the previously defined P-region of voltage-activated K+ channels. In this study we investigate the effect of mutations in the signature sequence of the Shaker channel on K+ selectivity determined under bi-ionic conditions. Nonconservative substitutions of two threonine residues and the tyrosine residue leave selectivity intact. In contrast, mutations at some positions render the channel nonselective among monovalent cations. These findings are consistent with a proposal that the signature sequence contributes to a selectivity filter. Furthermore, the results illustrate that the hydroxyl groups at the third and fourth positions, and the aromatic group at position seven, are not essential in determining K+ selectivity.