We have developed a method for high-throughput isothermal amplification of single DNA molecules in a droplet-based microfluidic system. DNA amplification in droplets was analyzed using an intercalating fluorochrome, allowing fast and accurate "digital" quantification of the template DNA based on the Poisson distribution of DNA molecules in droplets. The clonal amplified DNA in each 2 pL droplet was further analyzed by measuring the enzymatic activity of the encoded proteins after fusion with a 15 pL droplet containing an in vitro translation system.
In mammals exclusively, the pore-forming Ca2+ release-activating Ca2+ (CRAC) channel subunit, Orai1, occurs in two forms due to alternative translation initiation. The longer, mammal-specific Orai1α contains an additional 63 amino acids upstream of the conserved start site for Orai1β, which occurs at methionine 64 in Orai1α. Orai1 participates in the generation of three distinct Ca2+ currents, including two store-operated currents, Icrac, which involves the Ca2+-sensing protein STIM1 activation of Orai1 channels, and Isoc, which involves a tertiary interaction among Orai1, the transient receptor potential (TRP) family member TRPC1, and STIM1. Orai1 is also a pore-forming subunit of an arachidonic acid (leukotriene C4)-regulated current Iarc that involves interactions of Orai1, Orai3, and STIM1. Here, we evaluated the roles of the two Orai1 forms in the Ca2+ currents Icrac, Isoc, and Iarc. We found that Orai1α and Orai1β are largely interchangeable for Icrac and Isoc, although Orai1α exhibits stronger inhibition by Ca2+. Only the mammalian-specific Orai1α functions in the arachidonic acid-regulated current, Iarc. Thus, alternative translation initiation of Orai1 message produces at least three types of Ca2+ channels with distinct signaling and regulatory properties.
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