Subunit folding and assembly steps are interspersed during Shaker potassium channel biogenesis

In the voltage-dependent Shaker K+ channel, distinct regions of the protein form the voltage sensor, contribute to the permeation pathway, and recognize compatible subunits for assembly. To investigate channel biogenesis, we disrupted the formation of these discrete functional domains with mutations, including an amino-terminal deletion, Delta97-196, which is likely to disrupt subunit oligomerization; D316K and K374E, which prevent proper folding of the voltage sensor; and E418K and C462K, which are likely to disrupt pore formation. We determined whether these mutant subunits undergo three previously identified assembly events as follows: 1) tetramerization of Shaker subunits, 2) assembly of Shaker (alpha) and cytoplasmic beta subunits, and 3) association of the amino and carboxyl termini of adjacent Shaker subunits. Delta97-196 subunits failed to establish any of these quaternary interactions. The Delta97-196 deletion also prevented formation of the pore. The other mutant subunits assembled into tetramers and associated with the beta subunit but did not establish proximity between the amino and carboxyl termini of adjacent subunits. The results indicate that oligomerization mediated by the amino terminus is required for subsequent pore formation and either precedes or is independent of folding of the voltage sensor. In contrast, the amino and carboxyl termini of adjacent subunits associate late during biogenesis. Because subunits with folding defects oligomerize, we conclude that Shaker channels need not assemble from pre-folded monomers. Furthermore, association with native subunits can weakly promote the proper folding of some mutant subunits, suggesting that steps of folding and assembly alternate during channel biogenesis.