Rearrangements in the Relative Orientation of Cytoplasmic Domains Induced by a Membrane-anchored Protein Mediate Modulations in Kv Channel Gating

Abstract

Interdomain interactions between intracellular N and C termini have been described for various K ؉ channels, including the voltage-gated Kv2.1, and suggested to affect channel gating. However, no channel regulatory protein directly affecting N/C interactions has been demonstrated. Most Kv2.1 channel interactions with regulatory factors occur at its C terminus. The vesicular SNARE that is also present at a high concentration in the neuronal plasma membrane, VAMP2, is the only protein documented to affect Kv2.1 gating by binding to its N terminus. As its binding target has been mapped near a site implicated in Kv2.1 N/C interactions, we hypothesized that VAMP2 binding to the N terminus requires concomitant conformational changes in the C terminus, which wraps around the N terminus from the outside, to give VAMP2 access. Here, we first determined that the Kv2.1 N terminus, although crucial, is not sufficient to convey functional interaction with VAMP2, and that, concomitant to its binding to the "docking loop" at the Kv2.1 N terminus, VAMP2 binds to the proximal part of the Kv2.1 C terminus, C1a. Next, using computational biology approaches (ab initio modeling, docking, and molecular dynamics simulations) supported by molecular biology, biochemical, electrophysiological, and fluorescence resonance energy transfer analyses, we mapped the interaction sites on both VAMP2 and Kv2.1 and found that this interaction is accompanied by rearrangements in the relative orientation of Kv2.1 cytoplasmic domains. We propose that VAMP2 modulates Kv2.1 inactivation by interfering with the interaction between the docking loop and C1a, a mechanism for gating regulation that may pertain also to other Kv channels.Interdomain interactions between intracellularly located N and C termini have been described for various K ϩ channels, including inwardly rectifying Kir2.3 and Kir6.2 (1, 2), small conductance Ca 2ϩ -activated (hSK3) (3), and voltage-gated Kv2.1 (4) and Kv4.1 (5) channels. In the case of Kv2.1, two modes of interaction have been proposed: an association of the distal part of Kv2.1 C terminus (termed CTA domain; amino acids (aa) 741-853) 4 with aa 67 and 75 of the Kv2.1 N terminus (4); or an association between the proximal part of the Kv2.1 C terminus (aa 444 -477) and the predicted loop structure (aa 55-71) in the N-terminal T1 domain (6). In addition, involvement of the S4-S5 linker in this interaction has been suggested (7). Although these studies propose two different C-terminal sites, they indicate a specific loop in the N terminus of Kv2.1 (6, 8), which could be functionally related to the Shaker and Shal docking loops in the lateral part of their T1 domains (9, 10). These latter loops are responsible for the subfamily-specific association with ␤-subunits (Kv␤ and KChIP, respectively). Further, the interaction between the N and C cytoplasmic termini (N/C interaction) of Kv2.1 has been shown to be dynamic and voltage-dependent and to involve structural rearrangements between these domains, which could affect both activati...

Regulation of Neuronal M-Channel Gating in an Isoform-Specific Manner: Functional Interplay between Calmodulin and Syntaxin 1A

“…Notably, N-C close associations that are involved in the gating and activation of many channel types have been demonstrated (Varnum and Zagotta, 1997;Schulte et al, 1998;Tucker and Ashcroft, 1999;Qu et al, 2000;Lippiat et al, 2002;Tsuboi et al, 2004;Kobrinsky et al, 2006). Furthermore, using FRET analysis, our laboratory has recently demonstrated that a Kv2.1 regulatory protein modulates channel gating via effects on the association between the termini (Lvov et al, 2009). Here, pulldown assays (Fig.…”

“…Channel gating modulators have been shown to alter the relative interactions between intracellular channel domains (Lippiat et al, 2002;Tsuboi et al, 2004;Lvov et al, 2009). Thus, we set out to investigate whether the Q2 gating downregulation by Syx is accompanied by specific changes in the N-C interactions.…”

“…We used the FRET approach to monitor the N-C proximity in functional intact channels under physiological conditions. Cross talk was assessed by the apparent FRET efficiency (FRET) between eCFP (C) and eYFP (Y) (as done previously; Lvov et al, 2009), genetically attached to the distal ends of NTs and CTs, respectively, of Q2, Q3*, and the chimeric channels. The double-labeled channels retained WT voltage sensitivity [ Fig.…”

“…FRET experiments were performed as described previously (Lvov et al, 2009). Oocytes were injected with the following mRNA concentrations (in ng/oocyte): 45 double-labeled Q2, Q3*, or mutant channels, 0.75 Syx, or 5 CaM.…”

Regulation of neuronal KCNQ2 channel by Src: dual rearrangement of cytosolic termini underlies bidirectional gating regulation

“…Indeed, channel gating modulators have been shown to alter N-terminus-C-terminus interactions (Lippiat et al, 2002;Tsuboi et al, 2004;Lvov et al, 2009;Etzioni et al, 2011). Specifically, constitutive N-terminus-C-terminus interactions have been characterized in Q2 and Q3, and have been shown in Q2 to be modulated by CaM and syntaxin, accompanying changes in Q2 Po (Etzioni et al, 2011).…”