Youxing Jiang

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.

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.

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.

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.

STRUCTURE OF THE RCK DOMAIN FROM THE E. COLI K+ CHANNEL AND DEMONSTRATION OF ITS PRESENCE IN THE HUMAN BK CHANNEL

The intracellular C-terminal domain structure of a six-transmembrane K+ channel from Escherichia coli has been solved by X-ray crystallography at 2.4 A resolution. The structure is representative of a broad class of domains/proteins that regulate the conductance of K+ (here referred to as RCK domains) in prokaryotic K+ transporters and K+ channels. The RCK domain has a Rossmann-fold topology with unique positions, not commonly conserved among Rossmann-fold proteins, composing a well-conserved salt bridge and a hydrophobic dimer interface. Structure-based amino acid sequence alignments and mutational analysis are used to demonstrate that an RCK domain is also present and is an important component of the gating machinery in eukaryotic large-conductance Ca2+ activated K+ channels.

Functional analysis of an archaebacterial voltage-dependent K + channel

All living organisms use ion channels to regulate the transport of ions across cellular membranes. Certain ion channels are classed as voltage-dependent because they have a voltage-sensing structure that induces their pores to open in response to changes in the cell membrane voltage. Until recently, the voltage-dependent K+, Ca2+ and Na+ channels were regarded as a unique development of eukaryotic cells, adapted to accomplish specialized electrical signalling, as exemplified in neurons. Here we present the functional characterization of a voltage-dependent K+ (KV) channel from a hyperthermophilic archaebacterium from an oceanic thermal vent. This channel possesses all the functional attributes of classical neuronal KV channels. The conservation of function reflects structural conservation in the voltage sensor as revealed by specific, high-affinity interactions with tarantula venom toxins, which evolved to inhibit eukaryotic KV channels.

Atomic structure of a Na+- and K+-conducting channel.

Ion selectivity is one of the basic properties that define an ion channel. Most tetrameric cation channels, which include the K+, Ca2+, Na+ and cyclic nucleotide-gated channels, probably share a similar overall architecture in their ion-conduction pore, but the structural details that determine ion selection are different. Although K+ channel selectivity has been well studied from a structural perspective, little is known about the structure of other cation channels. Here we present crystal structures of the NaK channel from Bacillus cereus, a non-selective tetrameric cation channel, in its Na+- and K+-bound states at 2.4 Å and 2.8 Å resolution, respectively. The NaK channel shares high sequence homology and a similar overall structure with the bacterial KcsA K+ channel, but its selectivity filter adopts a different architecture. Unlike a K+ channel selectivity filter, which contains four equivalent K+-binding sites, the selectivity filter of the NaK channel preserves the two cation-binding sites equivalent to sites 3 and 4 of a K+ channel, whereas the region corresponding to sites 1 and 2 of a K+ channel becomes a vestibule in which ions can diffuse but not bind specifically. Functional analysis using an 86Rb flux assay shows that the NaK channel can conduct both Na+ and K+ ions. We conclude that the sequence of the NaK selectivity filter resembles that of a cyclic nucleotide-gated channel and its structure may represent that of a cyclic nucleotide-gated channel pore.

Structure of the gating ring from the human large-conductance Ca(2+)-gated K(+) channel.

Large-conductance Ca2+-gated K+ (BK) channels are essential for many biological processes such as smooth muscle contraction and neurotransmitter release. This group of channels can be activated synergistically by both voltage and intracellular Ca2+, with the large carboxy-terminal intracellular portion being responsible for Ca2+ sensing. Here we present the crystal structure of the entire cytoplasmic region of the human BK channel in a Ca2+-free state. The structure reveals four intracellular subunits, each comprising two tandem RCK domains, assembled into a gating ring similar to that seen in the MthK channel and probably representing its physiological assembly. Three Ca2+ binding sites including the Ca2+ bowl are mapped onto the structure based on mutagenesis data. The Ca2+ bowl, located within the second RCK domain, forms an EF-hand-like motif and is strategically positioned close to the assembly interface between two subunits. The other two Ca2+ (or Mg2+) binding sites, Asp 367 and Glu 374/Glu 399, are located on the first RCK domain. The Asp 367 site has high Ca2+ sensitivity and is positioned in the groove between the amino- and carboxy-terminal subdomains of RCK1, whereas the low-affinity Mg2+-binding Glu 374/Glu 399 site is positioned on the upper plateau of the gating ring and close to the membrane. Our structure also contains the linker connecting the transmembrane and intracellular domains, allowing us to dock a voltage-gated K+ channel pore of known structure onto the gating ring with reasonable accuracy and generate a structural model for the full BK channel.

Structural Insight into the Ion-Exchange Mechanism of the Sodium/Calcium Exchanger

Maintaining Equilibrium Na+/Ca2+ exchangers (NCX) are membrane transporters that maintain the homeostasis of cytosolic Ca2+ and play an essential role in Ca2+ signaling. Despite a long history of physiological work and a large body of functional data, the structural basis underlying the ion exchange mechanism of NCX is poorly understood. Liao et al. (p. 686; see the Perspective by Abramson et al.) present a high-resolution crystal structure of an NCX from Methanococcus jannaschii and demonstrate that this archaeal NCX catalyzes Na+/Ca2+-exchange reactions similar to its eukaryotic counterpart. The structure clarifies the mechanism of ion exchange proteins and reveals the basis for the stoichiometry, cooperativity, and bidirectionality of the reaction. The arrangement of four central ion-binding sites with differing specificities suggests how a membrane transporter protein works. Sodium/calcium (Na+/Ca2+) exchangers (NCX) are membrane transporters that play an essential role in maintaining the homeostasis of cytosolic Ca2+ for cell signaling. We demonstrated the Na+/Ca2+-exchange function of an NCX from Methanococcus jannaschii (NCX_Mj) and report its 1.9 angstrom crystal structure in an outward-facing conformation. Containing 10 transmembrane helices, the two halves of NCX_Mj share a similar structure with opposite orientation. Four ion-binding sites cluster at the center of the protein: one specific for Ca2+ and three that likely bind Na+. Two passageways allow for Na+ and Ca2+ access to the central ion-binding sites from the extracellular side. Based on the symmetry of NCX_Mj and its ability to catalyze bidirectional ion-exchange reactions, we propose a structure model for the inward-facing NCX_Mj.

Novel insights into K(+) selectivity from high-resolution structures of an open K(+) channel pore.

K+ channels are highly selective for K+ over Na+. Here we present several crystal structures of the MthK K+ channel pore at up to 1.45-Å resolution. The MthK selectivity filter maintains a conductive conformation even in the absence of K+, allowing the channel to conduct Na+. The high-resolution structures, along with single-channel recordings, allow for an accurate analysis of how K+ competes with Na+ in a conductive selectivity filter. At high K+ concentrations, two K+ ions equivalently occupy the four sites in the selectivity filter, whereas at low K+/high Na+ concentrations, a single K+ ion remains bound in the selectivity filter, preferably at site 1 or site 3. This single K+ binding at low concentration effectively blocks the permeation of Na+, providing a structural basis for the anomalous mole-fraction effect, a key property of multi-ion pores.