The mechanisms by which the transmembrane domains of a potassium channel move in order to gate the channel are still not properly understood. A comparison of the ‘closed’ structure of the KcsA channel and the ‘open’ state of the MthK channel suggests that the second transmembrane domain hinges at a highly conserved glycine residue (1). This glycine residue is conserved in all known Kv, BK and Kir channels with the exception of Kir5.1 and Kir4.1 which contain either serine or threonine residues at this position. Kir5.1 selectively heteromultimerises with Kir4.1 to form novel heteromeric channels with unique functional and biophysical properties. In particular Kir4.1-Kir5.1 heteromeric channels exhibit a ‘bursting’ single channel behaviour with multiple subconductance states. In this study we have examined the functional consequences of site-directed mutations at this position in both Kir4.1 and Kir5.1 subunits expressed as a tandemly linked heteromeric channel (Kir4.1-Kir5.1). Wild-type and mutant Kir channel mRNAs were injected into Xenopus oocytes; macroscopic channel properties were examined using two-electrode voltage clamp, whilst single-channel gating was recorded using cell-attached and excised inside-out patches. In comparison to wild-type channels Kir4.1(T154G)-Kir5.1 and Kir4.1(T154G)-Kir5.1(S157G) mutant channels had a reduced open probability (Po= 31.1 ± 8%, n=26 and 26.3 ± 3.4%, n=27, respectively). The recent determination of X-ray crystal structures for the KirBac1.1 channel and the intracellular domains of Kir3.1 combined with the high degree of sequence conservation between different family members enables 3D homology modelling studies to be performed. Using both the KirBac1.1 and Kir3.1 structures as templates we generated a 3D homology model of a heteromeric Kir4.1/Kir5.1 channel and used this model to address the role of this non-conserved gating ‘hinge’. The 3D model suggests that the side-chains of residues T154 in Kir4.1 and S157 in Kir5.1 are in close proximity with the pore loops of the channel which form the selectivity filter. Recent studies have indicated that the ‘gate’ of the Kir channel may involve a complex interaction between the intracellular gate at the ‘helix bundle crossing’ and a second gate within the selectivity filter. Our studies suggest that these residues may contribute to the unique single-channel properties of Kir4.1-Kir5.1 heteromeric channels by influencing a potential gate within the selectivity filter.