SUBUNIT FOLDING AND ASSEMBLY STEPS ARE INTERSPERSED DURING SHAKER POTASSIUM CHANNEL BIOGENESIS

In the voltage-dependent Shaker K+ channel, distinct regions of the pr otein form the voltage sensor, contribute to the permeation pathway, a nd recognize compatible subunits for assembly. To investigate channel biogenesis, we disrupted the formation of these discrete functional do mains with mutations, including an aminoterminal deletion, Delta 97-19 6, which is likely to disrupt subunit oligomerization; D316K and K374E , which prevent proper folding of the voltage sensor; and E418K and C4 62K which are likely to disrupt pore formation. We determined whether these mutant subunits undergo three previously identified assembly eve nts as follows: 1) tetramerization of Shaker subunits, 2) assembly of Shaker (alpha) and cytoplasmic beta subunits, and 3) association of th e amino and carboxyl termini of adjacent Shaker subunits. Delta 97-196 subunits failed to establish any of these quaternary interactions, Th e Delta 97-196 deletion also prevented formation of the pore. The othe r mutant subunits assembled into tetramers and associated with the bet a subunit but did not establish proximity between the amino and carbox yl termini of adjacent subunits. The results indicate that oligomeriza tion mediated by the amino terminus is required for subsequent pore fo rmation and either precedes or is independent of folding of the voltag e sensor. In contrast, the amino and carboxyl termini of adjacent subu nits associate late during biogenesis. Because subunits with folding d efects oligomerize, we conclude that Shaker channels need not assemble from pre-folded monomers. Furthermore, association with native subuni ts can weakly promote the proper folding of some mutant subunits, sugg esting that steps of folding and assembly alternate during channel bio genesis.