TY - JOUR T1 - Acquired Cadmium Resistance in Metallothionein-I/II(–/–) Knockout Cells: Role of the T-Type Calcium Channel Cacnα<sub>1G</sub> in Cadmium Uptake JF - Molecular Pharmacology JO - Mol Pharmacol SP - 629 LP - 639 DO - 10.1124/mol.105.014241 VL - 69 IS - 2 AU - Elaine M. Leslie AU - Jie Liu AU - Curtis D. Klaassen AU - Michael P. Waalkes Y1 - 2006/02/01 UR - http://molpharm.aspetjournals.org/content/69/2/629.abstract N2 - Metallothioneins (MTs) are cytoplasmic proteins that sequester certain divalent cations and are considered a primary cellular defense against the toxic transition metal cadmium (Cd2+). 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, Cd2+-resistant MT(–/–) (CdR) and CdR revertant (CdR-rev) cell lines were developed and characterized to investigate non–MT-mediated cellular protection mechanisms. Resistance to Cd2+ was approximately 70-fold higher in CdR than the parental MT(–/–) cell line (IC50 = 20 versus 0.3 μM, respectively) and was stable in the absence of Cd2+ for 35 days. Accumulation of Cd2+ by the CdR cell line was reduced by approximately 95% compared with parental cells, primarily because of a decreased Cd2+ uptake. Cd2+ 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 Cd2+ 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 reverse transcription-polymerase chain reaction analysis indicated that the Cacnα1G T-type Ca2+ channel was expressed at a reduced level in CdR compared with the parental MT(–/–) cell line, suggesting it is important for Cd2+ uptake. The CdR1-rev cell line was found to have a Cd2+ uptake and sensitivity level in between that of the CdR1 and MT(–/–) cell lines. Consistent with this was an intermediate expression of Cacnα1G in the CdR-rev cell line. These data suggest that decreased expression of Cacnα1G protects cells from Cd2+ exposure by limiting Cd2+ uptake. ER -