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Mol Pharmacol. 2006 Feb;69(2):629-39. Epub 2005 Nov 10.

Acquired cadmium resistance in metallothionein-I/II(-/-) knockout cells: role of the T-type calcium channel Cacnalpha1G in cadmium uptake.

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Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute and National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.


Metallothioneins (MTs) are cytoplasmic proteins that sequester certain divalent cations and are considered a primary cellular defense against the toxic transition metal cadmium (Cd(2+)). 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(2+)-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(2+) was approximately 70-fold higher in CdR than the parental MT(-/-) cell line (IC(50) = 20 versus 0.3 microM, respectively) and was stable in the absence of Cd(2+) for 35 days. Accumulation of Cd(2+) by the CdR cell line was reduced by approximately 95% compared with parental cells, primarily because of a decreased Cd(2+) uptake. Cd(2+) uptake by the MT(-/-) parental cell line was independent of sodium, energy, and electrogenic potential. Uptake was saturable (K(m) = 65 nM; V(max) = 4.9 pmol/mg/min) and pH-dependent (maximal at pH 6.5-7). Potent inhibitors of Cd(2+) uptake included Zn(2+) (IC(50) = 7 microM), Mn(2+) (IC(50) = 0.4 microM), and the T-type Ca(2+) channel antagonist mibefradil (IC(50) = 5 microM), whereas other metals (including Fe(2+)) and L-type Ca(2+) channel antagonists had little effect. Immunoblot and real-time reverse transcription-polymerase chain reaction analysis indicated that the Cacnalpha(1G) T-type Ca(2+) channel was expressed at a reduced level in CdR compared with the parental MT(-/-) cell line, suggesting it is important for Cd(2+) uptake. The CdR1-rev cell line was found to have a Cd(2+) uptake and sensitivity level in between that of the CdR1 and MT(-/-) cell lines. Consistent with this was an intermediate expression of Cacnalpha(1G) in the CdR-rev cell line. These data suggest that decreased expression of Cacnalpha(1G) protects cells from Cd(2+) exposure by limiting Cd(2+) uptake.

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