Both N- and C-terminal regions contribute to the assembly and functional expression of homo- and heteromultimeric voltage-gated K+ channels

Abstract

The functional diversity of voltage-gated K+ channels may be partially determined by the mechanisms that permit or limit the assembly of molecularly diverse K+ channel subunits. To determine possible amino acid sequence domains required for subunit assembly and expression, we have constructed 15 N- and C-terminal interstitial or truncation deletion mutations in mKv1.1 (MBK1), a mouse Shaker-like K+ channel. We injected Xenopus oocytes with cRNA encoding each of these mutants and coinjected each mutant cRNA with cRNA for wild-type mKv1.3, another mouse Shaker-like K+ channel that can form heteromultimers with mKv1.1. We found that the last five amino acids of the C-terminus of mKv1.1 contribute to functional expression by (1) rescuing the function of mutants with a large truncation of the C-terminus (delta 424–495), and (2) contributing to the slow inactivation kinetics (time constant of 2– 3 sec) of wild-type mKv1.1 whole-cell K+ currents. All C-terminal deletion mutants were able to express at least as heteromultimers with mKv1.3, suggesting that the C-terminus is not required for channel assembly. In contrast, nine different interstitial or truncation mutants in which part of a highly conserved, large (80–99 amino acid residues) domain within the N-terminus had been deleted were unable to express either homomultimers or heteromultimers. The relatively small sizes and nonoverlapping distributions of the interstitial deletions enable us to suggest that the structural integrity of this entire N- terminal domain is required for subunit assembly and functional expression of this and probably other Shaker-like K+ channel proteins.

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

“…The surface expression level in COS-1 cells was determined by using a whole-cell [ 3 H]NEMagitoxin-binding assay and was similar for both WT and ⌬C mutant channels, although slightly higher for the WT channel (Table 1), consistent with previous results for other Shaker-like K ϩ channels (10,37). The yield of correctly folded truncated Shaker channels after affinity purification was 42% of the total protein, compared with Ϸ30% for the WT channel (17).…”

“…It is known that in Shaker-like K ϩ channels, C termini are involved in a variety of processes that range from channel gating (10,11) and voltage sensitivity (4) to the binding of the membrane-associated guanylate kinases (12). They also participate in channel assembly in Kv 2.1 (13,14).…”

“…They also participate in channel assembly in Kv 2.1 (13,14). All members of the Shaker subfamily of Kv channels display a remarkable amino acid sequence conservation within the distal region of their C termini, characterized by the -T͞SDV motif (10,12).…”

Voltage-gated ion channels in the axon initial segment of human cortical pyramidal cells and their relationship with chandelier cells

“…They contribute to the repolarization of single-action potentials, they modulate action potential duration and frequency, and they maintain the internodal resting potential (29,34,45,46). Juxtaparanodal Kv channels are composed of various heteromultimeric combinations of pore-forming Kv1 ␣ (Kv 1.1, 1.2, 1.4, and 1.6) and the associated cytoplasmic Kv␤2 subunits (42,44,47,48). In the juxtaparanodal region, Kv1.1 and Kv1.2 have been shown to colocalize with, and to interact and͞or cluster with, Caspr2, a member of the neurexin superfamily (49,50).…”

Shaker K+ Channel T1 Domain Self-tetramerizes to a Stable Structure

“…Recently we and others have identified a conserved molecular domain, the T1 domain, encoded within the cytoplasmic N terminus of the ␣-subunit protein that plays an important role in the assembly of K ϩ channel subunit proteins (5)(6)(7)(8)(9)(10). Our studies have suggested that the T1 domain, translated by itself, can self-assemble (6).…”