scholarly journals Voltage-Controlled Gating at the Intracellular Entrance to a Hyperpolarization-Activated Cation Channel

2002 ◽  
Vol 119 (1) ◽  
pp. 83-91 ◽  
Author(s):  
Brad S. Rothberg ◽  
Ki Soon Shin ◽  
Prashant S. Phale ◽  
Gary Yellen
Keyword(s):  
Open Channel ◽  
Cardiac Cells ◽  
Hcn Channels ◽  
Hcn Channel ◽  
Channel One ◽  
Blocking Rate ◽  
Activation Gate ◽  
Flow Through ◽  
Controlled Structure ◽  
Pore Lining

Hyperpolarization-activated cation (HCN) channels regulate pacemaking activity in cardiac cells and neurons. Our previous work using the specific HCN channel blocker ZD7288 provided evidence for an intracellular activation gate for these channels because it appears that ZD7288, applied from the intracellular side, can enter and leave HCN channels only at voltages where the activation gate is opened (Shin, K.S., B.S. Rothberg, and G. Yellen. 2001. J. Gen. Physiol. 117:91–101). However, the ZD7288 molecule is larger than the Na+ or K+ ions that flow through the open channel. In the present study, we sought to resolve whether the voltage gate at the intracellular entrance to the pore for ZD7288 also can be a gate for permeant ions in HCN channels. Single residues in the putative pore-lining S6 region of an HCN channel (cloned from sea urchin; spHCN) were substituted with cysteines, and the mutants were probed with Cd2+ applied to the intracellular side of the channel. One mutant, T464C, displayed rapid irreversible block when Cd2+ was applied to opened channels, with an apparent blocking rate of ∼3 × 105 M−1s−1. The blocking rate was decreased for channels held at more depolarized voltages that close the channels, which is consistent with the Cd2+ access to this residue being gated from the intracellular side of the channel. 464C channels could be recovered from Cd2+ inhibition in the presence of a dithiol applied to the intracellular side. The rate of this recovery also was reduced when channels were held at depolarized voltages. Finally, Cd2+ could be trapped inside channels that were composed of WT/464C tandem-linked subunits, which could otherwise recover spontaneously from Cd2+ inhibition. Thus, Cd2+ escape is also gated at the intracellular side of the channel. Together, these results are consistent with a voltage-controlled structure at the intracellular side of the spHCN channel that can gate the flow of cations through the pore.

scholarly journals Inner activation gate in S6 contributes to the state-dependent binding of cAMP in full-length HCN2 channel

2012 ◽  
Vol 140 (1) ◽  
pp. 29-39 ◽  
Author(s):  
Shengjun Wu ◽  
Weihua Gao ◽  
Changan Xie ◽  
Xinping Xu ◽  
Christina Vorvis ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Cyclic Nucleotide ◽  
Binding Domain ◽  
Hcn Channels ◽  
Channel Blockers ◽  
Structural Elements ◽  
Hcn Channel ◽  
Channel Interaction ◽  
State Dependent ◽  
Activation Gate

Recently, applications of the patch-clamp fluorometry (PCF) technique in studies of cyclic nucleotide–gated (CNG) and hyperpolarization-activated, cyclic nucleotide–regulated (HCN) channels have provided direct evidence for the long-held notion that ligands preferably bind to and stabilize these channels in an open state. This state-dependent ligand–channel interaction involves contributions from not only the ligand-binding domain but also other discrete structural elements within the channel protein. This insight led us to investigate whether the pore of the HCN channel plays a role in the ligand–whole channel interaction. We used three well-characterized HCN channel blockers to probe the ion-conducting passage. The PCF technique was used to simultaneously monitor channel activity and cAMP binding. Two ionic blockers, Cs+ and Mg2+, effectively block channel conductance but have no obvious effect on cAMP binding. Surprisingly, ZD7288, an open channel blocker specific for HCN channels, significantly reduces the activity-dependent increase in cAMP binding. Independent biochemical assays exclude any nonspecific interaction between ZD7288 and isolated cAMP-binding domain. Because ZD7228 interacts with the inner pore region, where the activation gate is presumably located, we did an alanine scanning of the intracellular end of S6, from T426 to A435. Mutations of three residues, T426, M430, and H434, which are located at regular intervals on the S6 α-helix, enhance cAMP binding. In contrast, mutations of two residues in close proximity, F431A and I432A, dampen the response. Our results demonstrate that movements of the structural elements near the activation gate directly affect ligand binding affinity, which is a simple mechanistic explanation that could be applied to the interpretation of ligand gating in general.

scholarly journals Movements near the Gate of a Hyperpolarization-activated Cation Channel

2003 ◽  
Vol 122 (5) ◽  
pp. 501-510 ◽  
Author(s):  
Brad S. Rothberg ◽  
Ki Soon Shin ◽  
Gary Yellen
Keyword(s):  
Cardiac Cells ◽  
Hcn Channels ◽  
The Novel ◽  
Cadmium Ions ◽  
Metal Interactions ◽  
Cysteine Substitution ◽  
Kv Channels ◽  
Nanomolar Concentration ◽  
Activation Gate ◽  
Transmembrane Regions

Hyperpolarization-activated cation (HCN) channels regulate pacemaking activity in cardiac cells and neurons. Like the related depolarization-activated K+ channels (Kv channels), HCN channels use an intracellular activation gate to regulate access to an inner cavity, lined by the S6 transmembrane regions, which leads to the selectivity filter near the extracellular surface. Here we describe two types of metal interactions with substituted cysteines in the S6, which alter the voltage-controlled movements of the gate. At one position (L466), substitution of cysteine in all four subunits allows Cd2+ ions at nanomolar concentration to stabilize the open state (a “lock-open” effect). This effect depends on native histidines at a nearby position (H462); the lock-open effect can be abolished by changing the histidines to tyrosines, or enhanced by changing them to cysteines. Unlike a similar effect in Kv channels, this effect depends on a Cd2+ bridge between 462 and 466 in the same subunit. Cysteine substitution at another position (Q468) produces two effects of Cd2+: both a lock-open effect and a dramatic slowing of channel activation—a “lock-closed” effect. The two effects can be separated, because the lock-open effect depends on the histidine at position 462. The novel lock-closed effect results from stabilization of the closed state by the binding of up to four Cd2+ ions. During the opening conformational change, the S6 apparently moves from one position in which the 468C cysteines can bind four Cd2+ ions, possibly as a cluster of cysteines and cadmium ions near the central axis of the pore, to another position (or flexible range of positions) where either 466C or 468C can bind Cd2+ in association with the histidine at 462.

Simulation of Supercritical Bend Flow through an Open Channel

2016 ◽  
Vol 15 (0) ◽  
pp. 22
Author(s):  
Rupak Saha ◽  
Sanchayan Mukherjee ◽  
Ajay Kumar Das ◽  
Kanhu Keshab Jena ◽  
Manajit Mandal ◽  
...  
Keyword(s):  
Open Channel ◽  
Flow Through ◽  
Bend Flow

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.

Predictions of bulk velocity for open channel flow through submerged vegetation

2020 ◽  
Vol 32 (4) ◽  
pp. 795-799
Author(s):  
Wei-jie Wang ◽  
Xiao-yu Cui ◽  
Fei Dong ◽  
Wen-qi Peng ◽  
Zhen Han ◽  
...  
Keyword(s):  
Channel Flow ◽  
Open Channel ◽  
Open Channel Flow ◽  
Bulk Velocity ◽  
Submerged Vegetation ◽  
Flow Through

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.

Pool-orifice and pool-orifice-weir fishways

1989 ◽  
Vol 16 (5) ◽  
pp. 774-777 ◽  
Author(s):  
N. Rajaratnam ◽  
C. Katopodis ◽  
A. Mainali
Keyword(s):  
Open Channel ◽  
Open Channel Flow ◽  
Technical Note ◽  
Total Flow ◽  
Total Flow Rate ◽  
Vertical Slot ◽  
Flow Hydraulics ◽  
Orifice Flow ◽  
Flow Through ◽  
Submerged Orifice

This technical note presents a method of analyzing the flow in pool-orifice fishways by dividing it into vertical slot and submerged orifice flow regimes. For a pool-orifice-weir fishway, with flow through the orifice as well as over the weir, a method has been suggested for predicting the total flow rate in the fishway. Experimental observations are presented in support of these methods. Key words: open-channel flow, hydraulics, fishways, turbulent flow.

scholarly journals Blocker State Dependence and Trapping in Hyperpolarization-Activated Cation Channels

2001 ◽  
Vol 117 (2) ◽  
pp. 91-102 ◽  
Author(s):  
Ki Soon Shin ◽  
Brad S. Rothberg ◽  
Gary Yellen
Keyword(s):  
Molecular Basis ◽  
State Dependence ◽  
Activation Gate ◽  
Voltage Dependent ◽  
The Difference ◽  
Pore Lining ◽  
Selective Blocker ◽  
Inside Out ◽  
Differential Affinity ◽  
Structural Determinant

Hyperpolarization-activated cation currents (Ih) are key determinants of repetitive electrical activity in heart and nerve cells. The bradycardic agent ZD7288 is a selective blocker of these currents. We studied the mechanism for ZD7288 blockade of cloned Ih channels in excised inside-out patches. ZD7288 blockade of the mammalian mHCN1 channel appeared to require opening of the channel, but strong hyperpolarization disfavored blockade. The steepness of this voltage-dependent effect (an apparent valence of ∼4) makes it unlikely to arise solely from a direct effect of voltage on blocker binding. Instead, it probably indicates a differential affinity of the blocker for different channel conformations. Similar properties were seen for ZD7288 blockade of the sea urchin homologue of Ih channels (SPIH), but some of the blockade was irreversible. To explore the molecular basis for the difference in reversibility, we constructed chimeric channels from mHCN1 and SPIH and localized the structural determinant for the reversibility to three residues in the S6 region likely to line the pore. Using a triple point mutant in S6, we also revealed the trapping of ZD7288 by the closing of the channel. Overall, the observations led us to hypothesize that the residues responsible for ZD7288 block of Ih channels are located in the pore lining, and are guarded by an intracellular activation gate of the channel.

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