scholarly journals Voltage Sensor Movement and cAMP Binding Allosterically Regulate an Inherently Voltage-independent Closed−Open Transition in HCN Channels

2007 ◽  
Vol 129 (2) ◽  
pp. 175-188 ◽  
Author(s):  
Shan Chen ◽  
Jing Wang ◽  
Lei Zhou ◽  
Meena S. George ◽  
Steven A. Siegelbaum
Keyword(s):  
Cyclic Nucleotide ◽  
Voltage Sensor ◽  
Hcn Channels ◽  
Nucleotide Binding Domain ◽  
Voltage Range ◽  
Slow Kinetics ◽  
Rapid Kinetics ◽  
Hcn1 Channels ◽  
Rate Limiting ◽  
Allosteric Model

The hyperpolarization-activated cyclic nucleotide-modulated cation (HCN) channels are regulated by both membrane voltage and the binding of cyclic nucleotides to a cytoplasmic, C-terminal cyclic nucleotide-binding domain (CNBD). Here we have addressed the mechanism of this dual regulation for HCN2 channels, which activate with slow kinetics that are strongly accelerated by cAMP, and HCN1 channels, which activate with rapid kinetics that are weakly enhanced by cAMP. Surprisingly, we find that the rate of opening of HCN2 approaches a maximal value with extreme hyperpolarization, indicating the presence of a voltage-independent kinetic step in the opening process that becomes rate limiting at very negative potentials. cAMP binding enhances the rate of this voltage-independent opening step. In contrast, the rate of opening of HCN1 is much greater than that of HCN2 and does not saturate with increasing hyperpolarization over the voltage range examined. Domain-swapping chimeras between HCN1 and HCN2 reveal that the S4–S6 transmembrane region largely determines the limiting rate in opening kinetics at negative voltages. Measurements of HCN2 tail current kinetics also reveal a voltage-independent closing step that becomes rate limiting at positive voltages; the rate of this closing step is decreased by cAMP. These results are consistent with a cyclic allosteric model in which a closed–open transition that is inherently voltage independent is subject to dual allosteric regulation by voltage sensor movement and cAMP binding. This mechanism accounts for several properties of HCN channel gating and has potentially important physiological implications.

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 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 Domain IV voltage-sensor movement is both sufficient and rate limiting for fast inactivation in sodium channels

2013 ◽  
Vol 142 (2) ◽  
pp. 101-112 ◽  
Author(s):  
Deborah L. Capes ◽  
Marcel P. Goldschen-Ohm ◽  
Manoel Arcisio-Miranda ◽  
Francisco Bezanilla ◽  
Baron Chanda
Keyword(s):  
Sodium Channels ◽  
Voltage Sensor ◽  
Fast Inactivation ◽  
Voltage Range ◽  
Voltage Gated Sodium Channels ◽  
Channel Opening ◽  
The Individual ◽  
Electrical Impulses ◽  
Voltage Sensing Domain ◽  
Rate Limiting

Voltage-gated sodium channels are critical for the generation and propagation of electrical signals in most excitable cells. Activation of Na+ channels initiates an action potential, and fast inactivation facilitates repolarization of the membrane by the outward K+ current. Fast inactivation is also the main determinant of the refractory period between successive electrical impulses. Although the voltage sensor of domain IV (DIV) has been implicated in fast inactivation, it remains unclear whether the activation of DIV alone is sufficient for fast inactivation to occur. Here, we functionally neutralize each specific voltage sensor by mutating several critical arginines in the S4 segment to glutamines. We assess the individual role of each voltage-sensing domain in the voltage dependence and kinetics of fast inactivation upon its specific inhibition. We show that movement of the DIV voltage sensor is the rate-limiting step for both development and recovery from fast inactivation. Our data suggest that activation of the DIV voltage sensor alone is sufficient for fast inactivation to occur, and that activation of DIV before channel opening is the molecular mechanism for closed-state inactivation. We propose a kinetic model of sodium channel gating that can account for our major findings over a wide voltage range by postulating that DIV movement is both necessary and sufficient for fast inactivation.

scholarly journals Structural insights into the mechanisms of CNBD channel function

2017 ◽  
Vol 150 (2) ◽  
pp. 225-244 ◽  
Author(s):  
Zachary M. James ◽  
William N. Zagotta
Keyword(s):  
Cyclic Nucleotide ◽  
Ion Selectivity ◽  
K Channel ◽  
Hcn Channels ◽  
Nucleotide Binding Domain ◽  
Physiological Processes ◽  
Structural Framework ◽  
Voltage Dependent ◽  
Compare And Contrast ◽  
Structural Insights

Cyclic nucleotide-binding domain (CNBD) channels are a family of ion channels in the voltage-gated K+ channel superfamily that play crucial roles in many physiological processes. CNBD channels are structurally similar but functionally very diverse. This family includes three subfamilies: (1) the cyclic nucleotide-gated (CNG) channels, which are cation-nonselective, voltage-independent, and cyclic nucleotide-gated; (2) the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are weakly K+ selective, hyperpolarization-activated, and cyclic nucleotide-gated; and (3) the ether-à-go-go-type (KCNH) channels, which are strongly K+ selective, depolarization-activated, and cyclic nucleotide-independent. Recently, several high-resolution structures have been reported for intact CNBD channels, providing a structural framework to better understand their diverse function. In this review, we compare and contrast the recent structures and discuss how they inform our understanding of ion selectivity, voltage-dependent gating, and cyclic nucleotide–dependent gating within this channel family.

scholarly journals Allosteric Regulation of the Cyclic Nucleotide-Binding Domain in HCN Channels

2015 ◽  
Vol 108 (2) ◽  
pp. 192a-193a
Author(s):  
Hannah A. DeBerg ◽  
Shahidul M. Islam ◽  
Michael C. Puljung ◽  
Benoit Roux ◽  
William N. Zagotta ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Allosteric Regulation ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Hcn Channels ◽  
Nucleotide Binding Domain ◽  
Cyclic Nucleotide Binding ◽  
Cyclic Nucleotide Binding Domain

scholarly journals A synthetic peptide that prevents cAMP regulation in mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Andrea Saponaro ◽  
Francesca Cantini ◽  
Alessandro Porro ◽  
Annalisa Bucchi ◽  
Dario DiFrancesco ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Rational Design ◽  
Structural Model ◽  
Hcn Channels ◽  
Nucleotide Binding Domain ◽  
Novel Approach ◽  
A Cell ◽  
The Central Nervous System ◽  
The Brain ◽  
Adrenergic Activation

Binding of TRIP8b to the cyclic nucleotide binding domain (CNBD) of mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels prevents their regulation by cAMP. Since TRIP8b is expressed exclusively in the brain, we envisage that it can be used for orthogonal control of HCN channels beyond the central nervous system. To this end, we have identified by rational design a 40-aa long peptide (TRIP8bnano) that recapitulates affinity and gating effects of TRIP8b in HCN isoforms (hHCN1, mHCN2, rbHCN4) and in the cardiac current If in rabbit and mouse sinoatrial node cardiomyocytes. Guided by an NMR-derived structural model that identifies the key molecular interactions between TRIP8bnano and the HCN CNBD, we further designed a cell-penetrating peptide (TAT-TRIP8bnano) which successfully prevented β-adrenergic activation of mouse If leaving the stimulation of the L-type calcium current (ICaL) unaffected. TRIP8bnano represents a novel approach to selectively control HCN activation, which yields the promise of a more targeted pharmacology compared to pore blockers.

scholarly journals Transfer of Voltage Independence from a Rat Olfactory Channel to the Drosophila Ether-à-go-go K+ Channel

1997 ◽  
Vol 109 (3) ◽  
pp. 301-311 ◽  
Author(s):  
Chih-Yung Tang ◽  
Diane M. Papazian
Keyword(s):  
Cyclic Nucleotide ◽  
Voltage Sensor ◽  
K Channel ◽  
Voltage Range ◽  
Voltage Gated ◽  
Cyclic Nucleotide Gated Channels ◽  
Voltage Dependent ◽  
Relative Stabilization ◽  
Voltage Gated Ion Channels ◽  
S4 Segment

The S4 segment is an important part of the voltage sensor in voltage-gated ion channels. Cyclic nucleotide-gated channels, which are members of the superfamily of voltage-gated channels, have little inherent sensitivity to voltage despite the presence of an S4 segment. We made chimeras between a voltage-independent rat olfactory channel (rolf) and the voltage-dependent ether-à-go-go K+ channel (eag) to determine the basis of their divergent gating properties. We found that the rolf S4 segment can support a voltage-dependent mechanism of activation in eag, suggesting that rolf has a potentially functional voltage sensor that is silent during gating. In addition, we found that the S3-S4 loop of rolf increases the relative stability of the open conformation of eag, effectively converting eag into a voltage-independent channel. A single charged residue in the loop makes a significant contribution to the relative stabilization of the open state in eag. Our data suggest that cyclic nucleotide-gated channels such as rolf contain a voltage sensor which, in the physiological voltage range, is stabilized in an activated conformation that is permissive for pore opening.

scholarly journals Integrated Allosteric Model of Voltage Gating of Hcn Channels

2001 ◽  
Vol 117 (6) ◽  
pp. 519-532 ◽  
Author(s):  
Claudia Altomare ◽  
Annalisa Bucchi ◽  
Eva Camatini ◽  
Mirko Baruscotti ◽  
Carlo Viscomi ◽  
...  
Keyword(s):  
Rate Constants ◽  
Voltage Sensor ◽  
Voltage Gating ◽  
Hcn Channels ◽  
Open Probability ◽  
Voltage Sensors ◽  
Negative Voltage ◽  
Kinetics Of ◽  
Allosteric Model ◽  
Activation Delay

Hyperpolarization-activated (pacemaker) channels are dually gated by negative voltage and intracellular cAMP. Kinetics of native cardiac f-channels are not compatible with HH gating, and require closed/open multistate models. We verified that members of the HCN channel family (mHCN1, hHCN2, hHCN4) also have properties not complying with HH gating, such as sigmoidal activation and deactivation, activation deviating from fixed power of an exponential, removal of activation “delay” by preconditioning hyperpolarization. Previous work on native channels has indicated that the shifting action of cAMP on the open probability (Po) curve can be accounted for by an allosteric model, whereby cAMP binds more favorably to open than closed channels. We therefore asked whether not only cAMP-dependent, but also voltage-dependent gating of hyperpolarization-activated channels could be explained by an allosteric model. We hypothesized that HCN channels are tetramers and that each subunit comprises a voltage sensor moving between “reluctant” and “willing” states, whereas voltage sensors are independently gated by voltage, channel closed/open transitions occur allosterically. These hypotheses led to a multistate scheme comprising five open and five closed channel states. We estimated model rate constants by fitting first activation delay curves and single exponential time constant curves, and then individual activation/deactivation traces. By simply using different sets of rate constants, the model accounts for qualitative and quantitative aspects of voltage gating of all three HCN isoforms investigated, and allows an interpretation of the different kinetic properties of different isoforms. For example, faster kinetics of HCN1 relative to HCN2/HCN4 are attributable to higher HCN1 voltage sensors' rates and looser voltage-independent interactions between subunits in closed/open transitions. It also accounts for experimental evidence that reduction of sensors' positive charge leads to negative voltage shifts of Po curve, with little change of curve slope. HCN voltage gating thus involves two processes: voltage sensor gating and allosteric opening/closing.

scholarly journals Pathways of inter- and intrasubunit allosteric signaling in the C-linker disk and cyclic nucleotide-binding domain of HCN2 channels

2020 ◽  
Author(s):  
Christopher Pfleger ◽  
Jana Kusch ◽  
Mahesh Kondapuram ◽  
Tina Schwabe ◽  
Christian Sattler ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Cyclic Nucleotide ◽  
Signal Transmission ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Hcn Channels ◽  
Nucleotide Binding Domain ◽  
Membrane Hyperpolarization ◽  
Cyclic Nucleotide Binding ◽  
Cyclic Nucleotide Binding Domain

AbstractOpening of hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels is controlled by membrane hyperpolarization and binding of cyclic nucleotides to the tetrameric cyclic nucleotide-binding domain (CNBD), attached to the C-linker disk (CL). Confocal patch-clamp fluorometry revealed a pronounced cooperativity of ligand binding among protomers. However, by which pathways allosteric signal transmission occurs remained elusive. Here, we investigate how changes in the structural dynamics of the CL- CNBD of mouse HCN2 upon cAMP binding relate to inter- and intrasubunit signal transmission. Applying a rigidity theory-based approach, we identify two intersubunit and one intrasubunit pathways that differ in allosteric coupling strength between cAMP binding sites or towards the CL. These predictions agree with results from electrophysiological and patch-clamp fluorometry experiments. Our results map out distinct routes within the CL-CNBD that modulate different cAMP binding responses in HCN2 channels. They signify that functionally relevant submodules may exist within and across structurally discernable subunits in HCN channels.

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