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Glycosylation and Cell Surface Expression of Kv1.2 Potassium Channel are Regulated by Determinants in the Pore Region

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Abstract

Voltage-gated K+ channels contain six membrane spanning segments and a pore-forming domain. We used site-directed mutation to examine the role of specific amino acids in the extracellular region of the pore in Kv1.2. When expressed in CHO cells, a K+ current was not observed for mutants S356A, S360A, T383A and T384A. However, coexpression of the Kvβ2 subunit and the S360A mutant resulted in a robust peak current. Immunocytochemistry for Kv1.2 showed staining throughout the cytoplasm in cells coexpressing the β2 and S360A, whereas only the perinuclear region was stained in cells expressing the S360A mutant. Western blotting revealed that the major immunoreactive protein in wild-type- and mutant-expressing cells is 60-kDa, but 87-kDa bands were also detected in cells expressing wild-type Kv1.2 and cells coexpressing β2and S360A. These results suggest that amino acids in the pore region help regulate ion permeability or cellular trafficking by affecting glycosylation of Kv1.2.

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Abbreviations

RT-PCR:

reverse transcription-polymerase chain reaction

GAPDH:

glyceraldehyde-3-phosphate dehydrogenase

WT:

wild-type

DMEM:

Dulbecco’s modified Eagle’s medium

FCS:

fetal calf serum

EDTA:

ethylenediaminetetraacetic acid

PBS:

phosphate-buffered saline

PBST:

PBS containing 5% nonfat dry milk and 0.1% Tween 20

VGKC:

voltage-gated K+ channel

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

HEPES:

N-2-hydroxyethylpiperazine-N′-2-ethane sulfonic acid

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Acknowledgements

We thank Drs. M. Furuta, N. Hachiya, and K. Kaneko of the Department of Cortical Function Disorders, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan for recombinant gene construction and for helpful discussions; Dr. Y. Kubo of Tokyo Medical and Dental University, Tokyo, Japan for CHO cells; Dr. J.P. Adelman of Oregon Health Sciences University, Portland, OR, USA for the RBK2/SP6S plasmid; Dr. J.S. Trimmer of State University of New York, Stony Brook, NY, USA for a subclone encoding the rat Kvβ2 protein in the pRGB4 vector; and Ms. R. Sato of Showa Pharmaceutical University for technical assistance. This work was supported by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan (14572164).

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Authors and Affiliations

  1. Department of Pharmacotherapeutics, Showa Pharmaceutical University, Tokyo, Machida, Japan

    Tetsuhiro Fujita, Iku Utsunomiya, Jin Ren, Yousuke Matsushita, Miwa Kawai, Sachie Sasaki, Keiko Hoshi, Tadashi Miyatake & Kyoji Taguchi

  2. Department of Pharmacotherapeutics, Showa Pharmaceutical University, 3-3165 Higashitamagawagakuen, 194-8543, Tokyo, Machida, Japan

    Iku Utsunomiya

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  1. Tetsuhiro Fujita
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Correspondence to Iku Utsunomiya.

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Fujita, T., Utsunomiya, I., Ren, J. et al. Glycosylation and Cell Surface Expression of Kv1.2 Potassium Channel are Regulated by Determinants in the Pore Region. Neurochem Res 31, 589–596 (2006). https://doi.org/10.1007/s11064-006-9056-4

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