PKD Phosphorylation as Novel Pathway of KV11.1 Regulation

Annette Buur Steffensen; Sofia Hammami Bomholtz; Martin Nybo Andersen; Jesper Velgaard Olsen; N. Mutsaers; Pia Rengtved Lundegaard; Alicia Lundby; Nicole Schmitt

doi:10.1159/000491007

PKD Phosphorylation as Novel Pathway of K_V11.1 Regulation

Annette Buur Steffensen, Sofia Hammami Bomholtz, Martin Nybo Andersen, Jesper Velgaard Olsen, N. Mutsaers, Pia Rengtved Lundegaard, Alicia Lundby, Nicole Schmitt

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Abstract

BACKGROUND/AIMS: The voltage-gated potassium channel KV11.1 has been originally cloned from the brain and is expressed in a variety of tissues. The role of phosphorylation for channel function is a matter of debate. In this study, we aimed to elucidate the extent and role of protein kinase D mediated phosphorylation.

METHODS: We employed mass spectrometry, whole-cell patch clamp electrophysiology, confocal microscopy, site-directed mutagenesis, and western blotting.

RESULTS: Using brain tissue from rat and mouse, we mapped several phosphorylated KV11.1 residues by LC-MS mass spectrometry and identified protein kinase D (PKD1) as possible regulatory kinase. Co-expression of KV11.1 with PKD1 reduced current amplitudes without altering protein levels or surface expression of the channel. Based on LC-MS results from in vivo and HEK293 cell experiments we chose four KV11.1 mutant candidates for further functional analysis. Ablation of the putative PKD phosphorylation site in the mutant S284A increased the maximal current indicating S284 as a main PKD target in KV11.1.

CONCLUSIONS: Our data might help mitigating a long-standing controversy in the field regarding PKC regulation of KV11.1. We propose that PKD1 mediates the PKC effects on KV11.1 and we found that PKD targets S284 in the N-terminus of the channel.

Original language	English
Journal	Cellular Physiology and Biochemistry
Volume	47
Issue number	4
Pages (from-to)	1742-1750
Number of pages	9
ISSN	1015-8987
DOIs	https://doi.org/10.1159/000491007
Publication status	Published - 1 Jul 2018

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title = "PKD Phosphorylation as Novel Pathway of KV11.1 Regulation",

abstract = "BACKGROUND/AIMS: The voltage-gated potassium channel KV11.1 has been originally cloned from the brain and is expressed in a variety of tissues. The role of phosphorylation for channel function is a matter of debate. In this study, we aimed to elucidate the extent and role of protein kinase D mediated phosphorylation.METHODS: We employed mass spectrometry, whole-cell patch clamp electrophysiology, confocal microscopy, site-directed mutagenesis, and western blotting.RESULTS: Using brain tissue from rat and mouse, we mapped several phosphorylated KV11.1 residues by LC-MS mass spectrometry and identified protein kinase D (PKD1) as possible regulatory kinase. Co-expression of KV11.1 with PKD1 reduced current amplitudes without altering protein levels or surface expression of the channel. Based on LC-MS results from in vivo and HEK293 cell experiments we chose four KV11.1 mutant candidates for further functional analysis. Ablation of the putative PKD phosphorylation site in the mutant S284A increased the maximal current indicating S284 as a main PKD target in KV11.1.CONCLUSIONS: Our data might help mitigating a long-standing controversy in the field regarding PKC regulation of KV11.1. We propose that PKD1 mediates the PKC effects on KV11.1 and we found that PKD targets S284 in the N-terminus of the channel.",

author = "Steffensen, {Annette Buur} and Bomholtz, {Sofia Hammami} and Andersen, {Martin Nybo} and Olsen, {Jesper Velgaard} and N. Mutsaers and Lundegaard, {Pia Rengtved} and Alicia Lundby and Nicole Schmitt",

note = "{\textcopyright} 2018 The Author(s). Published by S. Karger AG, Basel.",

year = "2018",

month = jul,

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doi = "10.1159/000491007",

language = "English",

volume = "47",

pages = "1742--1750",

journal = "Cellular Physiology and Biochemistry",

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T1 - PKD Phosphorylation as Novel Pathway of KV11.1 Regulation

AU - Steffensen, Annette Buur

AU - Bomholtz, Sofia Hammami

AU - Andersen, Martin Nybo

AU - Olsen, Jesper Velgaard

AU - Mutsaers, N.

AU - Lundegaard, Pia Rengtved

AU - Lundby, Alicia

AU - Schmitt, Nicole

PY - 2018/7/1

Y1 - 2018/7/1

N2 - BACKGROUND/AIMS: The voltage-gated potassium channel KV11.1 has been originally cloned from the brain and is expressed in a variety of tissues. The role of phosphorylation for channel function is a matter of debate. In this study, we aimed to elucidate the extent and role of protein kinase D mediated phosphorylation.METHODS: We employed mass spectrometry, whole-cell patch clamp electrophysiology, confocal microscopy, site-directed mutagenesis, and western blotting.RESULTS: Using brain tissue from rat and mouse, we mapped several phosphorylated KV11.1 residues by LC-MS mass spectrometry and identified protein kinase D (PKD1) as possible regulatory kinase. Co-expression of KV11.1 with PKD1 reduced current amplitudes without altering protein levels or surface expression of the channel. Based on LC-MS results from in vivo and HEK293 cell experiments we chose four KV11.1 mutant candidates for further functional analysis. Ablation of the putative PKD phosphorylation site in the mutant S284A increased the maximal current indicating S284 as a main PKD target in KV11.1.CONCLUSIONS: Our data might help mitigating a long-standing controversy in the field regarding PKC regulation of KV11.1. We propose that PKD1 mediates the PKC effects on KV11.1 and we found that PKD targets S284 in the N-terminus of the channel.

AB - BACKGROUND/AIMS: The voltage-gated potassium channel KV11.1 has been originally cloned from the brain and is expressed in a variety of tissues. The role of phosphorylation for channel function is a matter of debate. In this study, we aimed to elucidate the extent and role of protein kinase D mediated phosphorylation.METHODS: We employed mass spectrometry, whole-cell patch clamp electrophysiology, confocal microscopy, site-directed mutagenesis, and western blotting.RESULTS: Using brain tissue from rat and mouse, we mapped several phosphorylated KV11.1 residues by LC-MS mass spectrometry and identified protein kinase D (PKD1) as possible regulatory kinase. Co-expression of KV11.1 with PKD1 reduced current amplitudes without altering protein levels or surface expression of the channel. Based on LC-MS results from in vivo and HEK293 cell experiments we chose four KV11.1 mutant candidates for further functional analysis. Ablation of the putative PKD phosphorylation site in the mutant S284A increased the maximal current indicating S284 as a main PKD target in KV11.1.CONCLUSIONS: Our data might help mitigating a long-standing controversy in the field regarding PKC regulation of KV11.1. We propose that PKD1 mediates the PKC effects on KV11.1 and we found that PKD targets S284 in the N-terminus of the channel.

U2 - 10.1159/000491007

DO - 10.1159/000491007

M3 - Journal article

C2 - 29949809

SN - 1015-8987

VL - 47

SP - 1742

EP - 1750

JO - Cellular Physiology and Biochemistry

JF - Cellular Physiology and Biochemistry

IS - 4

ER -

PKD Phosphorylation as Novel Pathway of KV11.1 Regulation

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PKD Phosphorylation as Novel Pathway of K_V11.1 Regulation