Kv7 Channels Can Function without Constitutive Calmodulin Tethering

M-channels are voltage-gated potassium channels composed of Kv7.2-7.5 subunits that serve as important regulators of neuronal excitability. Calmodulin binding is required for Kv7 channel function and mutations in Kv7.2 that disrupt calmodulin binding cause Benign Familial Neonatal Convulsions (BFNC), a dominantly inherited human epilepsy. On the basis that Kv7.2 mutants deficient in calmodulin binding are not functional, calmodulin has been defined as an auxiliary subunit of Kv7 channels. However, we have identified a presumably phosphomimetic mutation S511D that permits calmodulin-independent function. Thus, our data reveal that constitutive tethering of calmodulin is not required for Kv7 channel function

Surface Expression and Subunit Specific Control of Steady Protein Levels by the Kv7.2 Helix A-B Linker

12 p.Kv7.2 and Kv7.3 are the main components of the neuronal voltage-dependent M-current, which is a subthreshold potassium conductance that exerts an important control on neuronal excitability. Despite their predominantly intracellular distribution, these channels must reach the plasma membrane in order to control neuronal activity. Thus, we analyzed the amino acid sequence of Kv7.2 to identify intrinsic signals that may control its surface expression. Removal of the interlinker connecting helix A and helix B of the intracellular C-terminus produces a large increase in the number of functional channels at the plasma membrane. Moreover, elimination of this linker increased the steady-state amount of protein, which was not associated with a decrease of protein degradation. The magnitude of this increase was inversely correlated with the number of helix A - helix B linkers present in the tetrameric channel assemblies. In contrast to the remarkable effect on the amount of Kv7.2 protein, removal of the Kv7.2 linker had no detectable impact on the steady-state levels of Kv7.3 protein.This work was supported by grants from the VII European framework program managed by the Fondo de Investigaciones Sanitarias (PI071316), from the Spanish Ministry of Education (BFU2009-07581 and SAF2006-1450), the Spanish Ion Channel Initiative Consolider project (CSD2008-00005), and the Basque Government (SAIOTEK SA-2006/00023). A. Alaimo was partially funded by Fundacion Biofisica Bizkaia. PA and JFO held a FPI fellowship from the Spanish Ministry of Science and Innovation (BES-2008-002314). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Model for the Ca²⁺-dependent CaM/Q2AB interaction.

<p><i>(A)</i> Sequence alignment of segments A and B in which L339, R353 and S511 are underlined. The predicted secondary structure of Kv7.2 according to the GORV algorithm is indicated above the sequence (<a href="http://gor.bb.iastate.edu/" target="_blank">http://gor.bb.iastate.edu/</a>, h = alpha helix, e = extended, c = coiled). The circle beneath a residue indicates that it contacts the N-lobe, while those in contact with the C-lobe are indicated with a square, both of which are color coded according to the CaM surface contact. The contact surface area has been estimated using the Sobolev <i>et al.</i> algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Sobolev1" target="_blank">[52]</a>. <i>(B)</i> Interaction model. The Q2AB helices are depicted as rectangles, the CaM lobes as ovals. Binding to CaM is transient <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Mruk1" target="_blank">[23]</a>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-GomezPosada1" target="_blank">[24]</a>, and the interaction with helix A is critical for function <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Alaimo1" target="_blank">[25]</a>. Given the greater affinity for helix B <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Xu1" target="_blank">[22]</a>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Alaimo1" target="_blank">[25]</a>, it is more likely that CaM docks initially to this helix <i>via</i> the N-lobe, facilitating the interactions between the C-lobe and helix A. Subsequently, a dynamic equilibrium is established: 1.- In the absence of Ca²⁺ the N-lobe dominates the interaction and initially binds to helix B. 2.- Subsequently, the C-lobe engages, establishing an equilibrium between binding to helix A and helix B. 3.- In the presence of Ca²⁺ the C-lobe binds to the IQ site of helix A. 4.- The holo-N-lobe alternates between helix A and helix B. Upon calcification, the interaction between helix B and the N lobe is weakened and the binding between helix A and the C-lobe becomes more significant. Concomitantly, the global affinity in the presence of Ca²⁺ is reduced.</p