The potassium channel subunits Sloα1, Sloα2.1, Sloα2.2, and Sloα3 are encoded by the genes KCNMA1, KCNT2, KCNT1, and KCNU1, respectively. Each subunit contains 6 or 7 transmembrane domains, a signature potassium channel G(Y/F)G re-entrant pore loop, and a large intracellular C-terminal domain that comprises >65% of the protein. The best understood subunit is Sloα1, which encodes the tetrameric large-conductance Ca²⁺-activated K⁺ channel, which is activated by a combination of membrane depolarisation and intracellular Ca²⁺ and Mg²⁺. In contrast, Sloα2.1, α2.2, and α3 are not activated by these divalent cations but are instead regulated by different intracellular ions (Na⁺, Cl^–, ATP^4- and H⁺ [1]). Despite these differences, there are strong structural similarities between these subunits, in particular the presence of RCK domains that in Sloα1 are important in Ca²⁺/Mg²⁺ activation [2] and also in tetramerisation [3]. We have studied human Sloα1, Sloα2.2, and mouse Sloα3 expressed in Xenopus oocytes using two-electrode voltage clamp. Consistent with previous studies Sloα2.2 and α3 expressed poorly and required 100 to 1000-fold more RNA than α1 to obtain significant currents. We hypothesised that these subunits represent a discrete subfamily of K⁺ channels, that the conserved RCK domains enable heteromultimerisation when co-expressed, and that this may be important in native tissue where Sloα2.x and Sloα3 may not readily form homomeric channels. Indeed a novel current was observed when Sloα1 and Sloα2.2 was co-expressed in a previous study [4]. To test this we employed a dominant negative approach by mutating the G(Y/F)G motif to AAA [3] in Sloα1, α2.2, and α3 and co-injecting with wild-type Sloα1. Each of the dominant negative subunits (dnSloαX) almost completely abolished Sloα1 currents when co-injected into oocytes, but had no effect on Kv1.1, a member of a different K+ channel subfamily. The dnSloα2.2 and dnSloα3 exerted their effects on Sloα1 with low RNA doses with which wild-type subunits fail to generate significant Sloα2.2 and α3 currents. Furthermore, fluorescently labelled subunits colocalised when transfected into HEK cells and studied by confocal microscopy. The high levels (50ng) of Sloα2.2 and α3 RNA that are required to generate macroscopic currents suggest that they do not readily form functional homomeric channels. At low RNA doses (1ng) dnSloα2.2 and dnSloα3 subunits are synthesised and incorporate into tetramers with Sloα1 to exert a dominant-negative effect. These data suggest that the different Sloα subunits have the potential to heteromultimerise when co-expressed and that Sloα2.x and Sloα3 subunits are unlikely to exist as homomultimers in vivo.