Activation Gate Region Influences Stoichiometry of Heteromeric Shaker Family Channels

Abstract

Shaker family voltage-gated K+ channel subunits can typically form functional homotetrameric channels or heterotetrameric channels with closely related subunits. Assembly is governed by cytoplasmic T1 domain contacts that are compatible within, but not across, gene subfamilies (Shaker, Shab, Shal and Shaw). Shaker family regulatory subunits differ in that they can only function in heteromeric channels because of T1 self-incompatibility. Mammalian regulatory subunits (Kv6, Kv8, Kv9) are members of the Shab family and modify the functional properties of Kv2.1 and Kv2.2. Kv2.1 and Kv9.3 form functional channels in a 3:1 stoichiometry. Using a combination of TIRF microscopy and electrophysiology, we show here that Kv2.1subunits also exclusively form 3:1 heteromers with Kv6.4. These findings raise the question of how stoichiometry is determined: it is unclear how T1 domain compatibility rules alone could distinguish between formation of 3:1 and 2:2 heteromers. We hypothesized that the activation gate region of the inner pore may be important in refining the stoichiometry of regulatory subunit-containing heteromers because it forms a large contact interface between subunits. Additionally, the sequence of this region in the regulatory subunits has drifted from the highly conserved Shab family consensus. Reverting the gate sequence in Kv6.4 to the Shab family consensus allowed the formation of functional 2:2 heteromers between Kv6.4 and Kv2.1 that could be detected by electrophysiology and TIRF microscopy. Drift of the gate sequence consensus can be observed in all verified regulatory subunits, including these mammalian Shab channels and evolutionarily separate regulatory subunit expansions in the Shaker, Shal and Shaw subfamilies in cnidarians. We suggest that the evolution of T1 self-incompatibility is a key event in the evolution of the regulatory subunit phenotype, but that drift of the activation gate interface plays an important role in fixing heteromeric channel stoichiometry.