Small vertical movement of a K+ channel voltage sensor measured with luminescence energy transfer

214

249

Voltage-gated ion channels control generation and propagation of action potentials in excitable cells. Significant progress has been made in understanding structure and function of the voltage-gated ion channels, highlighted by the high-resolution open-state structure of the voltage-gated potassium channel, Kv1.2. However, because the structure of the closed state is unknown, the gating mechanism remains controversial. We adapted the ROSETTA membrane method to model the structures of the Kv1.2 and KvAP channels using homology, de novo, and domain assembly methods and selected the most plausible models using a limited number of experimental constraints. suggests gating movement that can be viewed as a sum of two previously suggested mechanisms: translation (2-4 Å) plus rotation (Ϸ180°) of the S4 segment as proposed in the original ''sliding helix'' or ''helical screw'' models coupled with a rolling motion of the S1-S3 segments around S4, similar to recent ''transporter'' models of gating. We propose a unified mechanism of voltagedependent gating for K v1.2 and KvAP in which this major conformational change moves the gating charge across the electric field in an analogous way for both channels.membrane protein ͉ ROSETTA method ͉ voltage-gated ion channel V oltage-gated potassium (Kv) channels are members of the voltage-gated ion channel superfamily (1, 2), which is important for initiation and propagation of action potentials in excitable cells. They are composed of four identical or homologous subunits, each containing six transmembrane segments: S1-S6. Segments S1-S4 form the voltage-sensing domain (VSD), and segments S5 and S6 connected by the P loop, which is involved in ion selectivity, comprise the pore-forming domain (PD). S4 has four gating-charge-carrying arginines (R1-R4) spaced at intervals of three amino acid residues, which are highly conserved and are thought to play a key role in coupling changes in membrane voltage to opening and closing of the pore (3-5). In the Kv channels Ϸ13 electronic charges cross the membrane electrical field per channel between the closed and open states (6-8).High-resolution structures of the bacterial potassium channel KvAP and the mammalian potassium channel K v 1.2 recently have been solved (9-11). Although the KvAP structures showed the VSD in a nonnative orientation with respect to the membrane and the PD, the K v 1.2 structure captured the VSD in a conformation that is thought to represent the open state of the channel. In addition, the structure reveals that the VSD from one subunit interacts closely with the PD of the adjacent subunit in a clockwise direction when the channel structure is viewed from the extracellular side of the membrane (9). However, the closedstate structure of these channels remains unknown, and the mechanism of action of the voltage sensor in translocating gating charge is a subject of controversy. The original ''sliding helix'' or ''helical screw'' models of gating posited that S4 moves outward along a clockwise spiral path through the protei...

Section: Model Of Kv12 In the Closedsupporting

confidence: 73%

mentioning

confidence: 99%

Voltage sensor conformations in the open and closed states in rosetta structural models of K ⁺ channels

Yarov‐Yarovoy¹,

Baker

Catterall³

2006

214

249

“…Ligand-induced changes in LRET and distances were measured as described in SI Text. The proportion of slow and fast populations was calculated as previously described (31), and details on the analysis are available in SI Text.…”

Section: Methodsmentioning

confidence: 99%

Structural insights into biased G protein-coupled receptor signaling revealed by fluorescence spectroscopy

Rahmeh

Damian

Cottet

et al. 2012

173

137

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Recent studies show that some GPCRs signal through both G protein and arrestin pathways in a ligand-specific manner. Ligands that direct signaling through a specific pathway are known as biased ligands. The arginine-vasopressin type 2 receptor (V2R), a prototypical peptide-activated GPCR, is an ideal model system to investigate the structural basis of biased signaling. Although the native hormone arginine-vasopressin leads to activation of both the stimulatory G protein (Gs) for the adenylyl cyclase and arrestin pathways, synthetic ligands exhibit highly biased signaling through either Gs alone or arrestin alone. We used purified V2R stabilized in neutral amphipols and developed fluorescence-based assays to investigate the structural basis of biased signaling for the V2R. Our studies demonstrate that the Gs-biased agonist stabilizes a conformation that is distinct from that stabilized by the arrestin-biased agonists. This study provides unique insights into the structural mechanisms of GPCR activation by biased ligands that may be relevant to the design of pathway-biased drugs.

“…However, the experiments providing structural constraints about the closed state conformation of the voltage sensor are more uncertain. Although it is generally accepted that the S1 and S2 helical segments do not move extensively upon gating (4)(5)(6)(7)(8), there is disagreement concerning the magnitude of movements associated with the S3 and S4 segments. For example, histidine scanning of the charged residues and cysteine scanning of the S4 and S3 segments indicate that the charges change exposure from the inside to the outside when the membrane is depolarized (9)(10)(11)(12)(13).…”

mentioning

confidence: 99%

Two atomic constraints unambiguously position the S4 segment relative to S1 and S2 segments in the closed state of Shaker K channel

Campos

Chanda

Roux

et al. 2007

Self Cite

161

218

It is now well established that the voltage-sensing S4 segment in voltage-dependent ion channels undergoes a conformational change in response to varying membrane potential. However, the magnitude of the movement of S4 relative to the membrane and the rest of the protein remains controversial. Here, by using histidine scanning mutagenesis in the Shaker K channel, we identified mutants I241H (S1 segment) and I287H (S2 segment) that generate inward currents at hyperpolarized potentials, suggesting that these residues are part of a hydrophobic plug that separates the water-accessible crevices. Additional experiments with substituted cysteine residues showed that, at hyperpolarized potentials, both I241C and I287C can spontaneously form disulphide and metal bridges with R362C, the position of the first charge-carrying residue in S4. These results constrain unambiguously the closedstate positions of the S4 segment with respect to the S1 and S2 segments, which are known to undergo little or no movement during gating. To satisfy these constraints, the S4 segment must undergo an axial rotation of Ϸ180°and a transmembrane (vertical) movement of Ϸ6.5 Å at the level of R362 in going from the open to the closed state of the channel, moving the gating charge across a focused electric field.gating current ͉ metal bridge ͉ omega current ͉ S-S bridge V oltage-gated channels are crucial players in excitability and cell homeostasis. Voltage-dependent sodium and potassium conductances are responsible for the generation and propagation of the nerve impulse (1). The salient property of these channels is the steep membrane potential dependence of their open probability. This voltage dependence is conferred by the voltage sensor that has been identified with their first four transmembrane segments (S1-S4). It is now clear that the four most extracellular basic residues of the S4 segment and the most intracellular acidic residue in the S2 segment in the Shaker K channel are the gating charge-carrying residues that move in the field in response to changes in membrane potential (2, 3). The movement and conformational change of segments S1-S4 within the voltage-sensing module in response to membrane depolarization is somehow coupled to the intracellular gate of the pore domain (S5-S6) to open and close the channel. A multitude of biophysical experiments have attempted to delineate the conformation of the voltage sensor and the position of its charges in the open and closed states. The structural restraints deduced from those experiments have subsequently been shown to be consistent with the overall conformation of the open state from the crystallographic structure of the Kv1.2 channel. However, the experiments providing structural constraints about the closed state conformation of the voltage sensor are more uncertain. Although it is generally accepted that the S1 and S2 helical segments do not move extensively upon gating (4-8), there is disagreement concerning the magnitude of movements associated with the S3 and S4 segments. For exampl...