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

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⁺ channels, including inwardly rectifying Kir2.3 and Kir6.2 (1, 2), small conductance Ca²⁺-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 β-subunits (Kvβ 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 activation and inactivation gating of the channel (4, 6, 7). These rearrangements can be clearly detected with fluorescence resonance energy transfer (FRET) (11). A similar N/C interaction has been shown to affect gating of the closely related Kv4.1 channel (5, 12).It is conceivable that the specific packaging of Kv2.1 cytoplasmic termini (a relatively long C terminus (>400 aa) wrapping the N terminus (<190 aa) from the outside (4)) not only supports multiple interactions between the termini but also reflects the fact that most of the interactions of the channel with intracellular and membrane-bound regulatory factors occur at the C terminus, including channel phosphorylation (13–15), clustering through a unique proxinal restriction and clustering signal (16), and protein-protein interactions with both the plasma membrane SNAREs, syntaxin 1A and SNAP-25 (17–19), and the MiRP2 (KCNE3) peptide (20). For the Kv2.1 N terminus, on the other hand, there are only two examples of protein-protein interactions: a transient association with KChAP (21), which does not affect channel function; and an interaction with the vesicular SNARE partner VAMP2 (synaptobrevin 2), which is also present at a high concentration in the neuronal plasma membrane and enhances channel inactivation (8). Specifically, VAMP2 has been shown to associate with the extension of a docking loop in the lateral part of the T1 domain (8) near the site of interaction with the C terminus (4, 6). Thus, it is reasonable to hypothesize that interaction with VAMP2 will affect the N/C interaction, similar to proton-mediated Kir2.3 (1) and Kir1.1 (22) N/C interactions or the ATP-dependent Kir6.2 (2) N/C interaction. To date, no protein molecule that directly affects N/C interactions in a K⁺ channel has been demonstrated. Because VAMP2 was the first protein documented to affect Kv2.1 channel gating by binding to a specific N-terminal site, which is probably masked by the C terminus, we have put forward the idea that its interaction with the Kv2.1 N terminus requires conformational changes in the C terminus that will enable its access to the N terminus.Here we endeavored to gain a mechanistic and structural understanding of the Kv2.1-VAMP2 interaction. Based on our evidence, we propose that VAMP2 modulates Kv2.1 gating by interfering with the Kv2.1 cytoplasmic N/C interaction.