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Cardiac G protein-activated Kir (GIRK) channels may assemble as heterotetrameric polypeptides from two subunits, Kir3.1 and Kir3.4. For a functional comparison with native channels in the CNS we investigated all possible combinations of heteromeric channel formation from brain Kir3.1, Kir3.2, Kir3.3, and Kir3.4 subunits in mRNA-injected Xenopus oocytes. Analysis of macroscopic current amplitudes and channel gating kinetics indicated that individual subunits or combinations of Kir3.2, Kir3.3, and Kir3.4 formed functional channels ineffectively. Each of these subunits gave rise to prominent currents with distinct characteristics only in the presence of Kir3.1 subunits. Functional expression of concatemeric constructs between Kir3.1 and Kir3.2/3.4 subunits as well as coimmunoprecipitations with subunit-specific antibodies confirmed heteromeric channel formation. Mutational swapping between subunits of a single pore loop residue (Kir3.1F137S; Kir3.3S114F; a phenylalanine confers slow channel gating in Kir3.1 subunits) revealed that Kir3.1 subunits are an important constituent for native heteromeric channels and dominate their functional properties. However, homomeric channels from Kir3.1 subunits in vivo may not exist due to the spatial conflict of bulky phenylalanines in the pore structure.