Objectives: 

Manganese entry has been widely used as a surrogate for calcium influx given its quenching effect on fura-2. Despite the nature of the store-operated calcium entry (SOCE) channels has not been completely elucidated yet, current reports involve both Orai1 and the transient receptor potential (TRP) channels. TRP channels are non-selective channels permeable to monovalent and divalent cations, including manganese. In contrast, Orai1 has been characterized as a highly selective calcium channel. The present study is aimed to investigate the permeability of Orai1 to manganese and its regulation by extracellular calcium.

Materials: 

HEK-293 cells were obtained from ATCC and cultured in DMEM media. Manganese entry, measured by the slope of the quenching of fura-2 fluorescence at the isoemissive wavelength of 360 nm, was determined by fluorimetry. Orai1 expression was modified by siRNA and DNA technology and tested by Western blotting.

Results: 

Detectable Orai1 mRNA was found in HEK-293 cells. Activation of SOCE in HEK-293 cells by the SERCA inhibitor thapsigargin (TG) resulted in a sustained quenching of fura-2 fluorescence due to manganese entry. In the presence of extracellular calcium, simultaneous overexpression of Orai1 and STIM1, as well as Orai1 expression silencing, did not significantly modify TG-evoked manganese entry. In a calcium-free medium overexpression of Orai1 and STIM1 significantly enhanced manganese entry (p<0.05 Student´s t-test; n=6).

Conclusions: 

In conclusion, our results indicate that the permeability of Orai1 to manganese is regulated by the extracellular calcium concentration.

Supported by MINECO (BFU2010-21043-C02-01) and Junta de Extremadura-FEDER (GR10010). IJ and ND were supported by BFU2007-60104 and PRE09020.