Abstract
Kv7.2 (KCNQ2) is the principal molecular component of the slow voltage gated M-channel, which strongly influences neuronal excitability. Calmodulin (CaM) binds to two intracellular C-terminal segments of Kv7.2 channels, helices A and B, and it is required for exit from the endoplasmic reticulum. However, the molecular mechanisms by which CaM controls channel trafficking are currently unknown. Here we used two complementary approaches to explore the molecular events underlying the association between CaM and Kv7.2 and their regulation by Ca2+. First, we performed a fluorometric assay using dansylated calmodulin (D-CaM) to characterize the interaction of its individual lobes to the Kv7.2 CaM binding site (Q2AB). Second, we explored the association of Q2AB with CaM by NMR spectroscopy, using 15N-labeled CaM as a reporter. The combined data highlight the interdependency of the N- and C-lobes of CaM in the interaction with Q2AB, suggesting that when CaM binds Ca2+ the binding interface pivots between the N-lobe whose interactions are dominated by helix B and the C-lobe where the predominant interaction is with helix A. In addition, Ca2+ makes CaM binding to Q2AB more difficult and, reciprocally, the channel weakens the association of CaM with Ca2+.
Highlights
Calmodulin (CaM) orchestrates cell function by interacting with a large number of different proteins, conferring them Ca2+dependent regulation [1;2]
Upon Ca2+ binding, CaM changes its conformation from the closed configuration to an open one, exposing the hydrophobic surfaces within the N- and C-lobes for Ca2+-dependent interactions with target proteins [8]
The structural malleability of CaM is extraordinary, as highlighted by the complexes with two variants of the SK2 potassium channel, where different sets of amino acid residues contribute to the hydrophobic interface at CaM, even though the interacting amino acids on the targets remain the same [6;9]
Summary
Calmodulin (CaM) orchestrates cell function by interacting with a large number of different proteins, conferring them Ca2+dependent regulation [1;2]. The lobes of CaM are relatively rigid when Ca2+ is bound, segmental inter-domain motion has been described [7] Such structural flexibility explains how CaM is capable of interacting with target proteins with different structural features [8]. Upon Ca2+ binding, CaM changes its conformation from the closed configuration to an open one, exposing the hydrophobic surfaces within the N- and C-lobes for Ca2+-dependent interactions with target proteins [8]. The structure of helix B from Kv7.4 complexed with holo-CaM was recently resolved [22], the association of CaM with the Kv7.2 binding domain clearly differs from its interaction with the individual segments [25]. Helix A, an essential component of the Kv7 CaM binding domain, is not present in the resolved complex, this structure provides valuable information on how CaM interacts with the channel. By comparing the residues affected by these interactions, as seen by NMR, and known disease causing mutations, we elaborate a model of the Ca2+-dependent regulation
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