Regulation of Shaker K+ channel inactivation gating by the cAMP-dependent protein kinase

doi:10.1016/0896-6273(94)90317-4

Neuron

Volume 12, Issue 5, May 1994, Pages 1097-1109

https://doi.org/10.1016/0896-6273(94)90317-4 Get rights and content

Abstract

In response to depolarization of the membrane potential, Shaker K⁺ channels undergo a series of voltage-dependent conformational changes, from resting to open conformations followed by a rapid transition into a long-lived closed conformation, the N-type inactivated state. Application of phosphatases to the cytoplasmic side of Shaker channels in excised inside-out patches slows N-type inactivation gating. Subsequent application of the purified catalytic subunit of the cAMP-dependent protein kinase (PKA) and ATP reverses the effect, accelerating N-type inactivation back to its initial rapid rate. Macroscopic and single-channel experiments indicate that N-type inactivation is selectively modulated. There was little or no effect on the voltage dependence and kinetics of activation. Comparison of site-directed mutant channels shows that a C-terminal consensus site for PKA phosphorylation is responsible for the modulation. Since a cell's integrative characteristics can be determined by the rate of inactivation of its voltage-dependent channels, modulation of these rates by phosphorylation is likely to have functional consequences.

References (68)

G.A. Gonzalez et al.
Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133
Cell
(1989)
T. Hoshi et al.
Two types of inactivation in Shaker K⁺ channels: effects of alterations in the carboxy-terminal region
Neuron
(1991)
R.L. Huganir et al.
Regulation of neurotransmitter receptor desensitization by protein phosphorylation
Neuron
(1990)
M. Li et al.
Functional modulation of brain sodium channels by cAMP-dependent phosphorylation
Neuron
(1992)
K.J. Loechner et al.
Control of potassium currents and cyclic AMP levels by autoactive neuropeptides in Aplysia neurons
Brain Res.
(1990)
O. Moran et al.
Modulation of a Shaker potassiu m A-channel by protein kinase C activation
FEBS Lett.
(1991)
R.B. Pearson et al.
Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations
Meth. Enzymol.
(1991)
E. Perozo et al.
Phosphorylation affects voltage gating of the delayed rectifier K⁺ channel by electrostatic interactions
Neuron
(1990)
M. Sokabe et al.
Quantitative video microscopy of patch clamped membranes stress, strain, capacitance, and stretch channel activation
Biophys. J.
(1991)
L.C. Timpe et al.
Four cDNA clones from the Shaker locus induce kinetically distinct A-type potassium currents in Xenopus oocytes
Neuron
(1988)

C. Ukomadu et al.

μl Na⁺ channels expressed transiently in human embryonic kidney cells: biochemical and biophysical properties

Neuron

(1992)

B. Zhang et al.

The regulatory role of known tyrosine autophosphorylation sites of the insulin receptor kinase domain

J. Biol. Chem.

(1991)

J.Y. Zhou et al.

Multiple gating modes and the effect of modulating factors on the 01 sodium channel

Neuron

(1991)

R.W. Aldrich et al.

Mechanism of frequency-dependent broadening of molluscan neurone soma spikes

J. Physiol.

(1979)

R.W. Aldrich et al.

A reinterpretation of mammalian sodium channel gating based on single channel recording

Nature

(1983)

R.W. Aldrich et al.

Differences in gating among amino-terminal variants of Shaker potassium channels

Cold Spring Harbor Symp. Quant. Biol.

(1990)

C.K. Augustine et al.

Phosphorylation modulates potassium conductance and gating current of perfused giant axons of squid

J. Gen. Physiol.

(1990)

J.A. Benson et al.

Serotonin increases an anomalously rectifying K⁺ current in the Aplysia neuron R15

F. Bezanilla et al.

Inactivation of the sodium channel. I. Sodium current experiments

J. Gen. Physiol.

(1977)

J.S. Camardo et al.

Modulation of a specific potassium channel in sensory neurons of Aplysia by serotonin and cAMP-dependent protein phosphorylation

S.K. Chung et al.

Protein kinase activity closely associated with a reconstituted calcium-activated potassium channel

Science

(1991)

J.E. Coleman

Structure and mechanism of alkaline phosphatase

Annu. Rev. Biophys. Biomol. Struct.

(1992)

W.J. Conover

Practical Nonparametric Statistics

D. Ewald et al.

Modulation of single Ca²⁺-dependent K⁺ channel activity by protein phosphorylation

Nature

(1985)

B.A. Goldsmith et al.

cAMP modulates multiple K* currents, increasing spike duration and excitability in Aplysia sensory neurons

P. Greengard et al.

Enhancement of the glutamate response by cAMP-dependent protein kinase in hippocampal neurons

Science

(1991)

O.P. Hamill et al.

Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches

Pflügers Arch.

(1981)

B. Hochner et al.

Modulation of a transient K⁺ current in the pleural sensory neurons of Aplysia by serotoni n and cAMP: implications for spike broadening

T. Hoshi et al.

Biophysical and molecular mechanisms of Shaker potassium channel inactivation

Science

(1990)

R.L. Huganir et al.

cAMP-dependent protein kinase phosphorylates the nicotinic acetylcholine receptor from Torpedo calitornica

R.L. Huganir et al.

Phosphorylation of the nicotinic acetylcholine receptor regulates its rate of desensitization

Nature

(1986)

E. Isacoff et al.

Putative receptor for the cytoplasmic inactivation gate in the Shaker K⁺ channel

Nature

(1991)

L.K. Kaczmarek et al.

Neuromodulation: The Biochemical Control of Neuronal Excitability

(1987)

L.K. Kaczmarek et al.

A voltage clamp analysis of currents underlying cAMP-induced membrane modulation in isolated peptidergic neurons of Aplysia

J. Neurophysiol.

(1984)

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Neuron

Regulation of Shaker K+ channel inactivation gating by the cAMP-dependent protein kinase

Abstract

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Brain Res.

FEBS Lett.

Meth. Enzymol.

Neuron

Biophys. J.

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J. Biol. Chem.

Neuron

Mechanism of frequency-dependent broadening of molluscan neurone soma spikes

J. Physiol.

A reinterpretation of mammalian sodium channel gating based on single channel recording

Nature

Differences in gating among amino-terminal variants of Shaker potassium channels

Cold Spring Harbor Symp. Quant. Biol.

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J. Gen. Physiol.

Serotonin increases an anomalously rectifying K+ current in the Aplysia neuron R15

Inactivation of the sodium channel. I. Sodium current experiments

J. Gen. Physiol.

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Protein kinase activity closely associated with a reconstituted calcium-activated potassium channel

Science

Structure and mechanism of alkaline phosphatase

Annu. Rev. Biophys. Biomol. Struct.

Practical Nonparametric Statistics

Modulation of single Ca2+-dependent K+ channel activity by protein phosphorylation

Nature

cAMP modulates multiple K* currents, increasing spike duration and excitability in Aplysia sensory neurons

Enhancement of the glutamate response by cAMP-dependent protein kinase in hippocampal neurons

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Pflügers Arch.

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Biophysical and molecular mechanisms of Shaker potassium channel inactivation

Science

cAMP-dependent protein kinase phosphorylates the nicotinic acetylcholine receptor from Torpedo calitornica

Phosphorylation of the nicotinic acetylcholine receptor regulates its rate of desensitization

Nature

Putative receptor for the cytoplasmic inactivation gate in the Shaker K+ channel

Nature

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A voltage clamp analysis of currents underlying cAMP-induced membrane modulation in isolated peptidergic neurons of Aplysia

J. Neurophysiol.

Regulation of Shaker K⁺ channel inactivation gating by the cAMP-dependent protein kinase

Serotonin increases an anomalously rectifying K⁺ current in the Aplysia neuron R15

Modulation of single Ca²⁺-dependent K⁺ channel activity by protein phosphorylation

Modulation of a transient K⁺ current in the pleural sensory neurons of Aplysia by serotoni n and cAMP: implications for spike broadening

Putative receptor for the cytoplasmic inactivation gate in the Shaker K⁺ channel