scholarly journals Effects of Ultraviolet Modification on the Gating Energetics of Cyclic Nucleotide–Gated Channels

2000 ◽  
Vol 116 (2) ◽  
pp. 253-282 ◽  
Author(s):  
Thomas R. Middendorf ◽  
Richard W. Aldrich
Keyword(s):  
Cyclic Nucleotide ◽  
Channel Gating ◽  
Quantum Yields ◽  
Open Probability ◽  
Current Increase ◽  
Cyclic Nucleotide Gated Channels ◽  
Channel Opening ◽  
High Concentrations ◽  
Current Reduction ◽  
Opposing Effects

Middendorf et al. (Middendorf, T.R., R.W. Aldrich, and D.A. Baylor. 2000. J. Gen. Physiol. 116:227–252) showed that ultraviolet light decreases the current through cloned cyclic nucleotide–gated channels from bovine retina activated by high concentrations of cGMP. Here we probe the mechanism of the current reduction. The channels' open probability before irradiation, Po(0), determined the sign of the change in current amplitude that occurred upon irradiation. UV always decreased the current through channels with high initial open probabilities [Po(0) > 0.3]. Manipulations that promoted channel opening antagonized the current reduction by UV. In contrast, UV always increased the current through channels with low initial open probabilities [Po(0) ≤ 0.02], and the magnitude of the current increase varied inversely with Po(0). The dual effects of UV on channel currents and the correlation of both effects with Po(0) suggest that the channels contain two distinct classes of UV target residues whose photochemical modification exerts opposing effects on channel gating. We present a simple model based on this idea that accounts quantitatively for the UV effects on the currents and provides estimates for the photochemical quantum yields and free energy costs of modifying the UV targets. Simulations indicate that UV modification may be used to produce and quantify large changes in channel gating energetics in regimes where the associated changes in open probability are not measurable by existing techniques.

scholarly journals The HCN domain couples voltage gating and cAMP response in hyperpolarization-activated cyclic nucleotide-gated channels

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Alessandro Porro ◽  
Andrea Saponaro ◽  
Federica Gasparri ◽  
Daniel Bauer ◽  
Christine Gross ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Channel Gating ◽  
Hcn Channels ◽  
Structural Mechanism ◽  
Cyclic Nucleotide Gated Channels ◽  
Channel Opening ◽  
Mode Of Operation ◽  
Voltage Dependent ◽  
Upward Displacement ◽  
Voltage Sensing Domain

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control spontaneous electrical activity in heart and brain. Binding of cAMP to the cyclic nucleotide-binding domain (CNBD) facilitates channel opening by relieving a tonic inhibition exerted by the CNBD. Despite high resolution structures of the HCN1 channel in the cAMP bound and unbound states, the structural mechanism coupling ligand binding to channel gating is unknown. Here we show that the recently identified helical HCN-domain (HCND) mechanically couples the CNBD and channel voltage sensing domain (VSD), possibly acting as a sliding crank that converts the planar rotational movement of the CNBD into a rotational upward displacement of the VSD. This mode of operation and its impact on channel gating are confirmed by computational and experimental data showing that disruption of critical contacts between the three domains affects cAMP- and voltage-dependent gating in three HCN isoforms.

scholarly journals Tetracaine Reports a Conformational Change in the Pore of Cyclic Nucleotide–gated Channels

1997 ◽  
Vol 110 (5) ◽  
pp. 591-600 ◽  
Author(s):  
Anthony A. Fodor ◽  
Kevin D. Black ◽  
William N. Zagotta
Keyword(s):  
Conformational Change ◽  
Local Anesthetic ◽  
Cyclic Nucleotide ◽  
State Dependence ◽  
Wild Type ◽  
Α Subunit ◽  
Allosteric Transition ◽  
State Dependent ◽  
Channel Opening ◽  
High Concentrations

Local anesthetics are a diverse group of clinically useful compounds that act as pore blockers of both voltage- and cyclic nucleotide–gated (CNG) ion channels. We used the local anesthetic tetracaine to probe the nature of the conformational change that occurs in the pore of CNG channels during the opening allosteric transition. When applied to the intracellular side of wild-type rod CNG channels expressed in Xenopus oocytes from the α subunit, the local anesthetic tetracaine exhibits state-dependent block, binding with much higher affinity to closed states than to open states. Here we show that neutralization of a glutamic acid in the conserved P region (E363G) eliminated this state dependence of tetracaine block. Tetracaine blocked E363G channels with the same effectiveness at high concentrations of cGMP, when the channel spent more time open, and at low concentrations of cGMP, when the channel spent more time closed. In addition, Ni2+, which promotes the opening allosteric transition, decreased the effectiveness of tetracaine block of wild-type but not E363G channels. Similar results were obtained in a chimeric CNG channel that exhibits a more favorable opening allosteric transition. These results suggest that E363 is accessible to internal tetracaine in the closed but not the open configuration of the pore and that the conformational change that accompanies channel opening includes a change in the conformation or accessibility of E363.

scholarly journals Ligand-binding domain subregions contributing to bimodal agonism in cyclic nucleotide–gated channels

2011 ◽  
Vol 137 (6) ◽  
pp. 591-603 ◽  
Author(s):  
Wai-Fung Wong ◽  
Kerry S.C. Chan ◽  
Matthew S. Michaleski ◽  
Adam Haesler ◽  
Edgar C. Young
Keyword(s):  
Steady State ◽  
Ligand Binding ◽  
Cyclic Nucleotide ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Phosphate Binding ◽  
Open Probability ◽  
Cyclic Nucleotide Gated Channels ◽  
Functional Features ◽  
Apparent Attenuation

Cyclic nucleotide–gated (CNG) channels bind cGMP or cAMP in a cytoplasmic ligand–binding domain (BD), and this binding typically increases channel open probability (Po) without inducing desensitization. However, the catfish CNGA2 (fCNGA2) subtype exhibits bimodal agonism, whereby steady-state Po increases with initial cGMP-binding events (“pro” action) up to a maximum of 0.4, but decreases with subsequent cGMP-binding events (“con” action) occurring at concentrations >3 mM. We sought to clarify if low pro-action efficacy was either necessary or sufficient for con action to operate. To find BD residues responsible for con action or low pro-action efficacy or both, we constructed chimeric CNG channels: subregions of the fCNGA2 BD were substituted with corresponding sequence from the rat CNGA4 BD, which does not support con action. Constructs were expressed in frog oocytes and tested by patch clamp of cell-free membranes. For nearly all BD elements, we found at least one construct where replacing that element preserved robust con action, with a ratio of steady-state conductances, g(10 mM cGMP)/g(3 mM cGMP) < 0.75. When all of the BD sequence C terminal of strand β6 was replaced, g(10 mM cGMP)/g(3 mM cGMP) was increased to 0.95 ± 0.05 (n = 7). However, this apparent attenuation of con action could be explained by an increase in the efficacy of pro action for all agonists, controlled by a conserved “phosphate-binding cassette” motif that contacts ligand; this produces high Po values that are less sensitive to shifts in gating equilibrium. In contrast, substituting a single valine in the N-terminal helix αA abolished con action (g(30 mM cGMP)/g(3 mM cGMP) increased to 1.26 ± 0.24; n = 7) without large increases in pro-action efficacy. Our work dissociates the two functional features of low pro-action efficacy and con action, and moreover identifies a separate structural determinant for each.

scholarly journals Influence of Permeant Ions on Gating in Cyclic Nucleotide–gated Channels

2002 ◽  
Vol 121 (1) ◽  
pp. 61-72 ◽  
Author(s):  
Miguel Holmgren
Keyword(s):  
Cyclic Nucleotide ◽  
Single Channel ◽  
Hek293 Cells ◽  
Monovalent Cations ◽  
Open Probability ◽  
Cyclic Nucleotide Gated Channels ◽  
Open Conformation ◽  
Open Time ◽  
Main Effect ◽  
The Stability

Cyclic nucleotide–gated channels are key components in the transduction of visual and olfactory signals where their role is to respond to changes in the intracellular concentration of cyclic nucleotides. Although these channels poorly select between physiologically relevant monovalent cations, the gating by cyclic nucleotide is different in the presence of Na+ or K+ ions. This property was investigated using rod cyclic nucleotide–gated channels formed by expressing the subunit 1 (or α) in HEK293 cells. In the presence of K+ as the permeant ion, the affinity for cGMP is higher than the affinity measured in the presence of Na+. At the single channel level, subsaturating concentrations of cGMP show that the main effect of the permeant K+ ions is to prolong the time channels remain open without major changes in the shut time distribution. In addition, the maximal open probability was higher when K+ was the permeant ion (0.99 for K+ vs. 0.95 for Na+) due to an increase in the apparent mean open time. Similarly, in the presence of saturating concentrations of cAMP, known to bind but unable to efficiently open the channel, permeant K+ ions also prolong the time channels visit the open state. Together, these results suggest that permeant ions alter the stability of the open conformation by influencing of the O→C transition.

scholarly journals C-Linker of Cyclic Nucleotide–gated Channels Controls Coupling of Ligand Binding to Channel Gating

1999 ◽  
Vol 113 (1) ◽  
pp. 17-34 ◽  
Author(s):  
Pierre Paoletti ◽  
Edgar C. Young ◽  
Steven A. Siegelbaum
Keyword(s):  
Amino Acids ◽  
Ligand Binding ◽  
Cyclic Nucleotide ◽  
Transmembrane Domain ◽  
Open Probability ◽  
C Elegans ◽  
Cyclic Nucleotide Gated Channels ◽  
Ligand Gating ◽  
Conserved Amino Acids ◽  
Precise Role

Cyclic nucleotide–gated channels are composed of a core transmembrane domain, structurally homologous to the voltage-gated K+ channels, and a cytoplasmic ligand-binding domain. These two modules are joined by ∼90 conserved amino acids, the C-linker, whose precise role in the mechanism of channel activation by cyclic nucleotides is poorly understood. We examined cyclic nucleotide–gated channels from bovine photoreceptors and Caenorhabditis elegans sensory neurons that show marked differences in cyclic nucleotide efficacy and sensitivity. By constructing chimeras from these two channels, we identified a region of 30 amino acids in the C-linker (the L2 region) as an important determinant of activation properties. An increase in both the efficacy of gating and apparent affinity for cGMP and cAMP can be conferred onto the photoreceptor channel by the replacement of its L2 region with that of the C. elegans channel. Three residues within this region largely account for this effect. Despite the profound effect of the C-linker region on ligand gating, the identity of the C-linker does not affect the spontaneous, ligand-independent open probability. Based on a cyclic allosteric model of activation, we propose that the C-linker couples the opening reaction in the transmembrane core region to the enhancement of the affinity of the open channel for agonist, which underlies ligand gating.

scholarly journals Mechanism of Inhibition of Cyclic Nucleotide–Gated Channel by Protein Tyrosine Kinase Probed with Genistein

2001 ◽  
Vol 117 (3) ◽  
pp. 219-234 ◽  
Author(s):  
Elena Molokanova ◽  
Richard H. Kramer
Keyword(s):  
Tyrosine Phosphorylation ◽  
Cyclic Nucleotide ◽  
Tyrosine Kinases ◽  
Single Channel ◽  
Channel Gating ◽  
Open Probability ◽  
Inhibitory Interaction ◽  
Α Subunit ◽  
Protein Tyrosine ◽  
Cng Channel

Rod cyclic nucleotide–gated (CNG) channels are modulated by changes in tyrosine phosphorylation catalyzed by protein tyrosine kinases (PTKs) and phosphatases (PTPs). We used genistein, a PTK inhibitor, to probe the interaction between the channel and PTKs. Previously, we found that in addition to inhibiting tyrosine phosphorylation of the rod CNG channel α-subunit (RETα), genistein triggers a noncatalytic inhibitory interaction between the PTK and the channel. These studies suggest that PTKs affects RETα channels in two ways: (1) by catalyzing phosphorylation of the channel protein, and (2) by allosterically regulating channel activation. Here, we study the mechanism of noncatalytic inhibition. We find that noncatalytic inhibition follows the same activity dependence pattern as catalytic modulation (phosphorylation): the efficacy and apparent affinity of genistein inhibition are much higher for closed than for fully activated channels. Association rates with the genistein–PTK complex were similar for closed and fully activated channels and independent of genistein concentration. Dissociation rates were 100 times slower for closed channels, which is consistent with a much higher affinity for genistein–PTK. Genistein–PTK affects channel gating, but not single channel conductance or the number of active channels. By analyzing single channel gating during genistein–PTK dissociation, we determined the maximal open probability for normal and genistein–PTK-bound channels. genistein–PTK decreases open probability by increasing the free energy required for opening, making opening dramatically less favorable. Ni2+, which potentiates RETα channel gating, partially relieves genistein inhibition, possibly by disrupting the association between the genistein–PTK and the channel. Studies on chimeric channels containing portions of RETα, which exhibits genistein inhibition, and the rat olfactory CNG channel α-subunit, which does not, reveals that a domain containing S6 and flanking regions is the crucial for genistein inhibition and may constitute the genistein–PTK binding site. Thus, genistein–PTK stabilizes the closed state of the channel by interacting with portions of the channel that participate in gating.

scholarly journals A Cysteine Scan of the Inner Vestibule of Cyclic Nucleotide–gated Channels Reveals Architecture and Rearrangement of the Pore

2003 ◽  
Vol 121 (6) ◽  
pp. 563-583 ◽  
Author(s):  
Galen E. Flynn ◽  
William N. Zagotta
Keyword(s):  
Free Energy ◽  
Cyclic Nucleotide ◽  
Time Course ◽  
Mutant Channel ◽  
Cyclic Nucleotide Gated Channels ◽  
Structural Framework ◽  
Shaker Channels ◽  
Channel Opening ◽  
Helical Packing ◽  
Favorable Change

Cyclic nucleotide–gated (CNG) channels belong to the P-loop–containing family of ion channels that also includes KcsA, MthK, and Shaker channels. In this study, we investigated the structure and rearrangement of the CNGA1 channel pore using cysteine mutations and cysteine-specific modification. We constructed 16 mutant channels, each one containing a cysteine mutation at one of the positions between 384 and 399 in the S6 region of the pore. By measuring currents activated by saturating concentrations of the full agonist cGMP and the partial agonists cIMP and cAMP, we show that mutating S6 residues to cysteine caused both favorable and unfavorable changes in the free energy of channel opening. The time course of cysteine modification with 2-aminoethylmethane thiosulfonate hydrochloride (MTSEA) was complex. For many positions we observed decreases in current activated by cGMP and concomitant increases in current activated by cIMP and cAMP. A model where modification affected both gating and permeation successfully reproduced the complex time course of modification for most of the mutant channels. From the model fits to the time course of modification for each mutant channel, we quantified the following: (a) the bimolecular rate constant of modification in the open state, (b) the change in conductance, and (c) the change in the free energy of channel opening for modification of each cysteine. At many S6 cysteines, modification by MTSEA caused a decrease in conductance and a favorable change in the free energy of channel opening. Our results are interpreted within the structural framework of the known structures of KcsA and MthK. We conclude that: (a) MTSEA modification affects both gating and permeation, (b) the open configuration of the pore of CNGA1 channels is consistent with the structure of MthK, and (c) the modification of S6 residues disrupts the helical packing of the closed channel, making it easier for channels to open.

scholarly journals Intersubunit physical couplings fostered by the left flipper domain facilitate channel opening of P2X4 receptors

2017 ◽  
Vol 292 (18) ◽  
pp. 7619-7635 ◽  
Author(s):  
Jin Wang ◽  
Liang-Fei Sun ◽  
Wen-Wen Cui ◽  
Wen-Shan Zhao ◽  
Xue-Fei Ma ◽  
...  
Keyword(s):  
Channel Gating ◽  
Receptor Activation ◽  
P2x Receptors ◽  
Covalent Modification ◽  
P2x Receptor ◽  
Lower Body ◽  
Structural Element ◽  
Open Probability ◽  
Channel Opening ◽  
P2x4 Receptors

P2X receptors are ATP-gated trimeric channels with important roles in diverse pathophysiological functions. A detailed understanding of the mechanism underlying the gating process of these receptors is thus fundamentally important and may open new therapeutic avenues. The left flipper (LF) domain of the P2X receptors is a flexible loop structure, and its coordinated motions together with the dorsal fin (DF) domain are crucial for the channel gating of the P2X receptors. However, the mechanism underlying the crucial role of the LF domain in the channel gating remains obscure. Here, we propose that the ATP-induced allosteric changes of the LF domain enable it to foster intersubunit physical couplings among the DF and two lower body domains, which are pivotal for the channel gating of P2X4 receptors. Metadynamics analysis indicated that these newly established intersubunit couplings correlate well with the ATP-bound open state of the receptors. Moreover, weakening or strengthening these physical interactions with engineered intersubunit metal bridges remarkably decreased or increased the open probability of the receptors, respectively. Further disulfide cross-linking and covalent modification confirmed that the intersubunit physical couplings among the DF and two lower body domains fostered by the LF domain at the open state act as an integrated structural element that is stringently required for the channel gating of P2X4 receptors. Our observations provide new mechanistic insights into P2X receptor activation and will stimulate development of new allosteric modulators of P2X receptors.

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