scholarly journals Electromechanical coupling mechanism for activation and inactivation of an HCN channel

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gucan Dai ◽  
Teresa K. Aman ◽  
Frank DiMaio ◽  
William N. Zagotta
Keyword(s):  
Cyclic Nucleotide ◽  
Metal Ion ◽  
Electromechanical Coupling ◽  
Transmembrane Domain ◽  
Coupling Mechanism ◽  
Hcn Channel ◽  
Membrane Hyperpolarization ◽  
Channel Inactivation ◽  
Close Proximity ◽  
Voltage Dependent

AbstractPacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels exhibit a reversed voltage-dependent gating, activating by membrane hyperpolarization instead of depolarization. Sea urchin HCN (spHCN) channels also undergo inactivation with hyperpolarization which occurs only in the absence of cyclic nucleotide. Here we applied transition metal ion FRET, patch-clamp fluorometry and Rosetta modeling to measure differences in the structural rearrangements between activation and inactivation of spHCN channels. We found that removing cAMP produced a largely rigid-body rotation of the C-linker relative to the transmembrane domain, bringing the A’ helix of the C-linker in close proximity to the voltage-sensing S4 helix. In addition, rotation of the C-linker was elicited by hyperpolarization in the absence but not the presence of cAMP. These results suggest that — in contrast to electromechanical coupling for channel activation — the A’ helix serves to couple the S4-helix movement for channel inactivation, which is likely a conserved mechanism for CNBD-family channels.

scholarly journals NONCANONICAL ELECTROMECHANICAL COUPLING MECHANISM OF AN HCN CHANNEL

2021 ◽  
Author(s):  
Gucan Dai ◽  
Teresa K. Aman ◽  
Frank DiMaio ◽  
William N. Zagotta
Keyword(s):  
Cyclic Nucleotide ◽  
Metal Ion ◽  
Electromechanical Coupling ◽  
Transmembrane Domain ◽  
Coupling Mechanism ◽  
Hcn Channel ◽  
Membrane Hyperpolarization ◽  
Recovery From Inactivation ◽  
Close Proximity ◽  
Voltage Dependent

Pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels exhibit a reversed voltage-dependent gating, activating by membrane hyperpolarization instead of depolarization. Sea urchin HCN (spHCN) channels also undergo an inactivation with hyperpolarization which occurs only in the absence of cyclic nucleotide. Here we applied transition metal ion FRET and Rosetta modeling to measure differences in the structural rearrangements between activation and inactivation of spHCN channels. We found that removal of cAMP produced a largely rigid-body rotation of the C-linker relative to transmembrane domain, bringing the A′ helix in close proximity to the voltage-sensing S4 helix. In addition, rotation of the C-linker was elicited by hyperpolarization in the absence but not in the presence of cAMP. These results suggest the A′ helix functions like a mechanical clutch to engage inactivation. cAMP binding disengages the clutch, permitting the hyperpolarization-dependent activation mediated by the S5 helix. Furthermore, rearrangement of A′ helix during recovery from inactivation of spHCN channels was similar to that for the activation of KCNH channels, suggesting a conserved mechanism for this noncanonical electromechanical coupling in the CNBD channel family.

scholarly journals HCN domain is required for HCN channel expression and couples voltage- and cAMP-dependent gating mechanisms

2020 ◽  
Author(s):  
Ze-Jun Wang ◽  
Ismary Blanco ◽  
Sebastien Hayoz ◽  
Tinatin I. Brelidze
Keyword(s):  
Cyclic Nucleotide ◽  
Transmembrane Domain ◽  
Rhythmic Activity ◽  
Surface Expression ◽  
Hcn Channels ◽  
Hcn Channel ◽  
Structural Element ◽  
Functional Link ◽  
Membrane Hyperpolarization ◽  
Membrane Spanning

ABSTRACTHyperpolarization-activated cyclic nucleotide-gated (HCN) channels are major regulators of synaptic plasticity, and rhythmic activity in the heart and brain. Opening of HCN channels requires membrane hyperpolarization and is further facilitated by intracellular cyclic nucleotides (cNMPs). In HCN channels, membrane hyperpolarization is sensed by the membrane-spanning voltage sensor domain (VSD) and the cNMP-dependent gating is mediated by the intracellular cyclic nucleotide-binding domain (CNBD) connected to the pore-forming S6 transmembrane domain via the C-linker. Previous functional analysis of HCN channels suggested a direct or allosteric coupling between the voltage- and cNMP-dependent activation mechanisms. However, the specifics of the coupling were unclear. The first cryo-EM structure of an HCN1 channel revealed that a novel structural element, dubbed HCN domain (HCND), forms a direct structural link between the VSD and C-linker/CNBD. In this study, we investigated the functional significance of the HCND. Deletion of the HCND prevented surface expression of HCN2 channels. Based on the HCN1 structure analysis, we identified R237 and G239 residues on the S2 of the VSD that form direct interactions with I135 on the HCND. Disrupting these interactions abolished HCN2 currents. We then identified three residues on the C-linker/CNBD (E478, Q382 and H559) that form direct interactions with residues R154 and S158 on the HCND. Disrupting these interactions affected both voltage- and cAMP-dependent gating of HCN2 channels. These findings indicate that the HCND is necessary for the surface expression of HCN channels, and provides a functional link between the voltage- and cAMP-dependent mechanisms of HCN channel gating.

scholarly journals Insights into the molecular mechanism for hyperpolarization-dependent activation of HCN channels

2018 ◽  
Vol 115 (34) ◽  
pp. E8086-E8095 ◽  
Author(s):  
Galen E. Flynn ◽  
William N. Zagotta
Keyword(s):  
Ion Channels ◽  
Cyclic Nucleotide ◽  
Electromechanical Coupling ◽  
Canonical Model ◽  
Hcn Channels ◽  
Voltage Gated ◽  
Kv Channels ◽  
Recovery From Inactivation ◽  
Voltage Dependent ◽  
Pore Domain

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels are both voltage- and ligand-activated membrane proteins that contribute to electrical excitability and pace-making activity in cardiac and neuronal cells. These channels are members of the voltage-gated Kv channel superfamily and cyclic nucleotide-binding domain subfamily of ion channels. HCN channels have a unique feature that distinguishes them from other voltage-gated channels: the HCN channel pore opens in response to hyperpolarizing voltages instead of depolarizing voltages. In the canonical model of electromechanical coupling, based on Kv channels, a change in membrane voltage activates the voltage-sensing domains (VSD) and the activation energy passes to the pore domain (PD) through a covalent linker that connects the VSD to the PD. In this investigation, the covalent linkage between the VSD and PD, the S4-S5 linker, and nearby regions of spHCN channels were mutated to determine the functional role each plays in hyperpolarization-dependent activation. The results show that: (i) the S4-S5 linker is not required for hyperpolarization-dependent activation or ligand-dependent gating; (ii) the S4 C-terminal region (S4C-term) is not necessary for ligand-dependent gating but is required for hyperpolarization-dependent activation and acts like an autoinhibitory domain on the PD; (iii) the S5N-term region is involved in VSD–PD coupling and holding the pore closed; and (iv) spHCN channels have two voltage-dependent processes, a hyperpolarization-dependent activation and a depolarization-dependent recovery from inactivation. These results are inconsistent with the canonical model of VSD–PD coupling in Kv channels and elucidate the mechanism for hyperpolarization-dependent activation of HCN channels.

scholarly journals Minimal molecular determinants of isoform-specific differences in efficacy in the HCN channel family

2018 ◽  
Vol 150 (8) ◽  
pp. 1203-1213 ◽  
Author(s):  
Claudia P. Alvarez-Baron ◽  
Vadim A. Klenchin ◽  
Baron Chanda
Keyword(s):  
Cyclic Nucleotide ◽  
Rhythmic Activity ◽  
Structural Similarity ◽  
Hcn Channels ◽  
Hcn Channel ◽  
Nucleotide Binding Domain ◽  
Functional Differences ◽  
Molecular Determinants ◽  
Voltage Dependent ◽  
Hcn1 Channels

Hyperpolarization-activated, cyclic nucleotide–gated (HCN) channels generate rhythmic activity in the heart and brain. Isoform-specific functional differences reflect the specializations required for the various roles that they play. Despite a high sequence and structural similarity, HCN isoforms differ greatly in their response to cyclic nucleotides. Cyclic AMP (cAMP) enhances the activity of HCN2 and HCN4 isoforms by shifting the voltage dependence of activation to more depolarized potentials, whereas HCN1 and HCN3 isoforms are practically insensitive to this ligand. Here, to determine the molecular basis for increased cAMP efficacy in HCN2 channels, we progressively mutate residues in the C-linker and cyclic nucleotide–binding domain (CNBD) of the mouse HCN2 to their equivalents in HCN1. We identify two clusters of mutations that determine the differences in voltage-dependent activation between these two isoforms. One maps to the C-linker region, whereas the other is in proximity to the cAMP-binding site in the CNBD. A mutant channel containing just five mutations (M485I, G497D, S514T, V562A, and S563G) switches cAMP sensitivity of full-length HCN2 to that of HCN1 channels. These findings, combined with a detailed analysis of various allosteric models for voltage- and ligand-dependent gating, indicate that these residues alter the ability of the C-linker to transduce signals from the CNBD to the pore gates of the HCN channel.

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 HCN domain is required for HCN channel cell-surface expression and couples voltage- and cAMP-dependent gating mechanisms

2020 ◽  
Vol 295 (24) ◽  
pp. 8164-8173
Author(s):  
Ze-Jun Wang ◽  
Ismary Blanco ◽  
Sebastien Hayoz ◽  
Tinatin I. Brelidze
Keyword(s):  
Cell Surface ◽  
Cyclic Nucleotide ◽  
Rhythmic Activity ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Hcn Channels ◽  
Hcn Channel ◽  
Structural Element ◽  
Functional Link ◽  
Membrane Hyperpolarization

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are major regulators of synaptic plasticity and rhythmic activity in the heart and brain. Opening of HCN channels requires membrane hyperpolarization and is further facilitated by intracellular cyclic nucleotides (cNMPs). In HCN channels, membrane hyperpolarization is sensed by the membrane-spanning voltage sensor domain (VSD), and the cNMP-dependent gating is mediated by the intracellular cyclic nucleotide-binding domain (CNBD) connected to the pore-forming S6 transmembrane segment via the C-linker. Previous functional analysis of HCN channels has suggested a direct or allosteric coupling between the voltage- and cNMP-dependent activation mechanisms. However, the specifics of this coupling remain unclear. The first cryo-EM structure of an HCN1 channel revealed that a novel structural element, dubbed the HCN domain (HCND), forms a direct structural link between the VSD and C-linker–CNBD. In this study, we investigated the functional significance of the HCND. Deletion of the HCND prevented surface expression of HCN2 channels. Based on the HCN1 structure analysis, we identified Arg237 and Gly239 residues on the S2 of the VSD that form direct interactions with Ile135 on the HCND. Disrupting these interactions abolished HCN2 currents. We also identified three residues on the C-linker–CNBD (Glu478, Gln482, and His559) that form direct interactions with residues Arg154 and Ser158 on the HCND. Disrupting these interactions affected both voltage- and cAMP-dependent gating of HCN2 channels. These findings indicate that the HCND is necessary for the cell-surface expression of HCN channels and provides a functional link between voltage- and cAMP-dependent mechanisms of HCN channel gating.

scholarly journals Kinetic analysis of the action of Leiurus scorpion alpha-toxin on ionic currents in myelinated nerve.

1985 ◽  
Vol 86 (5) ◽  
pp. 739-762 ◽  
Author(s):  
G K Wang ◽  
G Strichartz
Keyword(s):  
Steady State ◽  
Ionic Currents ◽  
Na Channel ◽  
Dependent Manner ◽  
Na Current ◽  
Toxin Concentration ◽  
Toxin Molecule ◽  
Channel Inactivation ◽  
Na Currents ◽  
Voltage Dependent

The effects of a neurotoxin, purified from the venom of the scorpion Leiurus quinquestriatus, on the ionic currents of toad single myelinated fibers were studied under voltage-clamp conditions. Unlike previous investigations using crude scorpion venom, purified Leiurus toxin II alpha at high concentrations (200-400 nM) did not affect the K currents, nor did it reduce the peak Na current in the early stages of treatment. The activation of the Na channel was unaffected by the toxin, the activation time course remained unchanged, and the peak Na current vs. voltage relationship was not altered. In contrast, Na channel inactivation was considerably slowed and became incomplete. As a result, a steady state Na current was maintained during prolonged depolarizations of several seconds. These steady state Na currents had a different voltage dependence from peak Na currents and appeared to result from the opening of previously inactivated Na channels. The opening kinetics of the steady state current were exponential and had rates approximately 100-fold slower than the normal activation processes described for transitions from the resting state to the open state. In addition, the dependence of the peak Na current on the potential of preceding conditioning pulses was also dramatically altered by toxin treatment; this parameter reached a minimal value near a membrane potential of -50 mV and then increased continuously to a "plateau" value at potentials greater than +50 mV. The amplitude of this plateau was dependent on toxin concentration, reaching a maximum value equal to approximately 50% of the peak current; voltage-dependent reversal of the toxin's action limits the amplitude of the plateauing effect. The measured plateau effect was half-maximum at a toxin concentration of 12 nM, a value quite similar to the concentration producing half of the maximum slowing of Na channel inactivation. The results of Hill plots for these actions suggest that one toxin molecule binds to one Na channel. Thus, the binding of a single toxin molecule probably both produces the steady state currents and slows the Na channel inactivation. We propose that Leiurus toxin inhibits the conversion of the open state to inactivated states in a voltage-dependent manner, and thereby permits a fraction of the total Na permeability to remain at membrane potentials where inactivation is normally complete.

scholarly journals Crystal structures of murine norovirus-1 RNA-dependent RNA polymerase

2011 ◽  
Vol 92 (7) ◽  
pp. 1607-1616 ◽  
Author(s):  
Ji-Hye Lee ◽  
Intekhab Alam ◽  
Kang Rok Han ◽  
Sunyoung Cho ◽  
Sungho Shin ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Crystal Structures ◽  
Active Site ◽  
Metal Ion ◽  
Health Concern ◽  
Murine Norovirus ◽  
Active Site Residues ◽  
Rna Dependent Rna Polymerase ◽  
Close Proximity ◽  
Functional Features

Norovirus is one of the leading agents of gastroenteritis and is a major public health concern. In this study, the crystal structures of recombinant RNA-dependent RNA polymerase (RdRp) from murine norovirus-1 (MNV-1) and its complex with 5-fluorouracil (5FU) were determined at 2.5 Å resolution. Crystals with C2 symmetry revealed a dimer with half a dimer in the asymmetrical unit, and the protein exists predominantly as a monomer in solution, in equilibrium with a smaller population of dimers, trimers and hexamers. MNV-1 RdRp exhibited polymerization activity with a right-hand fold typical of polynucleotide polymerases. The metal ion modelled in close proximity to the active site was found to be coordinated tetrahedrally to the carboxyl groups of aspartate clusters. The orientation of 5FU observed in three molecules in the asymmetrical unit was found to be slightly different, but it was stabilized by a network of favourable interactions with the conserved active-site residues Arg185, Asp245, Asp346, Asp347 and Arg395. The information gained on the structural and functional features of MNV-1 RdRp will be helpful in understanding replication of norovirus and in designing novel therapeutic agents against this important pathogen.

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