scholarly journals α-Helical Structural Elements within the Voltage-Sensing Domains of a K+ Channel

1999 ◽  
Vol 115 (1) ◽  
pp. 33-50 ◽  
Author(s):  
Yingying Li-Smerin ◽  
David H. Hackos ◽  
Kenton J. Swartz
Keyword(s):  
Secondary Structure ◽  
K Channel ◽  
Structural Elements ◽  
Alanine Scanning Mutagenesis ◽  
Voltage Sensing ◽  
Tertiary Structures ◽  
Voltage Gated ◽  
Transmembrane Segments ◽  
Membrane Spanning ◽  
Pore Domain

Voltage-gated K+ channels are tetramers with each subunit containing six (S1–S6) putative membrane spanning segments. The fifth through sixth transmembrane segments (S5–S6) from each of four subunits assemble to form a central pore domain. A growing body of evidence suggests that the first four segments (S1–S4) comprise a domain-like voltage-sensing structure. While the topology of this region is reasonably well defined, the secondary and tertiary structures of these transmembrane segments are not. To explore the secondary structure of the voltage-sensing domains, we used alanine-scanning mutagenesis through the region encompassing the first four transmembrane segments in the drk1 voltage-gated K+ channel. We examined the mutation-induced perturbation in gating free energy for periodicity characteristic of α-helices. Our results are consistent with at least portions of S1, S2, S3, and S4 adopting α-helical secondary structure. In addition, both the S1–S2 and S3–S4 linkers exhibited substantial helical character. The distribution of gating perturbations for S1 and S2 suggest that these two helices interact primarily with two environments. In contrast, the distribution of perturbations for S3 and S4 were more complex, suggesting that the latter two helices make more extensive protein contacts, possibly interfacing directly with the shell of the pore domain.

scholarly journals Localization and Molecular Determinants of the Hanatoxin Receptors on the Voltage-Sensing Domains of a K+ Channel

2000 ◽  
Vol 115 (6) ◽  
pp. 673-684 ◽  
Author(s):  
Yingying Li-Smerin ◽  
Kenton J. Swartz
Keyword(s):  
K Channel ◽  
Alanine Scanning Mutagenesis ◽  
Voltage Sensing ◽  
Alanine Scanning ◽  
K Channels ◽  
Voltage Gated ◽  
Physical Location ◽  
Molecular Determinants ◽  
Central Pore ◽  
Pore Domain

Hanatoxin inhibits voltage-gated K+ channels by modifying the energetics of activation. We studied the molecular determinants and physical location of the Hanatoxin receptors on the drk1 voltage-gated K+ channel. First, we made multiple substitutions at three previously identified positions in the COOH terminus of S3 to examine whether these residues interact intimately with the toxin. We also examined a region encompassing S1–S3 using alanine-scanning mutagenesis to identify additional determinants of the toxin receptors. Finally, guided by the structure of the KcsA K+ channel, we explored whether the toxin interacts with the peripheral extracellular surface of the pore domain in the drk1 K+ channel. Our results argue for an intimate interaction between the toxin and the COOH terminus of S3 and suggest that the Hanatoxin receptors are confined within the voltage-sensing domains of the channel, at least 20–25 Å away from the central pore axis.

scholarly journals Coupling between Voltage Sensors and Activation Gate in Voltage-gated K+ Channels

2002 ◽  
Vol 120 (5) ◽  
pp. 663-676 ◽  
Author(s):  
Zhe Lu ◽  
Angela M. Klem ◽  
Yajamana Ramu
Keyword(s):  
Electrical Signal ◽  
K Channel ◽  
Coupling Mechanism ◽  
Excitable Cells ◽  
Voltage Sensing ◽  
K Channels ◽  
Voltage Sensors ◽  
Voltage Gated ◽  
Transmembrane Segments ◽  
Activation Gate

Current through voltage-gated K+ channels underlies the action potential encoding the electrical signal in excitable cells. The four subunits of a voltage-gated K+ channel each have six transmembrane segments (S1–S6), whereas some other K+ channels, such as eukaryotic inward rectifier K+ channels and the prokaryotic KcsA channel, have only two transmembrane segments (M1 and M2). A voltage-gated K+ channel is formed by an ion-pore module (S5–S6, equivalent to M1–M2) and the surrounding voltage-sensing modules. The S4 segments are the primary voltage sensors while the intracellular activation gate is located near the COOH-terminal end of S6, although the coupling mechanism between them remains unknown. In the present study, we found that two short, complementary sequences in voltage-gated K+ channels are essential for coupling the voltage sensors to the intracellular activation gate. One sequence is the so called S4–S5 linker distal to the voltage-sensing S4, while the other is around the COOH-terminal end of S6, a region containing the actual gate-forming residues.

scholarly journals Atomic Constraints between the Voltage Sensor and the Pore Domain in a Voltage-gated K+ Channel of Known Structure

2008 ◽  
Vol 131 (6) ◽  
pp. 549-561 ◽  
Author(s):  
Anthony Lewis ◽  
Vishwanath Jogini ◽  
Lydia Blachowicz ◽  
Muriel Lainé ◽  
Benoît Roux
Keyword(s):  
Structural Model ◽  
Voltage Sensor ◽  
K Channel ◽  
Structural Reorganization ◽  
Voltage Gated ◽  
Transmembrane Segments ◽  
Close Physical Proximity ◽  
Activated State ◽  
Dynamics Simulations ◽  
Pore Domain

In voltage-gated K+ channels (Kv), membrane depolarization promotes a structural reorganization of each of the four voltage sensor domains surrounding the conducting pore, inducing its opening. Although the crystal structure of Kv1.2 provided the first atomic resolution view of a eukaryotic Kv channel, several components of the voltage sensors remain poorly resolved. In particular, the position and orientation of the charged arginine side chains in the S4 transmembrane segments remain controversial. Here we investigate the proximity of S4 and the pore domain in functional Kv1.2 channels in a native membrane environment using electrophysiological analysis of intersubunit histidine metallic bridges formed between the first arginine of S4 (R294) and residues A351 or D352 of the pore domain. We show that histidine pairs are able to bind Zn2+ or Cd2+ with high affinity, demonstrating their close physical proximity. The results of molecular dynamics simulations, consistent with electrophysiological data, indicate that the position of the S4 helix in the functional open-activated state could be shifted by ∼7–8 Å and rotated counterclockwise by 37° along its main axis relative to its position observed in the Kv1.2 x-ray structure. A structural model is provided for this conformation. The results further highlight the dynamic and flexible nature of the voltage sensor.

scholarly journals Functionality of the voltage-gated proton channel truncated in S4

2009 ◽  
Vol 107 (5) ◽  
pp. 2313-2318 ◽  
Author(s):  
Souhei Sakata ◽  
Tatsuki Kurokawa ◽  
Morten H. H. Nørholm ◽  
Masahiro Takagi ◽  
Yoshifumi Okochi ◽  
...  
Keyword(s):  
Voltage Sensor ◽  
Proton Channel ◽  
Voltage Sensing ◽  
Proton Channels ◽  
Voltage Gated ◽  
Transmembrane Segments ◽  
Cysteine Scanning ◽  
Dog Pancreas ◽  
Voltage Gated Ion Channels ◽  
Channel Properties

The voltage sensor domain (VSD) is the key module for voltage sensing in voltage-gated ion channels and voltage-sensing phosphatases. Structurally, both the VSD and the recently discovered voltage-gated proton channels (Hv channels) voltage sensor only protein (VSOP) and Hv1 contain four transmembrane segments. The fourth transmembrane segment (S4) of Hv channels contains three periodically aligned arginines (R1, R2, R3). It remains unknown where protons permeate or how voltage sensing is coupled to ion permeation in Hv channels. Here we report that Hv channels truncated just downstream of R2 in the S4 segment retain most channel properties. Two assays, site-directed cysteine-scanning using accessibility of maleimide-reagent as detected by Western blotting and insertion into dog pancreas microsomes, both showed that S4 inserts into the membrane, even if it is truncated between the R2 and R3 positions. These findings provide important clues to the molecular mechanism underlying voltage sensing and proton permeation in Hv channels.

scholarly journals Molecular Coupling of S4 to a K+ Channel's Slow Inactivation Gate

2000 ◽  
Vol 116 (5) ◽  
pp. 623-636 ◽  
Author(s):  
Eli Loots ◽  
Ehud Y. Isacoff
Keyword(s):  
Transmembrane Domain ◽  
Direct Interaction ◽  
K Channel ◽  
Slow Inactivation ◽  
Domain Interaction ◽  
Voltage Sensing ◽  
Molecular Gates ◽  
Voltage Sensing Domain ◽  
Pore Domain ◽  
Bond Disruption

The mechanism by which physiological signals regulate the conformation of molecular gates that open and close ion channels is poorly understood. Voltage clamp fluorometry was used to ask how the voltage-sensing S4 transmembrane domain is coupled to the slow inactivation gate in the pore domain of the Shaker K+ channel. Fluorophores attached at several sites in S4 indicate that the voltage-sensing rearrangements are followed by an additional inactivation motion. Fluorophores attached at the perimeter of the pore domain indicate that the inactivation rearrangement projects from the selectivity filter out to the interface with the voltage-sensing domain. Some of the pore domain sites also sense activation, and this appears to be due to a direct interaction with S4 based on the finding that S4 comes into close enough proximity to the pore domain for a pore mutation to alter the nanoenvironment of an S4-attached fluorophore. We propose that activation produces an S4–pore domain interaction that disrupts a bond between the S4 contact site on the pore domain and the outer end of S6. Our results indicate that this bond holds the slow inactivation gate open and, therefore, we propose that this S4-induced bond disruption triggers inactivation.

scholarly journals A novel Hv1 inhibitor reveals a new mechanism of inhibition of a voltage-sensing domain

2021 ◽  
Vol 153 (9) ◽  
Author(s):  
Chang Zhao ◽  
Liang Hong ◽  
Saleh Riahi ◽  
Victoria T. Lim ◽  
Douglas J. Tobias ◽  
...  
Keyword(s):  
Md Simulations ◽  
Binding Pocket ◽  
Proton Channel ◽  
Voltage Sensing ◽  
Voltage Gated ◽  
Functional Studies ◽  
Mechanism Of Inhibition ◽  
Target Binding ◽  
Voltage Gated Ion Channels ◽  
Pore Domain

Voltage-gated sodium, potassium, and calcium channels consist of four voltage-sensing domains (VSDs) that surround a central pore domain and transition from a down state to an up state in response to membrane depolarization. While many types of drugs bind pore domains, the number of organic molecules known to bind VSDs is limited. The Hv1 voltage-gated proton channel is made of two VSDs and does not contain a pore domain, providing a simplified model for studying how small ligands interact with VSDs. Here, we describe a ligand, named HIF, that interacts with the Hv1 VSD in the up and down states. We find that HIF rapidly inhibits proton conduction in the up state by blocking the open channel, as previously described for 2-guanidinobenzimidazole and its derivatives. HIF, however, interacts with a site slowly accessible in the down state. Functional studies and MD simulations suggest that this interaction traps the compound in a narrow pocket lined with charged residues within the VSD intracellular vestibule, which results in slow recovery from inhibition. Our findings point to a “wrench in gears” mechanism whereby side chains within the binding pocket trap the compound as the teeth of interlocking gears. We propose that the use of screening strategies designed to target binding sites with slow accessibility, similar to the one identified here, could lead to the discovery of new ligands capable of interacting with VSDs of other voltage-gated ion channels in the down state.

Isolation of putative voltage-gated epithelial K-channel isoforms from rabbit kidney and LLC-PK1 cells

1992 ◽  
Vol 262 (1) ◽  
pp. F151-F157 ◽  
Author(s):  
G. V. Desir ◽  
H. A. Hamlin ◽  
E. Puente ◽  
R. F. Reilly ◽  
F. Hildebrandt ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Rabbit Kidney ◽  
K Channel ◽  
Kidney Cortex ◽  
Epithelial Cell Line ◽  
K Channels ◽  
Voltage Gated ◽  
Transmembrane Segments ◽  
Partial Length

Epithelial voltage-gated potassium (K) channels have been well studied using electrophysiological methods, but little is known about their structures. We tested the hypothesis that some of these channels belong to the Shaker gene family, which encodes voltage-gated K channels in excitable tissues. From published sequences of Shaker proteins in Drosophila, rat, and mouse brain, we chose regions that were conserved between species. Based on these protein sequences, degenerate oligonucleotides flanking the putative voltage sensor (S4) were synthesized and used as primers for the polymerase chain reaction. Five Shaker-like cDNAs were amplified from rabbit kidney cortex and three from LLC-PK1, an epithelial cell line derived from pig kidney. Each partial-length rabbit kidney cDNA is approximately 850 base pairs (bp) long. The deduced amino acid sequences contain five putative transmembrane segments and are 79-97% identical to two Shaker isoforms expressed in rat brain (RBK1 and RBK2). Sequence similarity is greatest in the putative transmembrane segments S1-S5. Importantly, the S4 segment, the putative voltage gate is highly conserved in all 5 cDNAs. Southern analysis of rabbit genomic DNA suggests that each isoform is encoded by a different gene. The partial length LLC-PK1 cDNAs are 450-bp long, and the deduced amino acid sequences are 77-99% identical to the rabbit cDNAs. This is, to our knowledge, the first demonstration that Shaker-like genes are expressed in renal epithelial cells. These genes most likely encode voltage-gated K channels involved in renal epithelial K transport.

scholarly journals Changes in Local S4 Environment Provide a Voltage-sensing Mechanism for Mammalian Hyperpolarization–activated HCN Channels

2003 ◽  
Vol 123 (1) ◽  
pp. 5-20 ◽  
Author(s):  
Damian C. Bell ◽  
Huan Yao ◽  
Renee C. Saenger ◽  
John H. Riley ◽  
Steven A. Siegelbaum
Keyword(s):  
Hcn Channels ◽  
Transmembrane Segment ◽  
Voltage Sensing ◽  
Voltage Gated ◽  
Transmembrane Segments ◽  
Pacemaker Channels ◽  
Gated Channel ◽  
Voltage Gated Channels ◽  
S4 Segment ◽  
Positively Charged

The positively charged S4 transmembrane segment of voltage-gated channels is thought to function as the voltage sensor by moving charge through the membrane electric field in response to depolarization. Here we studied S4 movements in the mammalian HCN pacemaker channels. Unlike most voltage-gated channel family members that are activated by depolarization, HCN channels are activated by hyperpolarization. We determined the reactivity of the charged sulfhydryl-modifying reagent, MTSET, with substituted cysteine (Cys) residues along the HCN1 S4 segment. Using an HCN1 channel engineered to be MTS resistant except for the chosen S4 Cys substitution, we determined the reactivity of 12 S4 residues to external or internal MTSET application in either the closed or open state of the channel. Cys substitutions in the NH2-terminal half of S4 only reacted with external MTSET; the rates of reactivity were rapid, regardless of whether the channel was open or closed. In contrast, Cys substitutions in the COOH-terminal half of S4 selectively reacted with internal MTSET when the channel was open. In the open state, the boundary between externally and internally accessible residues was remarkably narrow (∼3 residues). This suggests that S4 lies in a water-filled gating canal with a very narrow barrier between the external and internal solutions, similar to depolarization-gated channels. However, the pattern of reactivity is incompatible with either classical gating models, which postulate a large translational or rotational movement of S4 within a gating canal, or with a recent model in which S4 forms a peripheral voltage-sensing paddle (with S3b) that moves within the lipid bilayer (the KvAP model). Rather, we suggest that voltage sensing is due to a rearrangement in transmembrane segments surrounding S4, leading to a collapse of an internal gating canal upon channel closure that alters the shape of the membrane field around a relatively static S4 segment.

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