Glycosylation and cell surface expression of Kv1.2 potassium channel are regulated by determinants in the pore region.
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 Kvbeta2 subunit and the S360A mutant resulted in a robust peak current. Immunocytochemistry for Kv1.2 showed staining throughout the cytoplasm in cells coexpressing the beta2 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 beta2and S360A. These results suggest that amino acids in the pore region help regulate ion permeability or cellular trafficking by affecting glycosylation of Kv1.2.
Fujita
Tetsuhiro
T
Department of Pharmacotherapeutics, Showa Pharmaceutical University, Tokyo, Machida, Japan.
Utsunomiya
Iku
I
Ren
Jin
J
Matsushita
Yousuke
Y
Kawai
Miwa
M
Sasaki
Sachie
S
Hoshi
Keiko
K
Miyatake
Tadashi
T
Taguchi
Kyoji
K
D000595:Amino Acid Sequence; D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols; D002477:Cells; D006224:Cricetinae; D003412:Cricetulus; D003520:Cyclophosphamide; D004317:Doxorubicin; D006031:Glycosylation; D051663:Kv1.2 Potassium Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014750:Vincristine
D051663:Kv1.2 Potassium Channel; D021122:Protein Subunits; D014750:Vincristine; D004317:Doxorubicin; D003520:Cyclophosphamide; D011188:Potassium