scholarly journals Bipolar Switching by HCN Voltage Sensor Underlies Hyperpolarization Activation

2018 ◽  
Author(s):  
John Cowgill ◽  
Vadim A. Klenchin ◽  
Claudia Alvarez-Baron ◽  
Debanjan Tewari ◽  
Alexander Blair ◽  
...  
Keyword(s):  
Voltage Sensor ◽  
Hcn Channels ◽  
Hcn Channel ◽  
Sensor Interface ◽  
Bipolar Switching ◽  
Full Complement ◽  
Common Principle ◽  
Pore Opening ◽  
Voltage Gated Ion Channels ◽  
Common Architecture

SUMMARYDespite sharing a common architecture with archetypal voltage-gated ion channels (VGIC), the hyperpolarization- and cyclic AMP-activated ion (HCN) channels open upon hyperpolarization rather than depolarization. The basic motions of voltage sensor and pore gates are conserved implying that these domains are inversely coupled in HCN channels. Using structure-guided protein engineering, we systematically assembled an array of mosaic channels that display the full complement of voltage-activation phenotypes observed in the VGIC superfamily. Our studies reveal that the voltage-sensing S3b-S4 transmembrane segment of the HCN channel has an intrinsic ability to drive pore opening in either direction. Specific contacts at the pore-voltage sensor interface and unique interactions near the pore gate forces the HCN channel into a hERG-like inactivated state, thereby obscuring their opening upon depolarization. Our findings reveal an unexpected common principle underpinning voltage gating in the VGIC superfamily and identify the essential determinants of gating polarity.

scholarly journals Bipolar switching by HCN voltage sensor underlies hyperpolarization activation

2018 ◽  
Vol 116 (2) ◽  
pp. 670-678 ◽  
Author(s):  
John Cowgill ◽  
Vadim A. Klenchin ◽  
Claudia Alvarez-Baron ◽  
Debanjan Tewari ◽  
Alexander Blair ◽  
...  
Keyword(s):  
Voltage Sensor ◽  
Hcn Channels ◽  
Structural Element ◽  
Bipolar Switching ◽  
Primary Determinant ◽  
Full Complement ◽  
Common Principle ◽  
Pore Opening ◽  
Voltage Gated Ion Channels ◽  
Common Architecture

Despite sharing a common architecture with archetypal voltage-gated ion channels (VGICs), hyperpolarization- and cAMP-activated ion (HCN) channels open upon hyperpolarization rather than depolarization. The basic motions of the voltage sensor and pore gates are conserved, implying that these domains are inversely coupled in HCN channels. Using structure-guided protein engineering, we systematically assembled an array of mosaic channels that display the full complement of voltage-activation phenotypes observed in the VGIC superfamily. Our studies reveal that the voltage sensor of the HCN channel has an intrinsic ability to drive pore opening in either direction and that the extra length of the HCN S4 is not the primary determinant for hyperpolarization activation. Tight interactions at the HCN voltage sensor–pore interface drive the channel into an hERG-like inactivated state, thereby obscuring its opening upon depolarization. This structural element in synergy with the HCN cyclic nucleotide-binding domain and specific interactions near the pore gate biases the channel toward hyperpolarization-dependent opening. Our findings reveal an unexpected common principle underpinning voltage gating in the VGIC superfamily and identify the essential determinants of gating polarity.

scholarly journals Helix breaking transition in the S4 of HCN channel is critical for hyperpolarization-dependent gating

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Marina A Kasimova ◽  
Debanjan Tewari ◽  
John B Cowgill ◽  
Willy Carrasquel Ursuleaz ◽  
Jenna L Lin ◽  
...  
Keyword(s):  
Ion Channels ◽  
Voltage Sensor ◽  
Inward Rectification ◽  
Hcn Channels ◽  
Hcn Channel ◽  
Functional Studies ◽  
Gating Charge ◽  
Helical Turn ◽  
Voltage Gated Ion Channels ◽  
Dynamics Simulations

In contrast to most voltage-gated ion channels, hyperpolarization- and cAMP gated (HCN) ion channels open on hyperpolarization. Structure-function studies show that the voltage-sensor of HCN channels are unique but the mechanisms that determine gating polarity remain poorly understood. All-atom molecular dynamics simulations (~20 μs) of HCN1 channel under hyperpolarization reveals an initial downward movement of the S4 voltage-sensor but following the transfer of last gating charge, the S4 breaks into two sub-helices with the lower sub-helix becoming parallel to the membrane. Functional studies on bipolar channels show that the gating polarity strongly correlates with helical turn propensity of the substituents at the breakpoint. Remarkably, in a proto-HCN background, the replacement of breakpoint serine with a bulky hydrophobic amino acid is sufficient to completely flip the gating polarity from inward to outward-rectifying. Our studies reveal an unexpected mechanism of inward rectification involving a linker sub-helix emerging from HCN S4 during hyperpolarization.

scholarly journals Mode shift of the voltage sensors in Shaker K+ channels is caused by energetic coupling to the pore domain

2011 ◽  
Vol 137 (5) ◽  
pp. 455-472 ◽  
Author(s):  
Georges A. Haddad ◽  
Rikard Blunck
Keyword(s):  
Conformational Change ◽  
Mechanical Load ◽  
Voltage Sensor ◽  
Charge Movement ◽  
Gating Currents ◽  
Mode Shift ◽  
Voltage Sensors ◽  
Pore Opening ◽  
Voltage Gated Ion Channels ◽  
Pore Domain

The voltage sensors of voltage-gated ion channels undergo a conformational change upon depolarization of the membrane that leads to pore opening. This conformational change can be measured as gating currents and is thought to be transferred to the pore domain via an annealing of the covalent link between voltage sensor and pore (S4-S5 linker) and the C terminus of the pore domain (S6). Upon prolonged depolarizations, the voltage dependence of the charge movement shifts to more hyperpolarized potentials. This mode shift had been linked to C-type inactivation but has recently been suggested to be caused by a relaxation of the voltage sensor itself. In this study, we identified two ShakerIR mutations in the S4-S5 linker (I384N) and S6 (F484G) that, when mutated, completely uncouple voltage sensor movement from pore opening. Using these mutants, we show that the pore transfers energy onto the voltage sensor and that uncoupling the pore from the voltage sensor leads the voltage sensors to be activated at more negative potentials. This uncoupling also eliminates the mode shift occurring during prolonged depolarizations, indicating that the pore influences entry into the mode shift. Using voltage-clamp fluorometry, we identified that the slow conformational change of the S4 previously correlated with the mode shift disappears when uncoupling the pore. The effects can be explained by a mechanical load that is imposed upon the voltage sensors by the pore domain and allosterically modulates its conformation. Mode shift is caused by the stabilization of the open state but leads to a conformational change in the voltage sensor.

scholarly journals Coupling of Ca2+and voltage activation in BK channels through the αB helix/voltage sensor interface

2020 ◽  
Vol 117 (25) ◽  
pp. 14512-14521
Author(s):  
Yanyan Geng ◽  
Zengqin Deng ◽  
Guohui Zhang ◽  
Gonzalo Budelli ◽  
Alice Butler ◽  
...  
Keyword(s):  
Structural Changes ◽  
Cell Types ◽  
Bk Channels ◽  
Voltage Sensor ◽  
Membrane Excitability ◽  
Gating Currents ◽  
Sensor Interface ◽  
Pore Opening ◽  
Helical Turn ◽  
Voltage Activation

Large-conductance Ca2+and voltage-activated K+(BK) channels control membrane excitability in many cell types. BK channels are tetrameric. Each subunit is composed of a voltage sensor domain (VSD), a central pore-gate domain, and a large cytoplasmic domain (CTD) that contains the Ca2+sensors. While it is known that BK channels are activated by voltage and Ca2+, and that voltage and Ca2+activations interact, less is known about the mechanisms involved. We explore here these mechanisms by examining the gating contribution of an interface formed between the VSDs and the αB helices located at the top of the CTDs. Proline mutations in the αB helix greatly decreased voltage activation while having negligible effects on gating currents. Analysis with the Horrigan, Cui, and Aldrich model indicated a decreased coupling between voltage sensors and pore gate. Proline mutations decreased Ca2+activation for both Ca2+bowl and RCK1 Ca2+sites, suggesting that both high-affinity Ca2+sites transduce their effect, at least in part, through the αB helix. Mg2+activation also decreased. The crystal structure of the CTD with proline mutation L390P showed a flattening of the first helical turn in the αB helix compared to wild type, without other notable differences in the CTD, indicating that structural changes from the mutation were confined to the αB helix. These findings indicate that an intact αB helix/VSD interface is required for effective coupling of Ca2+binding and voltage depolarization to pore opening and that shared Ca2+and voltage transduction pathways involving the αB helix may be involved.

scholarly journals S4 Movement in a Mammalian HCN Channel

2003 ◽  
Vol 123 (1) ◽  
pp. 21-32 ◽  
Author(s):  
Sriharsha Vemana ◽  
Shilpi Pandey ◽  
H. Peter Larsson
Keyword(s):  
Voltage Sensor ◽  
K Channel ◽  
Hcn Channels ◽  
Dependent Manner ◽  
Hcn Channel ◽  
Voltage Range ◽  
Kv Channels ◽  
State Dependent ◽  
Voltage Dependent ◽  
Hcn1 Channels

Hyperpolarization-activated, cyclic nucleotide–gated ion channels (HCN) mediate an inward cation current that contributes to spontaneous rhythmic firing activity in the heart and the brain. HCN channels share sequence homology with depolarization-activated Kv channels, including six transmembrane domains and a positively charged S4 segment. S4 has been shown to function as the voltage sensor and to undergo a voltage-dependent movement in the Shaker K+ channel (a Kv channel) and in the spHCN channel (an HCN channel from sea urchin). However, it is still unknown whether S4 undergoes a similar movement in mammalian HCN channels. In this study, we used cysteine accessibility to determine whether there is voltage-dependent S4 movement in a mammalian HCN1 channel. Six cysteine mutations (R247C, T249C, I251C, S253C, L254C, and S261C) were used to assess S4 movement of the heterologously expressed HCN1 channel in Xenopus oocytes. We found a state-dependent accessibility for four S4 residues: T249C and S253C from the extracellular solution, and L254C and S261C from the internal solution. We conclude that S4 moves in a voltage-dependent manner in HCN1 channels, similar to its movement in the spHCN channel. This S4 movement suggests that the role of S4 as a voltage sensor is conserved in HCN channels. In addition, to determine the reason for the different cAMP modulation and the different voltage range of activation in spHCN channels compared with HCN1 channels, we constructed a COOH-terminal–deleted spHCN. This channel appeared to be similar to a COOH-terminal–deleted HCN1 channel, suggesting that the main functional differences between spHCN and HCN1 channels are due to differences in their COOH termini or in the interaction between the COOH terminus and the rest of the channel protein in spHCN channels compared with HCN1 channels.

scholarly journals The HCN Channel Voltage Sensor Undergoes A Large Downward Motion During Hyperpolarization

2019 ◽  
Author(s):  
Gucan Dai ◽  
Teresa K. Aman ◽  
Frank DiMaio ◽  
William N. Zagotta
Keyword(s):  
Ion Channels ◽  
Metal Ion ◽  
Voltage Sensor ◽  
The Body ◽  
Resting Membrane Potential ◽  
Hcn Channels ◽  
Hcn Channel ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Transmembrane Voltage

Voltage-gated ion channels (VGICs) underlie almost all electrical signaling in the body1. They change their open probability in response to changes in transmembrane voltage, allowing permeant ions to flow across the cell membrane. Ion flow through VGICs underlies numerous physiological processes in excitable cells1. In particular, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which operate at the threshold of excitability, are essential for pacemaking activity, resting membrane potential, and synaptic integration2. VGICs contain a series of positively-charged residues that are displaced in response to changes in transmembrane voltage, resulting in a conformational change that opens the pore3–6. These voltage-sensing charges, which reside in the S4 transmembrane helix of the voltage-sensor domain (VSD)3 and within the membrane’s electric field, are thought to move towards the inside of the cell (downwards) during membrane hyperpolarization7. HCN channels are unique among VGICs because their open probability is increased by membrane hyperpolarization rather than depolarization8–10. The mechanism underlying this “reverse gating” is still unclear. Moreover, although many X-ray crystal and cryo-EM structures have been solved for the depolarized state of the VSD, including that of HCN channels11, no structures have been solved at hyperpolarized voltages. Here we measure the precise movement of the charged S4 helix of an HCN channel using transition metal ion fluorescence resonance energy transfer (tmFRET). We show that the S4 undergoes a significant (~10 Å) downward movement in response to membrane hyperpolarization. Furthermore, by applying constraints determined from tmFRET experiments to Rosetta modeling, we reveal that the carboxyl-terminal part of the S4 helix exhibits an unexpected tilting motion during hyperpolarization activation. These data provide a long-sought glimpse of the hyperpolarized state of a functioning VSD and also a framework for understanding the dynamics of reverse gating in HCN channels. Our methods can be broadly applied to probe short-distance rearrangements in other ion channels and membrane proteins.

scholarly journals The Role of HCN Channels on Membrane Excitability in the Nervous System

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Daisuke Kase ◽  
Keiji Imoto
Keyword(s):  
Intracellular Signaling ◽  
Hcn Channels ◽  
Heart Cells ◽  
Hcn Channel ◽  
Membrane Excitability ◽  
Wide Range ◽  
Inward Currents ◽  
Range Of Functions ◽  
Channel Currents

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels were first reported in heart cells and are recently known to be involved in a variety of neural functions in healthy and diseased brains. HCN channels generate inward currents when the membrane potential is hyperpolarized. Voltage dependence of HCN channels is regulated by intracellular signaling cascades, which contain cyclic AMP, PIP2, and TRIP8b. In addition, voltage-gated potassium channels have a strong influence on HCN channel activity. Because of these funny features, HCN channel currents, previously called funny currents, can have a wide range of functions that are determined by a delicate balance of modulatory factors. These multifaceted features also make it difficult to predict and elucidate the functional role of HCN channels in actual neurons. In this paper, we focus on the impacts of HCN channels on neural activity. The functions of HCN channels reported previously will be summarized, and their mechanisms will be explained by using numerical simulation of simplified model neurons.

scholarly journals Components of gating charge movement and S4 voltage-sensor exposure during activation of hERG channels

2013 ◽  
Vol 141 (4) ◽  
pp. 431-443 ◽  
Author(s):  
Zhuren Wang ◽  
Ying Dou ◽  
Samuel J. Goodchild ◽  
Zeineb Es-Salah-Lamoureux ◽  
David Fedida
Keyword(s):  
Mammalian Cells ◽  
Voltage Sensor ◽  
Delayed Rectifier ◽  
Charge Movement ◽  
Α Subunit ◽  
Time Constants ◽  
Gating Charge ◽  
Activation Gating ◽  
Pore Opening ◽  
Herg Channels

The human ether-á-go-go–related gene (hERG) K+ channel encodes the pore-forming α subunit of the rapid delayed rectifier current, IKr, and has unique activation gating kinetics, in that the α subunit of the channel activates and deactivates very slowly, which focuses the role of IKr current to a critical period during action potential repolarization in the heart. Despite its physiological importance, fundamental mechanistic properties of hERG channel activation gating remain unclear, including how voltage-sensor movement rate limits pore opening. Here, we study this directly by recording voltage-sensor domain currents in mammalian cells for the first time and measuring the rates of voltage-sensor modification by [2-(trimethylammonium)ethyl] methanethiosulfonate chloride (MTSET). Gating currents recorded from hERG channels expressed in mammalian tsA201 cells using low resistance pipettes show two charge systems, defined as Q1 and Q2, with V1/2’s of −55.7 (equivalent charge, z = 1.60) and −54.2 mV (z = 1.30), respectively, with the Q2 charge system carrying approximately two thirds of the overall gating charge. The time constants for charge movement at 0 mV were 2.5 and 36.2 ms for Q1 and Q2, decreasing to 4.3 ms for Q2 at +60 mV, an order of magnitude faster than the time constants of ionic current appearance at these potentials. The voltage and time dependence of Q2 movement closely correlated with the rate of MTSET modification of I521C in the outermost region of the S4 segment, which had a V1/2 of −64 mV and time constants of 36 ± 8.5 ms and 11.6 ± 6.3 ms at 0 and +60 mV, respectively. Modeling of Q1 and Q2 charge systems showed that a minimal scheme of three transitions is sufficient to account for the experimental findings. These data point to activation steps further downstream of voltage-sensor movement that provide the major delays to pore opening in hERG channels.

scholarly journals Structural changes during HCN channel gating defined by high affinity metal bridges

2012 ◽  
Vol 140 (3) ◽  
pp. 279-291 ◽  
Author(s):  
Daniel C.H. Kwan ◽  
David L. Prole ◽  
Gary Yellen
Keyword(s):  
Structural Changes ◽  
Structural Model ◽  
Channel Gating ◽  
Structural Basis ◽  
Hcn Channels ◽  
Hcn Channel ◽  
Membrane Hyperpolarization ◽  
Linker Region ◽  
High Affinity ◽  
Gating Mechanism

Hyperpolarization-activated cyclic nucleotide–sensitive nonselective cation (HCN) channels are activated by membrane hyperpolarization, in contrast to the vast majority of other voltage-gated channels that are activated by depolarization. The structural basis for this unique characteristic of HCN channels is unknown. Interactions between the S4–S5 linker and post-S6/C-linker region have been implicated previously in the gating mechanism of HCN channels. We therefore introduced pairs of cysteines into these regions within the sea urchin HCN channel and performed a Cd2+-bridging scan to resolve their spatial relationship. We show that high affinity metal bridges between the S4–S5 linker and post-S6/C-linker region can induce either a lock-open or lock-closed phenotype, depending on the position of the bridged cysteine pair. This suggests that interactions between these regions can occur in both the open and closed states, and that these regions move relative to each other during gating. Concatenated constructs reveal that interactions of the S4–S5 linker and post-S6/C-linker can occur between neighboring subunits. A structural model based on these interactions suggests a mechanism for HCN channel gating. We propose that during voltage-dependent activation the voltage sensors, together with the S4–S5 linkers, drive movement of the lower ends of the S5 helices around the central axis of the channel. This facilitates a movement of the pore-lining S6 helices, which results in opening of the channel. This mechanism may underlie the unique voltage dependence of HCN channel gating.

scholarly journals hERG Channel Voltage Sensor Movement Precedes Pore Opening

2014 ◽  
Vol 106 (2) ◽  
pp. 139a
Author(s):  
Samuel J. Goodchild ◽  
David Fedida
Keyword(s):  
Voltage Sensor ◽  
Herg Channel ◽  
Pore Opening
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