Acquired Cadmium Resistance in Metallothionein-I/II(–/–) Knockout Cells: Role of the T-Type Calcium Channel Cacnα_1Gin Cadmium Uptake

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 2ϩ ). MT-I/II(Ϫ/Ϫ) knockout [MT(Ϫ/Ϫ)] cells are available and serve as an excellent tool to study non-MT-related mechanisms in metal tolerance. In the current study, Cd 2ϩ -resistant MT(Ϫ/Ϫ) (CdR) and CdR revertant (CdR-rev) cell lines were developed and characterized to investigate non-MT-mediated cellular protection mechanisms. Resistance to Cd 2ϩ was approximately 70-fold higher in CdR than the parental MT(Ϫ/Ϫ) cell line (IC 50 ϭ 20 versus 0.3 M, respectively) and was stable in the absence of Cd 2ϩ for 35 days. Accumulation of Cd 2ϩ by the CdR cell line was reduced by approximately 95% compared with parental cells, primarily because of a decreased Cd 2ϩ uptake. Cd 2ϩ uptake by the MT(Ϫ/Ϫ) parental cell line was independent of sodium, energy, and electrogenic potential.Uptake was saturable (K m ϭ 65 nM; V max ϭ 4.9 pmol/mg/min) and pH-dependent (maximal at pH 6.5-7).

The role of ZIP8 down‐regulation in cadmium‐resistant metallothionein‐null cells

“…A more detailed profiling of these multi-metal transporters in terms of metal specificity, affinity and indispensability may be required in a future study. Leslie et al (2006) reported that the expression of T-type calcium channel (Cacnα 1G ) was suppressed in cadmium-resistant cells in which cadmium uptake was suppressed. However, the expression of Cacnα 1G in A7, B5 and P cells showed no significant differences (data not shown).…”

“…To date, several candidate transporters and channels involved in cadmium uptake in mammalian cells have been proposed: calcium channel (Leslie et al, 2006), calcium transporter (Min et al, 2008), iron transporter (Ryu et al, 2004;Kim et al, 2007;Bannon et al, 2003), zinc transporter (Dalton et al, 2005;He et al, 2006;Wang et al, 2007;Liu et al, 2008) and magnesium transporter (Martine et al, 2008). However, the role of each transport system in cadmium uptake remains still elusive.…”

Cd2+Block and Permeation of CaV3.1 (α1G) T-Type Calcium Channels: Candidate Mechanism for Cd2+Influx

“…Previous studies also linked L-type calcium channels (Hinkle et al, 1987) and the calcium-selective transient receptor potential canonical 6 channel (Kovacs et al, 2011) to Cd 2ϩ uptake. Development of resistance to Cd 2ϩ in cell culture has been linked to down-regulation of Ca V 3.1, which suggests the involvement of this channel in Cd 2ϩ toxicity (Leslie et al, 2006). Given the large number of calcium channels expressed throughout the body, the importance of Ca 2ϩ signaling, and the large number of ions a channel can transport (ϳ10 5 ions/s), even slight permeability of a calcium channel to Cd 2ϩ might lead to significant Cd 2ϩ entry.…”

Elevated GSH Level Increases Cadmium Resistance through Down-Regulation of Sp1-Dependent Expression of the Cadmium Transporter ZIP8

“…Recent studies suggest that the calcium channel is involved in cadmium uptake (Leslie et al, 2006). However, siRNA-based ZIP8 knockdown significantly lowered cadmium uptake rate in SR3A cells.…”

“…A recent genetic study revealed that ZIP8 plays an important role in cadmium uptake and is known to be expressed in lung rather than the gastrointestinal tract (Begum et al, 2002;Dalton et al, 2005), and a subsequent biochemical study revealed that ZIP8 has a high affinity for cadmium and manganese (He et al, 2006). In addition to these transporters, recent studies have suggested that the calcium channel is involved in cadmium uptake, although the significance of these calcium channels in cellular defense against cadmium toxicity has not been well developed (Leslie et al, 2006). Glutathione (GSH) is an abundant intracellular thiol-containing compounds (1ϳ10 mM).…”