Folding and misfolding of potassium channel monomers during assembly and tetramerization

Abstract

Significance Ion channels exist in all organisms. Here, we connect the folding of potassium channel monomers to the kinetics of tetramerization. Rather than adopting a native-like conformation once inserted into the bilayer, monomers initially exist as a structurally heterogeneous ensemble in a protein-dense region. This early clustering of monomers may be a general phenomenon that assists in the assembly of multimeric membrane proteins by pre-localizing the subunits. Folding can occur along fast or slow (misfolded) pathways that can be modulated with mutations that trap monomers in a native-like state. In spite of its name, the C-terminal “tetramerization” domain in KcsA does not enhance tetramerization, suggesting it may play another role in channel function. The dynamics and folding of potassium channel pore domain monomers are connected to the kinetics of tetramer assembly. In all-atom molecular dynamics simulations of Kv1.2 and KcsA channels, monomers adopt multiple nonnative conformations while the three helices remain folded. Consistent with this picture, NMR studies also find the monomers to be dynamic and structurally heterogeneous. However, a KcsA construct with a disulfide bridge engineered between the two transmembrane helices has an NMR spectrum with well-dispersed peaks, suggesting that the monomer can be locked into a native-like conformation that is similar to that observed in the folded tetramer. During tetramerization, fluoresence resonance energy transfer (FRET) data indicate that monomers rapidly oligomerize upon insertion into liposomes, likely forming a protein-dense region. Folding within this region occurs along separate fast and slow routes, with τfold ∼40 and 1,500 s, respectively. In contrast, constructs bearing the disulfide bond mainly fold via the faster pathway, suggesting that maintaining the transmembrane helices in their native orientation reduces misfolding. Interestingly, folding is concentration independent despite the tetrameric nature of the channel, indicating that the rate-limiting step is unimolecular and occurs after monomer association in the protein-dense region. We propose that the rapid formation of protein-dense regions may help with the assembly of multimeric membrane proteins by bringing together the nascent components prior to assembly. Finally, despite its name, the addition of KcsA’s C-terminal “tetramerization” domain does not hasten the kinetics of tetramerization.