Gating of Cyclic Nucleotide Gated Channels is also Voltage Dependent

Cyclic nucleotide-gated channels (cng channels) in the sensory membrane of olfactory receptor cells, activated after the odorant-induced increase of cytosolic cAMP concentration, conduct the receptor current that elicits electrical excitation of the receptor neurons. We investigated properties of cng channels from frog and rat using inside-out and outside-out membrane patches excised from isolated olfactory receptor cells. Channels were activated by cAMP and cGMP with activation constants of 2.5-4.0 microM for cAMP and 1.0-1.8 for cGMP. Hill coefficients of dose-response curves were 1.4-1.8, indicating cooperativity of ligand binding. Selectivity for monovalent alkali cations and the Na/Li mole-fraction behavior identified the channel as a nonselective cation channel, having a cation-binding site of high field strength in the pore. Cytosolic pH effects suggest the presence of an additional titratable group which, when protonated, inhibits the cAMP-induced current with an apparent pK of 5.0-5.2. The pH effects were not voltage dependent. Several blockers of Ca2+ channels also blocked olfactory cng channels. Amiloride, D 600, and diltiazem inhibited the cAMP-induced current from the cytosolic side. Inhibition constants were voltage dependent with values of, respectively, 0.1, 0.3, and 1 mM at -60 mV, and 0.03, 0.02, and 0.2 mM at +60 mV. Our results suggest functional similarity between frog and rat cng channels, as well as marked differences to cng channels from photoreceptors and other tissues.

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Mechanism of Tetracaine Block of Cyclic Nucleotide-gated Channels

The Journal of General Physiology ◽

10.1085/jgp.109.1.3 ◽

1997 ◽

Vol 109 (1) ◽

pp. 3-14 ◽

Cited By ~ 52

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.

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

The Journal of General Physiology ◽

10.1085/jgp.109.3.301 ◽

1997 ◽

Vol 109 (3) ◽

pp. 301-311 ◽

Cited By ~ 45

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.

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Gating of cyclic nucleotide-gated channels is voltage dependent

Nature Communications ◽

10.1038/ncomms1972 ◽

2012 ◽

Vol 3 (1) ◽

Cited By ~ 13

Author(s):

Arin Marchesi ◽

Monica Mazzolini ◽

Vincent Torre

Keyword(s):

Cyclic Nucleotide ◽

Cyclic Nucleotide Gated Channels ◽

Voltage Dependent

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Hyperpolarization-activated cyclic-nucleotide-gated channels: pathophysiological, developmental, and pharmacological insights into their function in cellular excitability

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2018-0115 ◽

2018 ◽

Vol 96 (10) ◽

pp. 977-984 ◽

Cited By ~ 12

Author(s):

Valentina Spinelli ◽

Laura Sartiani ◽

Alessandro Mugelli ◽

Maria Novella Romanelli ◽

Elisabetta Cerbai

Keyword(s):

Cyclic Nucleotide ◽

Cardiac Cells ◽

Pacemaker Activity ◽

Loss Of Function ◽

Cyclic Nucleotide Gated Channels ◽

Mediated Effects ◽

Cellular Excitability ◽

Voltage Dependent ◽

New Compounds

The hyperpolarization-activated cyclic-nucleotide-gated (HCN) proteins are voltage-dependent ion channels, conducting both Na+ and K+, blocked by millimolar concentrations of extracellular Cs+ and modulated by cyclic nucleotides (mainly cAMP) that contribute crucially to the pacemaker activity in cardiac nodal cells and subsidiary pacemakers. Over the last decades, much attention has focused on HCN current, If, in non-pacemaker cardiac cells and its potential role in triggering arrhythmias. In fact, in addition to pacemakers, HCN current is constitutively present in the human atria and has long been proposed to sustain atrial arrhythmias associated to different cardiac pathologies or triggered by various modulatory signals (catecholamines, serotonin, natriuretic peptides). An atypical If occurs in diseased ventricular cardiomyocytes, its amplitude being linearly related to the severity of cardiac hypertrophy. The properties of atrial and ventricular If and its modulation by pharmacological interventions has been object of intense study, including the synthesis and characterization of new compounds able to block preferentially HCN1, HCN2, or HCN4 isoforms. Altogether, clues emerge for opportunities of future pharmacological strategies exploiting the unique properties of this channel family: the prevalence of different HCN subtypes in organs and tissues, the possibility to target HCN gain- or loss-of-function associated with disease, the feasibility of novel isoform-selective drugs, as well as the discovery of HCN-mediated effects for old medicines.

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

eLife ◽

10.7554/elife.49672 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 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.

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