A Single-Pore Residue Renders the Arabidopsis Root Anion Channel SLAH2 Highly Nitrate Selective

In contrast to animal cells, plants use nitrate as a major source of nitrogen.Following the uptake of nitrate, this major macronutrient is fed into the vasculaturefor long-distance transport. The Arabidopsis thaliana shootexpresses the anion channel SLOW ANION CHANNEL1 (SLAC1) and its homolog SLAC1HOMOLOGOUS3 (SLAH3), which prefer nitrate as substrate but cannot exclude chlorideions. By contrast, we identified SLAH2 as a nitrate-specific channel that isimpermeable for chloride. To understand the molecular basis for nitrate selection inthe SLAH2 channel, SLAC1 and SLAH2 were modeled to the structure of HiTehA, adistantly related bacterial member. Structure-guided site-directed mutationsconverted SLAC1 into a SLAH2-like nitrate-specific anion channel and vice versa. Ourfindings indicate that two pore-occluding phenylalanines constrict the pore. Theselectivity filter of SLAC/SLAH anion channels is determined by the polarity ofpore-lining residues located on alpha helix 3. Changing the polar character of asingle amino acid side chain (Ser-228) to a nonpolar residue turned thenitrate-selective SLAH2 into a chloride/nitrate-permeable anion channel. Thus, themolecular basis of the anion specificity of SLAC/SLAH anion channels seems to bedetermined by the presence and constellation of polar side chains that act in concertwith the two pore-occluding phenylalanines.