Alice Lee

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.

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.

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.

A Gating Charge Transfer Center in Voltage Sensors

Open and Closed Case Voltage-dependent ion channels are gated by voltage sensors that show a switchlike response to voltage differences across the membrane. Tao et al. (p. 67; see the cover) used mutagenesis, electrophysiology, and x-ray crystallography to gain insight into the molecular basis of this response in voltage-dependent potassium channels. An occluded site was identified that catalyzes translation of positive charges across the membrane. The closed channel appears to be associated with a distribution of conformations, depending on the degree of hyperpolarization of the membrane, whereas the open channel appears to be associated with a specific conformation. Thus, the transition of the ion channel from open to closed occurs over a very small voltage difference. An occluded site stabilizes charged amino acids as they cross the membrane field to achieve switchlike channel opening. Voltage sensors regulate the conformations of voltage-dependent ion channels and enzymes. Their nearly switchlike response as a function of membrane voltage comes from the movement of positively charged amino acids, arginine or lysine, across the membrane field. We used mutations with natural and unnatural amino acids, electrophysiological recordings, and x-ray crystallography to identify a charge transfer center in voltage sensors that facilitates this movement. This center consists of a rigid cyclic “cap” and two negatively charged amino acids to interact with a positive charge. Specific mutations induce a preference for lysine relative to arginine. By placing lysine at specific locations, the voltage sensor can be stabilized in different conformations, which enables a dissection of voltage sensor movements and their relation to ion channel opening.

Structure of the KvAP voltage-dependent K+ channel and its dependence on the lipid membrane.

Voltage-dependent ion channels gate open in response to changes in cell membrane voltage. This form of gating permits the propagation of action potentials. We present two structures of the voltage-dependent K+ channel KvAP, in complex with monoclonal Fv fragments (3.9 Å) and without antibody fragments (8 Å). We also studied KvAP with disulfide cross-bridges in lipid membranes. Analyzing these data in the context of the crystal structure of Kv1.2 and EPR data on KvAP we reach the following conclusions: (i) KvAP is similar in structure to Kv1.2 with a very modest difference in the orientation of its voltage sensor; (ii) mAb fragments are not the source of non-native conformations of KvAP in crystal structures; (iii) because KvAP contains separate loosely adherent domains, a lipid membrane is required to maintain their correct relative orientations, and (iv) the model of KvAP is consistent with the proposal of voltage sensing through the movement of an arginine-containing helix-turn-helix element at the protein-lipid interface.

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.

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