Cooperativity between calmodulin-binding sites in Kv7.2 channels

Abstract

SummaryAmong the multiple roles assigned to calmodulin (CaM), controlling the surface expression of Kv7.2 channels by binding to two discontinuous sites is a unique property of this Ca 2+ binding protein.Mutations that interfere with CaM binding or the sequestering of CaM prevent this M-channel component from exiting the endoplasmic reticulum (ER), which reduces M-current density in hippocampal neurons, enhancing excitability and offering a rational mechanism to explain some forms of benign familial neonatal convulsions (BFNC). Previously, we identified a mutation (S511D) that impedes CaM binding while allowing the channel to exit the ER, hinting that CaM binding may not be strictly required for Kv7.2 channel trafficking to the plasma membrane. Alternatively, this interaction with CaM might escape detection and, indeed, we now show that the S511D mutant contains functional CaM-binding sites that are not detected by classical biochemical techniques. Surface expression and function is rescued by CaM, suggesting that free CaM in HEK293 cells is limiting and reinforcing the hypothesis that CaM binding is required for ER exit. Within the CaM-binding domain formed by two sites (helix A and helix B), we show that CaM binds to helix B with higher apparent affinity than helix A, both in the presence and absence of Ca 2+ , and that the two sites cooperate. Hence, CaM can bridge two binding domains, anchoring helix A of one subunit to helix B of another subunit, in this way influencing the function of Kv7.2 channels.

Lack of correlation between surface expression and currents in epileptogenic AB-calmodulin binding domain Kv7.2 potassium channel mutants

“…The EC 50 (nM) values obtained were: WT, 27.1 ± 1.2; R333Q, 30.1 ± 2.8; K526N, 55.9 ± 1.5, in the presence of Ca 2+ and WT, 11.0 ± 0.5; R333Q, 12.6 ± 1.3; K526N, 37.3 ± 2.4, in the absence of Ca 2+ (n ≥ 3). The data for WT are from [36]. Asterisks indicate values significantly different (*** = p < 0.001) versus WT.…”

“…The low concentration and the GST tag help prevent protein aggregation, and, with the use of this very sensitive assay, quantitative data for the interaction was obtained [10,36,37]. Dose–response binding curves were constructed titrating D-CaM fluorescence after adding increasing concentrations of GST-fusion, in the absence (10 mM EGTA added; Figure 4(c) right ) or in the presence of a physiologically relevant concentration of Ca 2+ (3.9 μM free Ca 2+ ; Figure 4(c) left ).…”

Uncoupling PIP2-calmodulin regulation of Kv7.2 channels by an assembly destabilizing epileptogenic mutation

“…In addition, besides the precedents for bridged configurations in other channels (Schumacher et al, 2001;Sarhan et al, 2012;Villarroel et al, 2014), the crystallographic structure of Kv7.1 [AB/CaM] has been trapped with CaM embracing helices A and B from different subunits (Sachyani et al, 2014). We have previously shown by whole-cell recording that precluding CaM binding to helix A or to helix B results in non-functional channels, but, remarkably, both mutant channels complement each other, restoring function when coexpressed (Alaimo et al, 2013a). Furthermore, at submicromolar concentrations, CaM links two Kv7.2 AB domains in vitro, anchoring helix A of one subunit to helix B of another subunit (Alaimo et al, 2013a).…”

“…We have previously shown by whole-cell recording that precluding CaM binding to helix A or to helix B results in non-functional channels, but, remarkably, both mutant channels complement each other, restoring function when coexpressed (Alaimo et al, 2013a). Furthermore, at submicromolar concentrations, CaM links two Kv7.2 AB domains in vitro, anchoring helix A of one subunit to helix B of another subunit (Alaimo et al, 2013a). Therefore, we consider reasonable the hypothesis that, by bringing together the AB helices from adjacent subunits thanks to helix D coiled-coil formation, each CaM lobe can engage with the complementary helix from a neighboring subunit.…”

Unconventional calmodulin anchoring site within the AB module of Kv7.2 channels

“…Quantitative binding data support a similar model for Kv7.2 channels in which the initial docking step takes place between the N-lobe and helix B under low or resting Ca 2+ levels . The current view is that CaM engagement with helix A is essential for Kv7.2 channel function, and that anchoring helix B to the N-lobe facilitates C-lobe binding to helix A, which is located distantly from helix B in the same or in another subunit of the tetrameric channel (Alaimo et al, 2009(Alaimo et al, , 2013a.…”