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We have constructed a series of deletion mutants of Kv1.3, a Shaker-like, voltage-gated K+ channel, and examined the ability of these truncated mutants to form channels and to specifically suppress full-length Kv1.3 currents. These constructs were expressed heterologously in both Xenopus oocytes and a mouse cytotoxic T cell line. Our results show that a truncated mutant Kv1.3 must contain both the amino terminus and the first transmembrane-spanning segment, S1, to suppress full-length Kv1.3 currents. Amino-terminal-truncated DNA sequences from one subfamily suppress K+ channel expression of members of only the same subfamily. The first 141 amino acids of the amino-terminal of Kv1.3 are not necessary for channel formation. Deletion of these amino acids yields a current identical to that of full-length Kv1.3, except that it cannot be suppressed by a truncated Kv1.3 containing the amino terminus and S1. To test the ability of truncated Kv1.3 to suppress endogenous K+ currents, we constructed a plasmid that contained both truncated Kv1.3 and a selection marker gene (mouse CD4). Although constitutively expressed K+ currents in Jurkat (a human T cell leukemia line) and GH3 (an anterior pituitary cell line) cells cannot be suppressed by this double-gene plasmid, stimulated (up-regulated) Shaker-like K+ currents in GH3 cells can be suppressed.

Crystal structures of Kv channels have given us a reasonably complete view of the structure of a mature Kv channel. However, details of its structure acquisition are missing. We now report on the biogenesis of secondary structure of the transmembrane segments and intervening linkers of Kv1.3. Using a combination of accessibility assays, both cysteine pegylation (Tu et al., 2007; Lu and Deutsch, 2005) and N-linked glycosylation (Mingarro et al., 2000), we derive the following principles of folding for nascent sequences within their native contexts. First, native helical transmembrane sequences initially form helices only within the distal 20A of the ribosomal tunnel near the exit port. We refer to this region as the α-zone. Helix formation in transmembrane segments thus occurs vectorially from N- to C-terminus as each segment moves sequentially into the α-zone. Second, linker sequences also form compact structures inside the α-zone even in the absence of helix formation of their C-terminal flanking transmembrane segments. Third, helical structures, whether transmembrane or linker segments, formed in the tunnel retain their helicity in the translocon. These principles emerge from a diversity of native transmembrane and linker sequences that comprise Kv1.3 and may therefore be applicable to protein biogenesis in general.[Supported by NIH grant GM 52302].References: Tu et al., Biochemistry 46: 8075, 2007; Lu and Deutsch, Biochemistry 44: 8230, 2005; Mingarro et al., BMC Cell Biol. 1: 3, 2000.

Voltage-gated K+ channels are tetrameric, but how the four subunits assemble is not known. We analyzed inactivation kinetics and peak current levels elicited for a variety of wild-type and mutant Kv1.3 subunits, expressed singly, in combination, and as tandem constructs, to show that 1) the dominant pathway involves a dimerization of dimers, and 2) dimer-dimer interaction may involve interaction sites that differ from those involved in monomer-monomer association. Moreover, using nondenaturing gel electrophoresis, we detected dimers and tetramers, but not trimers, in the translation reaction of Kv1.3 monomers.