Pore-forming subunits of ion channels show channel activity in heterologous cells. However, recombinant and native channels often differ in their channel properties. These discrepancies are resolved by the identification of channel auxiliary subunits. In this review article, an auxiliary subunit of ligand-gated ion channels is defined using four criteria: (1) as a Non-pore-forming subunit, (2) direct and stable interaction with a pore-forming subunit, (3) modulation of channel properties and/or trafficking in heterologous cells, (4) necessity in vivo. We focus particularly on three classes of ionotropic glutamate receptors and their transmembrane interactors. Precise identification of auxiliary subunits and reconstruction of native glutamate receptors will open new directions to understanding the brain and its functions.