Reconstitution and characterization of two forms of cyclic nucleotide-gated channels from skeletal muscle

A cyclic nucleotide-gated channel present in skeletal muscle plasma membrane has previously been identified as being responsible for insulin-activated sodium entry into muscle cells (J. E. M. McGeoch and G. Guidotti. J. Biol. Chem. 267:832-841, 1992). We have isolated this channel activity to further study and characterize it. The channel was solubilized from rabbit skeletal muscle sarcolemma and functionally reconstituted into phospholipid vesicles, as assayed by patch-clamp analysis of the reconstituted proteins. Channel activity was isolated by 8-bromo-guanosine 3',5'-cyclic monophosphate affinity chromatography, producing two distinct peaks of cyclic nucleotide-gated channel activity. These two types of channel activity differ in guanosine 3',5'-cyclic monophosphate affinity and in the ability to be opened by adenosine 3',5'-cyclic monophosphate. The cyclic nucleotide-gated channel from rod outer segments also forms two peaks of activity when purified in this manner. The presence of two forms of channel activity could have implications for the mechanism of insulin-activated sodium entry.

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

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1997.273.6.e1140 ◽

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.

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The Complex Story of Plant Cyclic Nucleotide-Gated Channels

International Journal of Molecular Sciences ◽

10.3390/ijms22020874 ◽

2021 ◽

Vol 22 (2) ◽

pp. 874

Author(s):

Edwin Jarratt-Barnham ◽

Limin Wang ◽

Youzheng Ning ◽

Julia M. Davies

Keyword(s):

Arabidopsis Thaliana ◽

Stress Response ◽

Complex Formation ◽

Cyclic Nucleotides ◽

Cyclic Nucleotide ◽

Cation Channels ◽

Cyclic Monophosphate ◽

Cyclic Nucleotide Gated Channels ◽

Gated Channel

Plant cyclic nucleotide-gated channels (CNGCs) are tetrameric cation channels which may be activated by the cyclic nucleotides (cNMPs) adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP). The genome of Arabidopsis thaliana encodes 20 CNGC subunits associated with aspects of development, stress response and immunity. Recently, it has been demonstrated that CNGC subunits form heterotetrameric complexes which behave differently from the homotetramers produced by their constituent subunits. These findings have widespread implications for future signalling research and may help explain how specificity can be achieved by CNGCs that are known to act in disparate pathways. Regulation of complex formation may involve cyclic nucleotide-gated channel-like proteins.

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Novel Role of KT5720 on Regulating Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel Activity and Dorsal Root Ganglion Neuron Excitability

DNA and Cell Biology ◽

10.1089/dna.2013.2021 ◽

2013 ◽

Vol 32 (6) ◽

pp. 320-328 ◽

Cited By ~ 8

Author(s):

Qiuping Cheng ◽

Yanhong Zhou

Keyword(s):

Dorsal Root Ganglion ◽

Dorsal Root Ganglion Neuron ◽

Cyclic Nucleotide ◽

Dorsal Root ◽

Ganglion Neuron ◽

Channel Activity ◽

Neuron Excitability ◽

Gated Channel

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Characterization of a novel cyclic nucleotide-gated channel from zebrafish brain

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2006.07.074 ◽

2006 ◽

Vol 348 (2) ◽

pp. 441-449 ◽

Cited By ~ 11

Author(s):

Michelle L. Tetreault ◽

Diane Henry ◽

Diana M. Horrigan ◽

Gary Matthews ◽

Anita L. Zimmerman

Keyword(s):

Cyclic Nucleotide ◽

Zebrafish Brain ◽

Gated Channel

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Endoplasmic reticulum (ER) Ca2+-channel activity contributes to ER stress and cone death in cyclic nucleotide-gated channel deficiency

Journal of Biological Chemistry ◽

10.1074/jbc.m117.782326 ◽

2017 ◽

Vol 292 (27) ◽

pp. 11189-11205 ◽

Cited By ~ 10

Author(s):

Michael R. Butler ◽

Hongwei Ma ◽

Fan Yang ◽

Joshua Belcher ◽

Yun-Zheng Le ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Cyclic Nucleotide ◽

Channel Activity ◽

Gated Channel

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

The Journal of General Physiology ◽

10.1085/jgp.113.6.873 ◽

1999 ◽

Vol 113 (6) ◽

pp. 873-895 ◽

Cited By ~ 32

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.

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