Human TRPV6 Stable Cell Line - HEK293

Maternal-fetal calcium ion (Ca2+) transport across the placenta plays a crucial role in maintaining fetal skeletal mineralization. Mutations in the gene encoding Transient Receptor Potential Cation Channel, Subfamily V, Member 6 (TRPV6) have been identified as pathogenic mutations responsible for transient neonatal hyperparathyroidism-a condition etiologically linked to insufficient maternal-fetal Ca2+ transport across the placenta. Here, researchers identified two novel mutations in subjects presenting with transient neonatal hyperparathyroidism. The TRPV6 protein harboring the p.Arg390His mutation is localized to the outer edge of the first transmembrane domain (S1); this mutation results in impaired trafficking of the protein to the plasma membrane. In contrast, the TRPV6 protein carrying the p.Gly291Ser mutation-located within the sixth ankyrin repeat (AR) domain-exhibits channel properties comparable to those of the wild-type (WT) channel; however, it leads to elevated intracellular steady-state Ca2+ concentrations, which may trigger Ca2+ overload and subsequently induce cell death in cells expressing this mutant channel. These findings suggest that the AR6 domain contributes to TRPV6-mediated maintenance of intracellular Ca2+ concentrations and may play a novel role in regulating the activity of the Ca2+-selective TRPV6 channel.

To analyze the biological significance of the p.Gly291Ser and p.Arg390His mutations, researchers transfected HEK293T cells with expression vectors carrying the mutant forms of TRPV6 and performed whole-cell patch-clamp recordings 20–24 hours post-transfection. HEK293T cells expressing the p.Gly291Ser mutant generated inducible currents upon exposure to either a divalent-free (DVF) bath solution or a high-Ca²⁺ bath solution; the characteristics of these currents were similar to those observed in wild-type TRPV6-expressing HEK293T cells (Figure 1a, c). In this experiment, the researchers utilized an NMDG solution devoid of Ca²⁺ and Mg²⁺ to facilitate a clearer observation of Ca²⁺ currents. No differences were observed between wild-type TRPV6-expressing HEK293T cells and p.Arg390His mutant-expressing cells regarding their current-voltage relationships or reversal potentials (Figure 1b), suggesting that both exhibit similar ion selectivity. Furthermore, no significant differences were detected between the two groups in intracellular Ca²⁺-dependent inactivation, at least within the 20–30-second timeframe of the patch-clamp recordings (Figure 1d). These results suggest that the apparent channel properties of the Gly291Ser mutant have been preserved. In contrast, in cells expressing the p.Arg390His mutant, neither the DVF bath solution nor the high-Ca²⁺ bath solution was able to induce any detectable current (Figure 1c).

Figure 1. Whole-cell patch-clamp recordings in TRPV6-expressing HEK293T cells. (Suzuki Y, et al., 2020)