TRPC1 is involved in Ca2+ influx and cytotoxicity following Pb2+ exposure in human embryonic kidney cells

Abstract

Lead (Pb2+) is a divalent heavy metal ion which causes severe damage to almost all life forms and is therefore considered a notorious toxicant. Exposure to Pb2+ is associated with poor cognitive development in children at relatively low levels that previously were thought to be safe. The mechanism through which Pb2+ enters cells, however, is unclear. Previous studies have showed that Ca2+ release-activated Ca2+ protein 1 (Orai1), a component of store-operated Ca2+ channels (SOCs), contributes to Pb2+ cellular entry. Canonical transient receptor potential (TRPC1) channel 1 is a transient receptor potential (TRP) channel which is sometimes referred to as a SOC. The present study was designed to investigate the role of TRPC1 in Pb2+ entry and toxicity in human embryonic kidney cells (HEK293). Additionally, changes in intracellular Ca2+ concentration were determined through Fluo-4 and Mag-fluo-4 fluorescent Ca2+ imaging. Following Pb2+ exposure, there was a dose-dependent decrease in cell viability. Overexpression of TRPC1 increased Pb2+-induced cell death, while knockdown of this channel attenuated cell death. There was increased entry of Pb2+, as measured by inductively coupled plasma mass spectrometry (ICP-MS), following overexpression of TRPC1. Conversely, knockdown of TRPC1 led to a decrease in Pb2+ influx. Down-regulation of STIM1 by RNA interference attenuated the Pb2+ influx, and transfection with a mutant STIM1, which could not gate TRPC1, had a similar effect. Co-transfection of mutant STIM1 and mutant TRPC1, which restore the electrostatic interaction between STIM1 and TRPC1, resumed Pb2+ entry in HEK293 cells. Down-regulation of TRPC1 by RNA interference decreased Ca2+ influx whilst its overexpression increased Ca2+ entry in HEK293 cells. These results suggest that TRPC1 is involved in the cytotoxicity and entry of Pb2+ through molecular interactions with STIM1 and subsequent Ca2+ influx in HEK293 cells.