In this study, we probe the folding of K v AP, a voltage-gated K + (K v ) channel. The K v AP channel, though of archaebacterial origin, is structurally and functionally similar to eukaryotic K v channels. An advantage of the K v AP channel is that it can be folded in vitro from an extensively unfolded state and the folding can be controlled by temperature. We utilize these properties of the K v AP channel to separately study the membrane insertion and the tetramerization stages during folding. We use two quantitative assays: a Cys PEGylation assay to monitor membrane insertion and a cross-linking assay to monitor tetramerization. We show that during folding the K v AP polypeptide is rapidly inserted into the lipid bilayer with a "native-like" topology. We identify a segment at the C-terminus that is important for multimerization of the K v AP channel. We show that this C-terminal domain forms a dimer, which raises the possibility that the tetramerization of the K v AP channel proceeds through a dimer of dimers pathway. Our studies show that the in vitro folding of the K v AP channel mirrors aspects of the cellular assembly pathway for voltage-gated K + channels and therefore suggest that evolutionarily distinct K v channels share a common folding pathway. The pathway for the folding and assembly of a K v channel is of central importance as defects in this pathway have been implicated in the etiology of several disease states. Our studies indicate that the K v AP channel provides an experimentally tractable system for elucidating the folding mechanism of K v channels.
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