scholarly journals Opening Mechanism of a Cyclic Nucleotide–gated Channel Based on Analysis of Single Channels Locked in Each Liganded State

1999 ◽  
Vol 113 (6) ◽  
pp. 873-895 ◽  
Author(s):  
MariaLuisa Ruiz ◽  
Jeffrey W. Karpen
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Cyclic Nucleotide ◽  
Single Channel ◽  
Kinetic Mechanism ◽  
Fixed Number ◽  
Single Channels ◽  
Cyclic Nucleotide Gated Channels ◽  
Gated Channel ◽  
Conductance States

Cyclic nucleotide–gated channels contain four subunits, each with a binding site for cGMP or cAMP in the cytoplasmic COOH-terminal domain. Previous studies of the kinetic mechanism of activation have been hampered by the complication that ligands are continuously binding and unbinding at each of these sites. Thus, even at the single channel level, it has been difficult to distinguish changes in behavior that arise from a channel with a fixed number of ligands bound from those that occur upon the binding and unbinding of ligands. For example, it is often assumed that complex behaviors like multiple conductance levels and bursting occur only as a consequence of changes in the number of bound ligands. We have overcome these ambiguities by covalently tethering one ligand at a time to single rod cyclic nucleotide–gated channels (Ruiz, ML., and J.W. Karpen. 1997. Nature. 389:389–392). We find that with a fixed number of ligands locked in place the channel freely moves between three conductance states and undergoes bursting behavior. Furthermore, a thorough kinetic analysis of channels locked in doubly, triply, and fully liganded states reveals more than one kinetically distinguishable state at each conductance level. Thus, even when the channel contains a fixed number of bound ligands, it can assume at least nine distinct states. Such complex behavior is inconsistent with simple concerted or sequential allosteric models. The data at each level of liganding can be successfully described by the same connected state model (with different rate constants), suggesting that the channel undergoes the same set of conformational changes regardless of the number of bound ligands. A general allosteric model, which postulates one conformational change per subunit in both the absence and presence of ligand, comes close to providing enough kinetically distinct states. We propose an extension of this model, in which more than one conformational change per subunit can occur during the process of channel activation.

Divalent cations block the cyclic nucleotide-gated channel of olfactory receptor neurons

1993 ◽  
Vol 69 (5) ◽  
pp. 1758-1768 ◽  
Author(s):  
F. Zufall ◽  
S. Firestein
Keyword(s):  
Cyclic Amp ◽  
Calcium Channels ◽  
Cyclic Nucleotide ◽  
Olfactory Receptor ◽  
Single Channel ◽  
Divalent Cations ◽  
Olfactory Receptor Neurons ◽  
Gated Channel ◽  
Receptor Neurons ◽  
Single Channel Currents

1. The effects of external divalent cations on odor-dependent, cyclic AMP-activated single-channel currents from olfactory receptor neurons of the tiger salamander (Ambystoma tigrinum) were studied in inside-out membrane patches taken from dendritic regions of freshly isolated sensory cells. 2. Channels were reversibly activated by 100 microM cyclic AMP. In the absence of divalent cations, the channel had a linear current-voltage relation giving a conductance of 45 pS. With increasing concentrations of either Ca2+ or Mg2+ in the external solution, the channel displayed a rapid flickering behavior. At higher concentrations of divalent cations, the transitions were too rapid to be fully resolved and appeared as a reduction in mean unitary single-channel current amplitude. 3. This effect was voltage dependent, and on analysis was shown to be due to an open channel block by divalent ions. In the case of Mg2+, the block increased steadily with hyperpolarization. In contrast, for Ca2+ the block first increased with hyperpolarization and then decreased with further hyperpolarization beyond -70 mV, providing evidence for Ca2+ permeation of this channel. 4. This block is similar to that seen in voltage-gated calcium channels. Additionally, the cyclic nucleotide-gated channel shows some pharmacological similarities with L-type calcium channels, including a novel block of the cyclic nucleotide channel by nifedipine (50 microM). 5. Our results indicate that the sensory generator current simultaneously depends on the presence of the second messenger and on the membrane potential of the olfactory neuron.

Expression of a single gene produces both forms of skeletal muscle cyclic nucleotide-gated channels

1997 ◽  
Vol 273 (6) ◽  
pp. E1140-E1148
Author(s):  
Lorraine C. Santy ◽  
Guido Guidotti
Keyword(s):  
Skeletal Muscle ◽  
Cyclic Nucleotide ◽  
Single Gene ◽  
Rabbit Aorta ◽  
Rabbit Skeletal Muscle ◽  
Channel Activity ◽  
Cyclic Nucleotide Gated Channels ◽  
Gated Channel ◽  
Polymerase Chain ◽  
Two Populations

Cyclic nucleotide-gated cation channels in skeletal muscle are responsible for insulin-activated sodium entry into this tissue (J. E. M. McGeoch and G. Guidotti. J. Biol. Chem. 267: 832–841, 1992). These channels have previously been isolated from rabbit skeletal muscle by 8-bromoguanosine 3′,5′-cyclic monophosphate (8-BrcGMP) affinity chromatography, which separates them into two populations differing in nucleotide affinity [L. C. Santy and G. Guidotti. Am. J. Physiol. 271 ( Endocrinol. Metab. 34): E1051-E1060, 1996]. In this study, a polymerase chain reaction approach was used to identify skeletal muscle cyclic nucleotide-gated channel cDNAs. Rabbit skeletal muscle expresses the same cyclic nucleotide-gated channel as rabbit aorta (M. Biel, W. Altenhofen, R. Hullin, J. Ludwig, M. Freichel, V. Flockerzi, N. Dascal, U. B. Kaupp, and F. Hofmann. FEBS Lett. 329: 134–138, 1993). The entire cDNA for this gene was cloned from rabbit skeletal muscle and an antiserum to this protein produced. Expression of this cDNA produces a 63-kDa protein with cyclic nucleotide-gated channel activity. A similarly sized immunoreactive protein is present in sarcolemma. Purification of the expressed channels reveals that this single gene produces both native skeletal muscle channel populations.

scholarly journals Access of Quaternary Ammonium Blockers to the Internal Pore of Cyclic Nucleotide-gated Channels: Implications for the Location of the Gate

2006 ◽  
Vol 127 (5) ◽  
pp. 481-494 ◽  
Author(s):  
Jorge E. Contreras ◽  
Miguel Holmgren
Keyword(s):  
Binding Site ◽  
Quaternary Ammonium ◽  
Cyclic Nucleotide ◽  
Single Channel ◽  
Dependent Manner ◽  
Receptor Binding Site ◽  
Α Subunit ◽  
Cyclic Nucleotide Gated Channels ◽  
Closed State ◽  
Kv Channels

Cyclic nucleotide-gated (CNG) channels play important roles in the transduction of visual and olfactory information by sensing changes in the intracellular concentration of cyclic nucleotides. We have investigated the interactions between intracellularly applied quaternary ammonium (QA) ions and the α subunit of rod cyclic nucleotide-gated channels. We have used a family of alkyl-triethylammonium derivatives in which the length of one chain is altered. These QA derivatives blocked the permeation pathway of CNG channels in a concentration- and voltage-dependent manner. For QA compounds with tails longer than six methylene groups, increasing the length of the chain resulted in higher apparent affinities of ∼1.2 RT per methylene group added, which is consistent with the presence of a hydrophobic pocket within the intracellular mouth of the channel that serves as part of the receptor binding site. At the single channel level, decyltriethyl ammonium (C10-TEA) ions did not change the unitary conductance but they did reduce the apparent mean open time, suggesting that the blocker binds to open channels. We provide four lines of evidence suggesting that QA ions can also bind to closed channels: (1) the extent of C10-TEA blockade at subsaturating [cGMP] was larger than at saturating agonist concentration, (2) under saturating concentrations of cGMP, cIMP, or cAMP, blockade levels were inversely correlated with the maximal probability of opening achieved by each agonist, (3) in the closed state, MTS reagents of comparable sizes to QA ions were able to modify V391C in the inner vestibule of the channel, and (4) in the closed state, C10-TEA was able to slow the Cd2+ inhibition observed in V391C channels. These results are in stark contrast to the well-established QA blockade mechanism in Kv channels, where these compounds can only access the inner vestibule in the open state because the gate that opens and closes the channel is located cytoplasmically with respect to the binding site of QA ions. Therefore, in the context of Kv channels, our observations suggest that the regions involved in opening and closing the permeation pathways in these two types of channels are different.

Molecular cloning and single-channel properties of the cyclic nucleotide-gated channel from catfish olfactory neurons

Neuron ◽  
1992 ◽  
Vol 8 (1) ◽  
pp. 45-58 ◽  
Author(s):  
Evan H. Goulding ◽  
John Ngai ◽  
Richard H. Kramer ◽  
Suzanne Colicos ◽  
Richard Axel ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
Cyclic Nucleotide ◽  
Single Channel ◽  
Olfactory Neurons ◽  
Gated Channel ◽  
Channel Properties

scholarly journals Ligand binding and activation properties of the purified bacterial cyclic nucleotide–gated channel SthK

2018 ◽  
Vol 150 (6) ◽  
pp. 821-834 ◽  
Author(s):  
Philipp A.M. Schmidpeter ◽  
Xiaolong Gao ◽  
Vikrant Uphadyay ◽  
Jan Rheinberger ◽  
Crina M. Nimigean
Keyword(s):  
Lipid Bilayers ◽  
Conformational Changes ◽  
Cyclic Nucleotide ◽  
Single Channel ◽  
Sequence Similarity ◽  
Guanosine Monophosphate ◽  
Particle Analysis ◽  
Xenopus Laevis Oocytes ◽  
High Sequence Similarity ◽  
Protein Expression And Purification

Cyclic nucleotide–modulated ion channels play several essential physiological roles. They are involved in signal transduction in photoreceptors and olfactory sensory neurons as well as pacemaking activity in the heart and brain. Investigations of the molecular mechanism of their actions, including structural and electrophysiological characterization, are restricted by the availability of stable, purified protein obtained from accessible systems. Here, we establish that SthK, a cyclic nucleotide–gated (CNG) channel from Spirochaeta thermophila, is an excellent model for investigating the gating of eukaryotic CNG channels at the molecular level. The channel has high sequence similarity with its eukaryotic counterparts and was previously reported to be activated by cyclic nucleotides in patch-clamp experiments with Xenopus laevis oocytes. We optimized protein expression and purification to obtain large quantities of pure, homogeneous, and active recombinant SthK protein from Escherichia coli. A negative-stain electron microscopy (EM) single-particle analysis indicated that this channel is a promising candidate for structural studies with cryo-EM. Using radioactivity and fluorescence flux assays, as well as single-channel recordings in lipid bilayers, we show that the protein is partially activated by micromolar concentrations of cyclic adenosine monophosphate (cAMP) and that channel activity is increased by depolarization. Unlike previous studies, we find that cyclic guanosine monophosphate (cGMP) is also able to activate SthK, but with much lower efficiency than cAMP. The distinct sensitivities to different ligands resemble eukaryotic CNG and hyperpolarization-activated and cyclic nucleotide–modulated channels. Using a fluorescence binding assay, we show that cGMP and cAMP bind to SthK with similar apparent affinities, suggesting that the large difference in channel activation by cAMP or cGMP is caused by the efficacy with which each ligand promotes the conformational changes toward the open state. We conclude that the functional characteristics of SthK reported here will permit future studies to analyze ligand gating and discrimination in CNG channels.

scholarly journals Mechanism of Tetracaine Block of Cyclic Nucleotide-gated Channels

1997 ◽  
Vol 109 (1) ◽  
pp. 3-14 ◽  
Author(s):  
Anthony A. Fodor ◽  
Sharona E. Gordon ◽  
William N. Zagotta
Keyword(s):  
Conformational Changes ◽  
Cyclic Nucleotide ◽  
Channel Blockers ◽  
Response Curves ◽  
Apparent Affinity ◽  
Ion Channel Blockers ◽  
Cyclic Nucleotide Gated Channels ◽  
Closed State ◽  
State Dependent ◽  
Voltage Dependent

Local anesthetics are a diverse group of ion channel blockers that can be used to probe conformational changes in the pore. We examined the effects of the local anesthetic tetracaine on rod and olfactory cyclic nucleotide-gated channels expressed from subunit 1 in Xenopus oocytes. We found that 40 μM tetracaine effectively blocked the bovine rod channel but not the rat olfactory channel at saturating concentrations of cGMP. By testing chimeric channels containing regions of sequence from both rod and olfactory channels, we found that determinants of apparent affinity for tetracaine at saturating cGMP did not map to any one region of the channel sequence. Rather, the differences in apparent affinity could be explained by differences between the chimeras in the free energy of the opening allosteric transition. If a channel construct (such as the rod channel) spent appreciable time in the closed state at saturating cGMP, then it had a high apparent affinity for tetracaine. If, on the other hand, a channel construct (such as the olfactory channel) spent little time in the closed state at saturating cGMP, then it had a low apparent affinity for tetracaine. Furthermore, tetracaine became more effective at low concentrations of cGMP and at saturating concentrations of cAMP, conditions which permit the channels to spend more time in the closed configuration. These results were well fit by a model in which tetracaine binds more tightly to the closed channel than to the open channel. Dose-response curves for tetracaine in the presence of saturating cGMP are well fit with a Michaelis-Menten binding scheme Indicating that a single tetracaine molecule is sufficient to produce block. In addition, tetracaine block is voltage dependent with an effective zδ of +0.56. These data are consistent with a pore-block hypothesis. The finding that tetracaine is a state-dependent pore blocker suggests that the inner mouth of the pore of cyclic nucleotide-gated channels undergoes a conformational change during channel opening.

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.

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