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Voltage sensor activation and modulation in ion channels
KTH, School of Engineering Sciences (SCI), Theoretical Physics, Theoretical & Computational Biophysics. (Erik Lindahl)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Voltage-gated ion channels play fundamental roles in neural excitability, they are for instance responsible for every single heart beat in our bodies, and dysfunctional channels cause disease that can be even lethal. Understanding how the voltage sensor of these channels function is critical for drug design of compounds targeting neuronal excitability.

The opening and closing of the pore in voltage-gated potassium (Kv) channels is caused by the arginine-rich S4 helix of the voltage sensor domain (VSD) moving in response to an external potential. In fact, VSDs are remarkably efficient at turning membrane potential into conformational changes, which likely makes them the smallest existing biological engines. Exactly how this is accomplished is not yet fully known and an area of hot debate, especially due to the lack of structures of the resting and intermediate states along the activation pathway. In this thesis I study how the VSD activation works and show how toxic compounds modulate channel gating through direct interaction with these quite unexplored drug targets.

First, I show that a secondary structure transition from alpha- to 3(10)-helix in the S4 helix is an important part of the gating as this helix type is significantly more favorable compared to the -helix in terms of a lower free energy barrier. Second, I present new models for intermediate states along the whole voltage sensor cycle from closed to open and suggest a new gating model for S4, where it moves as a sliding 3(10)-helix. Interestingly, this 3(10)-helix is formed in the region of the single most conserved residue in Kv channels, the phenylalanine F233. Located in the hydrophobic core, it directly faces S4 and creates a structural barrier for the gating charges. Substituting this residue alters the deactivation free energy barrier and can either facilitate the relaxation of the voltage sensor or increase the free energy barrier, depending on the size of the mutant. These results are confirmed by new experimental data that supports that a rigid ring at the phenylalanine position is the rate-limiting factor for the deactivation gating process, while the activation is unaffected. Finally, we study how the activation can be modulated for pharmaceutical reasons. Neurotoxins such as hanatoxin and stromatoxin push S3b towards S4 helix limiting S4's flexibility. This makes it harder for the VSD to activate and might explain the stronger binding affinities in resting state.

All these results are highly important both for the general topic of biological macromolecules undergoing functionally critical conformational transitions, as well as the particular case of voltage-gated ion channels where understanding of the gating process is probably the key step to explain the effects of mutations or drug interactions.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2012. , xiii, 77 p.
Series
Trita-FYS, ISSN 0280-316X ; 2012:77
Keyword [en]
activation, deactivation, inactivation, voltage sensor, VSD, gating, Kv1.2-2.1, Shaker, F233, hydrophobic barrier
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-104742ISBN: 978-91-7501-498-2 (print)OAI: oai:DiVA.org:kth-104742DiVA: diva2:566870
Public defence
2012-12-07, FB53, AlbaNova universitetscentrum, Stockholm, 10:00 (English)
Opponent
Supervisors
Funder
Swedish e‐Science Research CenterScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20121115

Available from: 2012-11-15 Created: 2012-11-09 Last updated: 2013-04-15Bibliographically approved
List of papers
1. 310-Helix Conformation Facilitates the Transition of a Voltage Sensor S4 Segment toward the Down State
Open this publication in new window or tab >>310-Helix Conformation Facilitates the Transition of a Voltage Sensor S4 Segment toward the Down State
2011 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 100, no 6, 1446-1454 p.Article in journal (Refereed) Published
Abstract [en]

The activation of voltage-gated ion channels is controlled by the S4 helix, with arginines every third residue. The x-ray structures are believed to reflect an open-inactivated state, and models propose combinations of translation, rotation, and tilt to reach the resting state. Recently, experiments and simulations have independently observed occurrence of 3(10)-helix in S4. This suggests S4 might make a transition from alpha- to 3(10)-helix in the gating process. Here, we show 3(10)-helix structure between 01 and R3 in the S4 segment of a voltage sensor appears to facilitate the early stage of the motion toward a down state. We use multiple microsecond-steered molecular simulations to calculate the work required for translating S4 both as a-helix and transformed to 3(10)-helix. The barrier appears to be caused by salt-bridge reformation simultaneous to R4 passing the F233 hydrophobic lock, and it is almost a factor-two lower with 3(10)-helix. The latter facilitates translation because R2/R3 line up to face E183/E226, which reduces the requirement to rotate S4. This is also reflected in a lower root mean-square deviation distortion of the rest of the voltage sensor. This supports the 3(10) hypothesis, and could explain some of the differences between the open-inactivated- versus activated-states.

National Category
Biophysics Bioinformatics and Systems Biology Theoretical Chemistry
Research subject
SRA - E-Science (SeRC)
Identifiers
urn:nbn:se:kth:diva-33480 (URN)10.1016/j.bpj.2011.02.003 (DOI)000288889700008 ()2-s2.0-79953898210 (Scopus ID)
Funder
EU, European Research Council, 209825Swedish Research CouncilSwedish e‐Science Research Center
Note

QC 20150716

Available from: 2011-05-16 Created: 2011-05-09 Last updated: 2017-12-11Bibliographically approved
2. Tracking a complete voltage-sensor cycle with metal-ion bridges
Open this publication in new window or tab >>Tracking a complete voltage-sensor cycle with metal-ion bridges
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2012 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 22, 8552-8557 p.Article in journal (Refereed) Published
Abstract [en]

Voltage-gated ion channels open and close in response to changes in membrane potential, thereby enabling electrical signaling in excitable cells. The voltage sensitivity is conferred through four voltage-sensor domains (VSDs) where positively charged residues in the fourth transmembrane segment (S4) sense the potential. While an open state is known from the Kv1.2/2.1 X-ray structure, the conformational changes underlying voltage sensing have not been resolved. We present 20 additional interactions in one open and four different closed conformations based on metal-ion bridges between all four segments of the VSD in the voltage-gated Shaker K channel. A subset of the experimental constraints was used to generate Rosetta models of the conformations that were subjected to molecular simulation and tested against the remaining constraints. This achieves a detailed model of intermediate conformations during VSD gating. The results provide molecular insight into the transition, suggesting that S4 slides at least 12 angstrom along its axis to open the channel with a 3(10) helix region present that moves in sequence in S4 in order to occupy the same position in space opposite F290 from open through the three first closed states.

Keyword
electrophysiology, inactivation, Xenopus oocytes, voltage clamp, conformational transition
National Category
Other Physics Topics
Identifiers
urn:nbn:se:kth:diva-98329 (URN)10.1073/pnas.1116938109 (DOI)000304881700044 ()2-s2.0-84861838905 (Scopus ID)
Funder
Swedish Research CouncilEU, European Research CouncilSwedish e‐Science Research CenterScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20120625

Available from: 2012-06-25 Created: 2012-06-25 Last updated: 2017-12-07Bibliographically approved
3. The free energy barrier for arginine gating charge translation is altered by mutations in the voltage sensor domain.
Open this publication in new window or tab >>The free energy barrier for arginine gating charge translation is altered by mutations in the voltage sensor domain.
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 10, e45880- p.Article in journal (Refereed) Published
Abstract [en]

The gating of voltage-gated ion channels is controlled by the arginine-rich S4 helix of the voltage-sensor domain moving in response to an external potential. Recent studies have suggested that S4 moves in three to four steps to open the conducting pore, thus visiting several intermediate conformations during gating. However, the exact conformational changes are not known in detail. For instance, it has been suggested that there is a local rotation in the helix corresponding to short segments of a 3-helix moving along S4 during opening and closing. Here, we have explored the energetics of the transition between the fully open state (based on the X-ray structure) and the first intermediate state towards channel closing (C), modeled from experimental constraints. We show that conformations within 3 Å of the X-ray structure are obtained in simulations starting from the C model, and directly observe the previously suggested sliding 3-helix region in S4. Through systematic free energy calculations, we show that the C state is a stable intermediate conformation and determine free energy profiles for moving between the states without constraints. Mutations indicate several residues in a narrow hydrophobic band in the voltage sensor contribute to the barrier between the open and C states, with F233 in the S2 helix having the largest influence. Substitution for smaller amino acids reduces the transition cost, while introduction of a larger ring increases it, largely confirming experimental activation shift results. There is a systematic correlation between the local aromatic ring rotation, the arginine barrier crossing, and the corresponding relative free energy. In particular, it appears to be more advantageous for the F233 side chain to rotate towards the extracellular side when arginines cross the hydrophobic region.

Keyword
Shaker K+ Channel, Nuclear Magnetic-Resonance, Potassium Channel, Solid-State, Sodium-Channels, Resting State, Side-Chains, S4 Segment, Dynamics, Protein
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-104738 (URN)10.1371/journal.pone.0045880 (DOI)000310050200005 ()2-s2.0-84867669798 (Scopus ID)
Funder
EU, European Research Council, 209825Swedish Research Council, 2010-5107Swedish e‐Science Research CenterScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20121112

Available from: 2012-11-09 Created: 2012-11-09 Last updated: 2017-12-07Bibliographically approved
4. The conserved phenylalanine in the K+ channel voltage-sensor domain creates a barrier with unidirectional effects
Open this publication in new window or tab >>The conserved phenylalanine in the K+ channel voltage-sensor domain creates a barrier with unidirectional effects
2013 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 1, 75-84 p.Article in journal (Refereed) Published
Abstract [en]

Voltage-gated ion channels are crucial for regulation of electric activity of excitable tissues such as nerve cells, and play important roles in many diseases. During activation, the charged S4 segment in the voltage sensor domain translates across a hydrophobic core forming a barrier for the gating charges. This barrier is critical for channel function, and a conserved phenylalanine in segment S2 has previously been identified to be highly sensitive to substitutions. Here, we have studied the kinetics of Kv1-type potassium channels (Shaker and Kv1.2/2.1 chimera) through site-directed mutagenesis, electrophysiology, and molecular simulations. The F290L mutation in Shaker (F233L in Kv1.2/2.1) accelerates channel closure by at least a factor 50, although opening is unaffected. Free energy profiles with the hydrophobic neighbors of F233 mutated to alanine indicate that the open state with the fourth arginine in S4 above the hydrophobic core is destabilized by ∼17 kJ/mol compared to the first closed intermediate. This significantly lowers the barrier of the first deactivation step, although the last step of activation is unaffected. Simulations of wild-type F233 show that the phenyl ring always rotates toward the extracellular side both for activation and deactivation, which appears to help stabilize a well-defined open state.

Keyword
Potassium Channel, Gating Charge, Transition, Kv1.2, State, Conformation, Hanatoxin, Segment, Field, S4
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-104739 (URN)10.1016/j.bpj.2012.11.3827 (DOI)000313541200010 ()2-s2.0-84872151920 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceEU, European Research Council, 209825Swedish Foundation for Strategic Research Swedish Research CouncilSwedish e‐Science Research Center
Note

QC 20130212. Updated from manuscript to article in journal.

Available from: 2012-11-09 Created: 2012-11-09 Last updated: 2017-12-07Bibliographically approved
5. Modulation of the voltage sensor resting state through Hanatoxin and Stromatoxin
Open this publication in new window or tab >>Modulation of the voltage sensor resting state through Hanatoxin and Stromatoxin
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-104740 (URN)
Note

QS 2012

Available from: 2012-11-09 Created: 2012-11-09 Last updated: 2016-08-16Bibliographically approved

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