ABCMdb

PMID: 9141359

Miwa A, Ueda K, Okada Y
Protein kinase C-independent correlation between P-glycoprotein expression and volume sensitivity of Cl- channel.
J Membr Biol. 1997 May 1;157(1):63-9., 1997-05-01 [PubMed]

Sentences

No. Mutations Sentence Comment

19 ABCB1 p.Lys433Met ABCB1 p.Lys433Met To obtain KB cells expressing the ATP hydrolysis-deficient PGP mutant, K433M (Gill et al., 1992), in which methionine was substituted for lysine-433 in the first nucleotide-binding domain, KB-3-1 cells were transfected by the MDR1 K433M expression vector with LipofectAMINE (Gibco, Gaithersburg, MD) according to manufacture's direction. Login to comment 21 ABCB1 p.Lys433Met The expression plasmid for K433M was constructed by fusing the mutated MDR I cDNA to aminoglycosidase phosphotransferase (neomycin resistant: neo) gene with an encephalomyocarditis virus 5' untranslated sequence, known as an internal ribosome entry site (IRES). Login to comment 61 ABCB1 p.Lys433Met Immunoblot of membrane protein fraction (20 mg) from untransfected KB-3-1 (lane 1), KB/K433M (lane 2) and KB-G2 (lane 3) in a 7% SDS-PAGE. Login to comment 64 ABCB1 p.Lys433Met The KB cells transfected with cDNA of the K433M mutant were also found to exhibit the protein expression in the membrane fraction (lane 2). Login to comment 106 ABCB1 p.Lys433Met When the K433M mutant of PGP was expressed, the volume sensitivity of VSOR Cl-channel was again found to be augmented, as shown in Fig. 3B (solid squares). Login to comment 107 ABCB1 p.Lys433Met Discussion The MDRI gene product, P-glycoprotein (PGP), is a member of the ABC (ATP-binding cassette) superfamily and acts as an ATP-dependent, active transporter expel- solid square: K433M/PGP a open circle: no PGP A. Miwa et al.: Cl-Channel and P-Glycoprotein solid symbol: wild PGP • A 140 open symbol: no PGP ,•• •• 120 100 Z 80 v 60 c LE 40 circle: control triangle: TPA .^•:: }• o 20 1.0 1.5 B 140 120 a a100 80 c a> 'v 60 40 U 20 2.0 2.5 3.0 3.5 4.0 4.5 relative surface area Fig. 3. Login to comment 114 ABCB1 p.Lys433Met ABCB1 p.Lys433Met (B) Increased volume sensitivity of Cl-currents in KB cells expressing the K433M mutant of PGP (solid squares) compared to those in PGP-lacking KB-3-1 cells (open circles). Login to comment 115 ABCB1 p.Lys433Met The slopes estimated by regression analysis were 34.3 ± 1.4 and 55.9 ± 4.0 (P < 0.05) for all the points of PGP-lacking KB-3-1 (n = 43) and of K433M/PGP-expressing KB-3-1 (n = 25), respectively. Login to comment 124 ABCB1 p.Lys433Met Volume sensitivity of the Cl - channel was also augmented by the expression with the K433M mutant of PGP (Fig. 3B) which was shown to exhibit no ATPase activity (Loo & Clarke, 1995 but reported to induce the volume-activated Cl-channel activity in S 1 cells (Gill et al., 1992). Login to comment