Generation and Characterization of a Cell Line with Inducible Expression of Ca v 3.2 (T-Type) Channels

Abstract

Establishment of stable cell lines that constitutively express Ca²⁺ channels at high density and that are useful for in vitro studies may be complicated by problems with seal quality and duration during whole-cell patch-clamp electrophysiology. The current studies describe the generation and characterization of cells that express the human α1H T-type Ca²⁺ channel under the control of a tetracycline-inducible expression system. Western blot and immunostaining studies revealed that expression of the α1H protein occurred only in the presence of tetracycline. Using the whole-cell patch-clamp method, the cells displayed peak inward currents of 1.15 ± 0.14 nA in response to voltage-clamp steps. The T-type Ca²⁺ current was inhibited by the T-type Ca²⁺ channel antagonist, mibefradil, with an IC₅₀ of 160 nM. This cell line, with inducible channel expression, sealed with longer duration during whole-cell patch-clamp recording when compared with a cell line that constitutively expresses the α1H Ca²⁺ channel. Ca²⁺ influx through this channel could also be detected after the addition of extracellular Ca²⁺. The amount of Ca²⁺ influx was dependent on the [Ca]_o with an EC₅₀ of 4 mM. The Ca²⁺ influx was also inhibited by mibefradil with a potency (IC₅₀= 183 nM) similar to that observed in the voltage-clamp studies. Overall, this inducible α1H Ca²⁺ channel-expressing cell line is useful for the study of human T-type Ca²⁺ channel function, and offers advantages over a similar cell line that constitutively expresses the channel.