Subunit Folding and Assembly Steps Are Interspersed during Shaker Potassium Channel Biogenesis

Abstract

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 aminoterminal deletion, ⌬97-196, 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 (␣) and cytoplasmic ␤ subunits, and 3) association of the amino and carboxyl termini of adjacent Shaker subunits. ⌬97-196 subunits failed to establish any of these quaternary interactions. The ⌬97-196 deletion also prevented formation of the pore. The other mutant subunits assembled into tetramers and associated with the ␤ 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.

Dissection of synaptic excitability phenotypes by using a dominant-negative Shaker K + channel subunit

“…Expression phenotypes resembled those of Sh mutations both physiologically and behaviorally. The data indicate that SDN functions in vivo as a dominant-negative suppressor of Sh, which is consistent with in vitro evidence (33,34,41). The observation that GFPtagged SDN does not accumulate at postsynaptic membrane sites supports the view that truncated subunits disrupt channel biogenesis before surface membrane insertion (58).…”

“…The resultant UAS-SDN vectors encoded a predicted protein with the first 246 aa of the Sh K ϩ channel, with residues 1-29 replaced by GFP. This protein resembles Sh1-246, a dominant-negative construct that suppresses Sh function in vitro (33,34). Embryonic transformation was performed by using standard methods (35).…”

“…SDN is a truncation of the wild-type Sh ␣-subunit after the first transmembrane helix ( Fig. 1 A and B) and is modeled after an in vitro-characterized dominant-negative construct (33,34,41). It retains the T1 domain, a conserved N-terminal region necessary for subunit oligomerization (41) and axonal targeting (42).…”

Conformational changes in the C terminus of Shaker K + channel bound to the rat Kvβ2-subunit

“…The functional role of the N-terminal domain was extensively studied during the last decade; it was found to be responsible for the process known as ''N-type inactivation'' in Shaker-like K ϩ channels (1,4,5). The N-terminal T1 domain promotes tetramerization of the Kv␣ subunits (Kv␣) in early channel ontogeny (6)(7)(8) and provides a platform for the binding of the Kv␤ subunit (Kv␤) (9). The functional role of the C-terminal region in Shaker-like Kv channels, however, is less clear.…”

Ile-177 and Ser-180 in the S1 Segment Are Critically Important in Kv1.1 Channel Function

“…Indeed, the mutations did not alter the consensus sites for the processing and maturation of Kv1.1 protein in transfected cells (22,23,29,30). Moreover, most of the wild type or mutated Kv1.1 polypeptides formed heteromultimeric complexes with TR-Kv1.1HA (Fig.…”