Functional cardiac GIRK channels are heteromeric complexes of Kir3.1 and Kir3.4. The role(s) of the different subunits at present are largely unknown. Kir3.4 is able to form functional homomeric channels, whereas Kir3.1 homomeric channels do not exist. Overexpression of Kir3.4 in atrial myocytes results in GIRK current, that is activated by intracellular [Na⁺], whereas endogenous 3.1/3.4 complexes lack Na⁺-sensitivity (Mintert et al. J. Physiol. 585, 2007). Here, we constructed a concatemer of Kir3.4 and Kir3.1, linked by YFP (N-K4-YFP-K1-C). When expressed in HEK293 cells, N-K4-YFP-K1-C translocates to the plasma membrane and forms functional channels that can be activated by a co-expressed A₁ adenosine receptor. A₁ receptor activation causes dynamic interaction of the channel construct with G_bgmonitored as increase in FRET ratio between YFP and CFP fused to b₂. Expression of N-K4-YFP-K1-C in atrial myocytes permits a direct comparison of current carried by dimers of the construct and endogenous Kir3.1/Kir3.4. Receptor-activated current in N-K4-YFP-K1-C expressing cells was significantly less inward-rectifying as compared to native myocytes. Analogous to Kir3.4 overexpression, N-K4-YFP-K1-C resulted in a receptor-independent background current activated by [Na⁺]_i. In an in silico approach we generate a computer model of N-K4-YFP-K1-C and perform a MD simulation in membraneous environment. The simulation suggests stable binding of Na⁺ and PIP₂, consistent with our experimental findings. These data demonstrate N-K4-YFP-K1-C as a useful tool for investigating, how subunit composition affects properties of GIRK channel currents.