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Identification by mass spectrometry and functional characterization of two phosphorylation sites of KCNQ2/KCNQ3 channels.

ABSTRACT: Neuronal potassium channel subunits of the KCNQ (Kv7) family underlie M-current (I(M)), and may also underlie the slow potassium current at the node of Ranvier, I(Ks). I(M) and I(Ks) are outwardly rectifying currents that regulate excitability of neurons and myelinated axons, respectively. Studies of native I(M) and heterologously expressed Kv7 subunits suggest that, in vivo, KCNQ channels exist within heterogeneous, multicomponent protein complexes. KCNQ channel properties are regulated by protein phosphorylation, protein-protein interactions, and protein-lipid interactions within such complexes. To better understand the regulation of neuronal KCNQ channels, we searched directly for posttranslational modifications on KCNQ2/KCNQ3 channels in vivo by using mass spectrometry. Here we describe two sites of phosphorylation. One site, specific for KCNQ3, appears functionally silent in electrophysiological assays but is located in a domain previously shown to be important for subunit tetramerization. Mutagenesis and electrophysiological studies of the second site, located in the S4-S5 intracellular loop of all KCNQ subunits, reveal a mechanism of channel inhibition.

SUBMITTER: Surti TS

PROVIDER: S-EPMC1297712 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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Identification by mass spectrometry and functional characterization of two phosphorylation sites of KCNQ2/KCNQ3 channels.

Surti Toral S TS Huang Lan L Jan Yuh Nung YN Jan Lily Y LY Cooper Edward C EC

Proceedings of the National Academy of Sciences of the United States of America 20051130 49

Neuronal potassium channel subunits of the KCNQ (Kv7) family underlie M-current (I(M)), and may also underlie the slow potassium current at the node of Ranvier, I(Ks). I(M) and I(Ks) are outwardly rectifying currents that regulate excitability of neurons and myelinated axons, respectively. Studies of native I(M) and heterologously expressed Kv7 subunits suggest that, in vivo, KCNQ channels exist within heterogeneous, multicomponent protein complexes. KCNQ channel properties are regulated by prot ...[more]

PMID: 16319223

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Project description:Changes in extracellular pH occur during both physiological neuronal activity and pathological conditions such as epilepsy and stroke. Such pH changes are known to exert profound effects on neuronal activity and survival. Heteromeric KCNQ2/3 potassium channels constitute a potential target for modulation by H+ ions as they are expressed widely within the CNS and have been proposed to underlie the M-current, an important determinant of excitability in neuronal cells. Whole-cell and single-channel recordings demonstrated a modulation of heterologously expressed KCNQ2/3 channels by extracellular H+ ions. KCNQ2/3 current was inhibited by H+ ions with an IC50 of 52 nM (pH 7.3) at -60 mV, rising to 2 microM (pH 5.7) at -10 mV. Neuronal M-current exhibited a similar sensitivity. Extracellular H+ ions affected two distinct properties of KCNQ2/3 current: the maximum current attainable upon depolarization (Imax) and the voltage dependence of steady-state activation. Reduction of Imax was antagonized by extracellular K+ ions and affected by mutations within the outer-pore turret, indicating an outer-pore based process. This reduction of Imax was shown to be due primarily to a decrease in the maximum open-probability of single KCNQ2/3 channels. Single-channel open times were shortened by acidosis (pH 5.9), while closed times were increased. Acidosis also recruited a longer-lasting closed state, and caused a switch of single-channel activity from the full-conductance state ( approximately 8 pS) to a subconductance state ( approximately 5 pS). A depolarizing shift in the activation curve of macroscopic KCNQ2/3 currents and single KCNQ2/3 channels was caused by acidosis, while alkalosis caused a hyperpolarizing shift. Activation and deactivation kinetics were slowed by acidosis, indicating specific effects of H+ ions on elements involved in gating. Contrasting modulation of homomeric KCNQ2 and KCNQ3 currents revealed that high sensitivity to H+ ions was conferred by the KCNQ3 subunit.

Dataset Information

Identification by mass spectrometry and functional characterization of two phosphorylation sites of KCNQ2/KCNQ3 channels.

Publications

Identification by mass spectrometry and functional characterization of two phosphorylation sites of KCNQ2/KCNQ3 channels.

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