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First published on November 10, 2005; DOI: 10.1124/mol.105.014241

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Received for publication May 24, 2005.
Revised November 10, 2005.
Accepted for publication November 10, 2005.

Acquired Cadmium Resistance in Metallothionein-I/II(-/-) Knockout Cells: Role of the T-type Calcium Channel Cacn{alpha}1G in Cadmium Uptake

Elaine M. Leslie 1, Jie Liu 2, Curtis D. Klaassen 3, Michael P. Waalkes 2*

1 University of North Carolina at Chapel Hill 2 NCI at NIEHS 3 University of Kansas Medical Center

* Address correspondence to: E-mail: waalkes{at}niehs.nih.gov

Abstract

Metallothioneins (MTs) are cytoplasmic proteins that sequester certain divalent cations and are considered a primary cellular defense against the toxic transition metal cadmium (Cd). MT-I/II(-/-) knockout [MT(-/-)] cells are available and serve as an excellent tool to study non-MT related mechanisms in metal tolerance. In the current study, Cd resistant MT(-/-) (CdR) and CdR revertant (CdR-rev) cell lines were developed and characterized to investigate non-MT mediated cellular protection mechanisms. Resistance to Cd was approximately 70-fold higher in CdR than the parental MT(-/-) cell line (IC50 20 µM versus 0.3 µM, respectively) and was stable in the absence of Cd for 35 days. Accumulation of Cd by the CdR cell line was reduced by approximately 95% compared with parental cells, primarily due to a decreased Cd uptake. Cd uptake by the MT(-/-) parental cell line was independent of sodium, energy and electrogenic potential. Uptake was saturable (Km 65 nM, Vmax 4.9 pmol/mg/min) and pH dependent (maximal at pH 6.5-7). Potent inhibitors of Cd uptake included Zn2+ (IC50 7 µM), Mn2+ (IC50 0.4 µM) and the T-type Ca2+ channel antagonist mibefradil (IC50 5 µM), whereas other metals (including Fe2+) and L-type Ca2+ channel antagonists had little effect. Immunoblot and real time RT-PCR analysis indicated that the Cacn{alpha}1G T-type Ca2+ channel was expressed at a reduced level in CdR compared with the parental MT(-/-) cell line, suggesting it is important for Cd uptake. The CdR1-rev cell line was found to have a Cd uptake and sensitivity level in between the CdR1 and MT(-/-) cell lines. Consistent with this was an intermediate expression of Cacn{alpha}1G in the CdR-rev cell. These data suggest that decreased expression of Cacn{alpha}1G protects cells from Cd exposure by limiting Cd uptake.


Key words: Calcium (Votage-Gated Channels), Metals and chelators


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Elevated GSH Level Increases Cadmium Resistance through Down-Regulation of Sp1-Dependent Expression of the Cadmium Transporter ZIP8
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[Abstract] [Full Text] [PDF]


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