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 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.
Highlights
The Shaker Kϩ channel is a member of the superfamily of voltage-dependent cation channels that control the excitability
The ⌬97–196, E418K, and C462K proteins did not form functional channels and failed to mature (Fig. 1B). These results indicate that proper formation of the assembly domain, the voltage sensor, and the pore are required for maturation, whereas the inactivation ball and chain can be deleted with no apparent detrimental effects on biogenesis
Shaker protein was precipitated only with Shaker antibodies, and in the reciprocal experiment, the  subunit was precipitated only with the Kv2 antibody (Fig. 7). These results demonstrate that Shaker subunits with mutations in the voltage sensor or pore region assemble with  subunits and that the association is mediated by residues in the Shaker amino terminus
Summary
Mutagenesis and Expression of Shaker Protein—Point mutations and ⌬97–196, a deletion of amino acids 97–196, were engineered into a pBluescript II (Stratagene, La Jolla, CA) subclone of the Shaker B cDNA [33] using polymerase chain reaction methods [34, 35]. Oocytes were prepared and injected as described previously [37]. Ice-cold lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM HEPES-NaOH, pH 7.4), supplemented with protease inhibitors as described previously [28], was added directly to the tissue culture plates. Cross-linking—Solubilized Shaker protein was incubated with 100 M 3,3Ј-dithiobis(sulfosuccinimidyl-propionate) (DTSSP) (Pierce) for 30 min at room temperature. Intact Xenopus oocytes expressing Shaker protein were suspended in modified phosphate-buffered saline, pH 7.4 [5], and incubated with 1 mM bismaleimidohexane (BMH) (Pierce), a membrane-permeable cross-linking reagent, for 10 min at room temperature. Immunoprecipitation, Electrophoresis, and Fluorography—Immunoprecipitation of Shaker or Kv2 protein was performed as described previously [29] using an antibody directed against the Shaker--galactosidase fusion protein Gels were stained with Coomassie Blue, soaked in fluorographic enhancer, dried, and exposed to film
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