No abstract

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.

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.

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 channel's 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.

The dopamine transporter (DAT) is a target of amphetamine (AMPH) and cocaine. These psychostimulants attenuate DAT clearance efficiency, thereby increasing synaptic dopamine (DA) levels. Re-uptake rate is determined by the number of functional transporters at the cell surface as well as by their turnover rate. Here, we present evidence that DAT substrates, including AMPH and DA, cause internalization of human DAT, thereby reducing transport capacity. Acute treatment with AMPH reduced the maximal rate of Dopamine (DA) signaling in the central nervous system mediates a wide variety of physiologic functions such as movement, motivational control of voluntary behavior, and lactation (1, 2). The magnitude and duration of DA signaling is defined by the amount of vesicular release, the sensitivity of the DA receptors, and the efficiency of DA clearance. The DA transporter (DAT) is largely responsible for regulating DA clearance (3).Psychostimulants, such as cocaine and amphetamine (AMPH), induce DA overflow into the synaptic cleft by acting on the DAT, thereby enhancing dopaminergic transmission (4). Cocaine acts by inhibiting the re-uptake of released DA (5, 6). AMPH-like drugs, however, are thought to promote the release of the transmitter (carrier-mediated efflux) as well as to inhibit its uptake (7,8). Repeated administration of AMPH has been shown to sensitize monoaminergic synapses to subsequent psychostimulant challenge (9). Furthermore, administration of a single, high dose of AMPH acutely (1 h) decreased DAT function in vivo as assessed in striatal synaptosomes prepared from drug-treated rats (10). In contrast, administration of a high dose of cocaine had no effect on subsequent transporter activity (10).To explore the mechanism for the differential effects of AMPH and cocaine on the homeostatic uptake capacity of the human DAT (hDAT), we stably expressed a FLAG-tagged hDAT in EM4 cells (see Materials and Methods). The use of the FLAG fusion protein has provided the opportunity for confocal microscopy analysis of trafficking of the transporter in cells. Here, we report that AMPH caused the hDAT to redistribute intracellularly in a dynamindependent manner, consequently reducing subsequent DA transport capacity. These results provide a mechanism for the AMPHinduced elevation of synaptic DA mediated through a reduction of the number of transporters on the cell surface. Materials and MethodsCell Culture. We created a synthetic hDAT gene, which was tagged at the amino terminus with a FLAG epitope. The gene encodes a protein with an amino acid sequence identical to that of wild-type hDAT with the Met at position 1 replaced by MDYKDDDDKA, but the nucleotide sequence was altered to increase the number of unique restriction sites and to optimize codon utilization. The nucleotide sequence of this construct and its creation will be described elsewhere. The FLAG-tagged syntheticDAT was subcloned into a bicistronic expression vector that expresses the syntheticDAT from a cytomegalovirus promoter and the hygromycin resista...

Inward-rectifier potassium (K + ) channels conduct K + ions most efficiently in one direction, into the cell. Kir2 channels control the resting membrane voltage in many electrically excitable cells and heritable mutations cause periodic paralysis and cardiac arrhythmia. We present the crystal structure of Kir2.2 from chicken, which, excluding the unstructured amino and carboxyl termini, is 90% identical to human Kir2.2. Crystals containing rubidium (Rb + ), strontium (Sr 2+ ), and europium (Eu 3+ ) reveal binding sites along the ion conduction pathway that are both conductive and inhibitory. The sites correlate with extensive electrophysiological data and provide a structural basis for understanding rectification. The channel's extracellular surface, with large structured turrets and an unusual selectivity filter entryway, might explain the relative insensitivity of eukaryotic inward rectifiers to toxins. These same surface features also suggest a possible approach to the development of inhibitory agents specific to each member of the inward-rectifier K + channel family.In 1949 Bernard Katz introduced the term "anomalous rectification" to distinguish the K + currents he observed in frog skeletal muscle from the "delayed rectification" K + currents of the squid axon action potential (1,2). Today we know that "delayed rectifiers" are a subset of the large family of voltage-dependent K + (Kv) channels, whereas "anomalous rectifiers" are members of a different family of channels more commonly known as inward-rectifier K + (Kir) channels (3). The name "inward rectifier" refers to a fundamental ion-conduction property exhibited to a greater or lesser degree by all members of the family: Given an equal but opposite electrochemical driving force, K + conductance into the cell far exceeds conductance out of the cell. Thus, Kir channels are analogous to one-way conductors, or diodes, in solid-state electronic devices.Electrophysiological experiments have shown that inward rectification is a consequence of voltage-dependent pore blockage by intracellular multivalent cations, especially Mg 2+ and polyamines (4-8). At internal negative (hyperpolarizing) membrane voltages the blocking ions are cleared from the pore so that K + conducts, whereas at internal positive (depolarizing) membrane voltages the blocking ions are driven into the pore from the cytoplasm so that K + conduction is blocked. As a result, Kir channels are conductive when an excitable cell is at rest and non-conductive during excitation. This property is thought to foster energy efficiency because it enables Kir channels to regulate the resting membrane potential, but not dissipate the K + gradient during an action potential (3). Another longstanding puzzle in eukaryotic Kir channel studies is their relative insensitivity to natural toxins that typically inhibit other K + channels (22-24). Snake, spider and scorpion venoms, for example, contain numerous toxins against various Kv channels and Ca 2+ -activated K + channels (25)(26)(27). By contrast, Kir ...

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 argininecontaining helix-turn-helix element at the protein-lipid interface.membrane protein ͉ protein-lipid interface ͉ voltage-gated ion channel ͉ voltage sensor V oltage-dependent ion channels ''sense'' voltage differences across the cell membrane and open or close in response to its value (1). These channels contain a centrally located pore surrounded by four voltage sensors. Voltage-dependent K ϩ (Kv) channels are tetramers with four identical subunits, each with six transmembrane segments (S1-S6): S5 and S6 form the central pore at the interface between the subunits, and S1-S4 form the voltage sensors (2, 3). The voltage sensors have four to seven positively charged amino acids (usually arginine) on S4, known as gating charges, and fewer negatively charged amino acids (aspartate or glutamate) distributed on S1, S2, and S3. The voltage sensors undergo a conformational change when the pore gates open, coupling movement of the S4 gating charges within the membrane electric field to channel open probability.The first structure of a Kv channel, termed KvAP, from the archeabacterium Aeropyrum Pernix (4), was determined by crystallizing the channel as a complex with a monoclonal Fab fragment attached to its voltage sensors [Protein Data Bank (PDB) ID code 1ORQ] (5). In that structure the voltage sensors are in a non-native conformation, displaced toward the intracellular side of the transmembrane pore. Together with a second structure of the voltage sensor alone, termed the isolated voltage sensor (PDB ID code 1ORS), the following ideas about voltagedependent gating were proposed (5, 6): that the voltage sensor is a very mobile structure, presumably because it must carry charged amino acids through the membrane electric field; that the charge bearing S4 forms a helix-turn-helix with the Cterminal half of S3 (S3b), and that the helix-turn-helix moves at the protein-lipid interface a large distance. Experiments using avidin capture of biotin linked to the voltage sensor indicated that four of the S4 arginine amino acids translate 15-20 Å across the membrane. These struc...

Voltage-dependent ion channels open and conduct ions in response to changes in cell-membrane voltage. The voltage sensitivity of these channels arises from the motion of charged arginine residues located on the S4 helices of the channel's voltage sensors. In KvAP, a prokaryotic voltage-dependent K+ channel, the S4 helix forms part of a helical hairpin structure, the voltage-sensor paddle. We have measured the membrane depth of residues throughout the KvAP channel using avidin accessibility to different-length tethered biotin reagents. From these measurements, we have calibrated the tether lengths and derived the thickness of the membrane that forms a barrier to avidin penetration, allowing us to determine the magnitude of displacement of the voltage-sensor paddles during channel gating. Here we show that the voltage-sensor paddles are highly mobile compared to other regions of the channel and transfer the gating-charge arginines 15-20 A through the membrane to open the pore.