Reconstitution of muscarinic modulation of the KCNQ2/KCNQ3 K+ channels that underlie the neuronal M current

Mark S Shapiro, John P. Roche, Edward J. Kaftan, Humberto Cruzblanca, Ken Mackie, Bertil Hille

Research output: Contribution to journalArticle

156 Citations (Scopus)

Abstract

Channels from KCNQ2 and KCNQ3 genes have been suggested to underlie the neuronal M-type K+ current. The M current is modulated by muscarinic agonists via G-proteins and an unidentified diffusible cytoplasmic messenger. Using whole-cell clamp, we studied tsA-201 cells in which cloned KCNQ2/KCNQ3 channels were coexpressed with M1 muscarinic receptors. Heteromeric KCNQ2/KCNQ3 currents were modulated by the muscarinic agonist oxotremorine-M (oxo-M) in a manner having all of the characteristics of modulation of native M current in sympathetic neurons. Oxo-M also produced obvious intracellular Ca2+ transients, observed by using indo-1 fluorescence. However, modulation of the current remained strong even when Ca2+ signals were abolished by the combined use of strong intracellular Ca2+ buffers, an inhibitor of IP3 receptors, and thapsigargin to deplete Ca2+ stores. Muscarinic modulation was not blocked by staurosporine, a broad-spectrum protein kinase inhibitor, arguing against involvement of protein kinases. The modulation was not associated with a shift in the voltage dependence of channel activation. Homomeric KCNQ2 and KCNQ3 channels also expressed well and were modulated individually by oxo-M, suggesting that the motifs for modulation are present on both channel subtypes. Homomeric KCNQ2 and KCNQ3 currents were blocked, respectively, at very low and at high concentrations of tetraethylammonium ion. Finally, when KCNQ2 subunits were overexpressed by intranuclear DNA injection in sympathetic neurons, total M current was fully modulated by the endogenous neuronal muscarinic signaling mechanism. Our data further rule out Ca2+ as the diffusible messenger. The reconstitution of muscarinic modulation of the M current that uses cloned components should facilitate the elucidation of the muscarinic signaling mechanism.

Original languageEnglish (US)
Pages (from-to)1710-1721
Number of pages12
JournalJournal of Neuroscience
Volume20
Issue number5
StatePublished - Mar 1 2000
Externally publishedYes

Fingerprint

Cholinergic Agents
Muscarinic Agonists
Muscarinic M1 Receptors
Inositol 1,4,5-Trisphosphate Receptors
Neurons
Tetraethylammonium
Thapsigargin
Staurosporine
Protein Kinase Inhibitors
GTP-Binding Proteins
Protein Kinases
Buffers
Fluorescence
Injections
DNA
Genes
oxotremorine M

Keywords

  • Calcium
  • G-protein
  • K channel
  • M current
  • Muscarinic receptor
  • Patch clamp

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Shapiro, M. S., Roche, J. P., Kaftan, E. J., Cruzblanca, H., Mackie, K., & Hille, B. (2000). Reconstitution of muscarinic modulation of the KCNQ2/KCNQ3 K+ channels that underlie the neuronal M current. Journal of Neuroscience, 20(5), 1710-1721.

Reconstitution of muscarinic modulation of the KCNQ2/KCNQ3 K+ channels that underlie the neuronal M current. / Shapiro, Mark S; Roche, John P.; Kaftan, Edward J.; Cruzblanca, Humberto; Mackie, Ken; Hille, Bertil.

In: Journal of Neuroscience, Vol. 20, No. 5, 01.03.2000, p. 1710-1721.

Research output: Contribution to journalArticle

Shapiro, MS, Roche, JP, Kaftan, EJ, Cruzblanca, H, Mackie, K & Hille, B 2000, 'Reconstitution of muscarinic modulation of the KCNQ2/KCNQ3 K+ channels that underlie the neuronal M current', Journal of Neuroscience, vol. 20, no. 5, pp. 1710-1721.
Shapiro, Mark S ; Roche, John P. ; Kaftan, Edward J. ; Cruzblanca, Humberto ; Mackie, Ken ; Hille, Bertil. / Reconstitution of muscarinic modulation of the KCNQ2/KCNQ3 K+ channels that underlie the neuronal M current. In: Journal of Neuroscience. 2000 ; Vol. 20, No. 5. pp. 1710-1721.
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abstract = "Channels from KCNQ2 and KCNQ3 genes have been suggested to underlie the neuronal M-type K+ current. The M current is modulated by muscarinic agonists via G-proteins and an unidentified diffusible cytoplasmic messenger. Using whole-cell clamp, we studied tsA-201 cells in which cloned KCNQ2/KCNQ3 channels were coexpressed with M1 muscarinic receptors. Heteromeric KCNQ2/KCNQ3 currents were modulated by the muscarinic agonist oxotremorine-M (oxo-M) in a manner having all of the characteristics of modulation of native M current in sympathetic neurons. Oxo-M also produced obvious intracellular Ca2+ transients, observed by using indo-1 fluorescence. However, modulation of the current remained strong even when Ca2+ signals were abolished by the combined use of strong intracellular Ca2+ buffers, an inhibitor of IP3 receptors, and thapsigargin to deplete Ca2+ stores. Muscarinic modulation was not blocked by staurosporine, a broad-spectrum protein kinase inhibitor, arguing against involvement of protein kinases. The modulation was not associated with a shift in the voltage dependence of channel activation. Homomeric KCNQ2 and KCNQ3 channels also expressed well and were modulated individually by oxo-M, suggesting that the motifs for modulation are present on both channel subtypes. Homomeric KCNQ2 and KCNQ3 currents were blocked, respectively, at very low and at high concentrations of tetraethylammonium ion. Finally, when KCNQ2 subunits were overexpressed by intranuclear DNA injection in sympathetic neurons, total M current was fully modulated by the endogenous neuronal muscarinic signaling mechanism. Our data further rule out Ca2+ as the diffusible messenger. The reconstitution of muscarinic modulation of the M current that uses cloned components should facilitate the elucidation of the muscarinic signaling mechanism.",
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AU - Cruzblanca, Humberto

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N2 - Channels from KCNQ2 and KCNQ3 genes have been suggested to underlie the neuronal M-type K+ current. The M current is modulated by muscarinic agonists via G-proteins and an unidentified diffusible cytoplasmic messenger. Using whole-cell clamp, we studied tsA-201 cells in which cloned KCNQ2/KCNQ3 channels were coexpressed with M1 muscarinic receptors. Heteromeric KCNQ2/KCNQ3 currents were modulated by the muscarinic agonist oxotremorine-M (oxo-M) in a manner having all of the characteristics of modulation of native M current in sympathetic neurons. Oxo-M also produced obvious intracellular Ca2+ transients, observed by using indo-1 fluorescence. However, modulation of the current remained strong even when Ca2+ signals were abolished by the combined use of strong intracellular Ca2+ buffers, an inhibitor of IP3 receptors, and thapsigargin to deplete Ca2+ stores. Muscarinic modulation was not blocked by staurosporine, a broad-spectrum protein kinase inhibitor, arguing against involvement of protein kinases. The modulation was not associated with a shift in the voltage dependence of channel activation. Homomeric KCNQ2 and KCNQ3 channels also expressed well and were modulated individually by oxo-M, suggesting that the motifs for modulation are present on both channel subtypes. Homomeric KCNQ2 and KCNQ3 currents were blocked, respectively, at very low and at high concentrations of tetraethylammonium ion. Finally, when KCNQ2 subunits were overexpressed by intranuclear DNA injection in sympathetic neurons, total M current was fully modulated by the endogenous neuronal muscarinic signaling mechanism. Our data further rule out Ca2+ as the diffusible messenger. The reconstitution of muscarinic modulation of the M current that uses cloned components should facilitate the elucidation of the muscarinic signaling mechanism.

AB - Channels from KCNQ2 and KCNQ3 genes have been suggested to underlie the neuronal M-type K+ current. The M current is modulated by muscarinic agonists via G-proteins and an unidentified diffusible cytoplasmic messenger. Using whole-cell clamp, we studied tsA-201 cells in which cloned KCNQ2/KCNQ3 channels were coexpressed with M1 muscarinic receptors. Heteromeric KCNQ2/KCNQ3 currents were modulated by the muscarinic agonist oxotremorine-M (oxo-M) in a manner having all of the characteristics of modulation of native M current in sympathetic neurons. Oxo-M also produced obvious intracellular Ca2+ transients, observed by using indo-1 fluorescence. However, modulation of the current remained strong even when Ca2+ signals were abolished by the combined use of strong intracellular Ca2+ buffers, an inhibitor of IP3 receptors, and thapsigargin to deplete Ca2+ stores. Muscarinic modulation was not blocked by staurosporine, a broad-spectrum protein kinase inhibitor, arguing against involvement of protein kinases. The modulation was not associated with a shift in the voltage dependence of channel activation. Homomeric KCNQ2 and KCNQ3 channels also expressed well and were modulated individually by oxo-M, suggesting that the motifs for modulation are present on both channel subtypes. Homomeric KCNQ2 and KCNQ3 currents were blocked, respectively, at very low and at high concentrations of tetraethylammonium ion. Finally, when KCNQ2 subunits were overexpressed by intranuclear DNA injection in sympathetic neurons, total M current was fully modulated by the endogenous neuronal muscarinic signaling mechanism. Our data further rule out Ca2+ as the diffusible messenger. The reconstitution of muscarinic modulation of the M current that uses cloned components should facilitate the elucidation of the muscarinic signaling mechanism.

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