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

A pore residue of the KCNQ3 potassium M-channel subunit controls surface expression

KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) are the principal subunits underlying the potassium M-current, which exerts a strong control on neuronal excitability. KCNQ3 subunits coassemble with KCNQ2 to form functional heteromeric channels that are specifically transported to the axonal initial segment and nodes of Ranvier. In contrast, there is no evidence for functional homomeric KCNQ3 channels in neurons, and it appears that these are inefficiently trafficked to the plasma membrane. Among eukaryotic potassium channels, the KCNQ3 subunit is unusual because it has an alanine in place of a threonine at the pore inner vestibule, three residues upstream of the GYG signature sequence of the selectivity filter. This residue is critical for the potentiation of the current after heteromerization, but the mechanism is unknown. We report that the presence of this uncommon residue at position 315 has a strong impact on the stability of the homotetramers and on channel trafficking. Wild-type KCNQ3 expressed alone is retained within the endoplasmic reticulum, and this mechanism is overcome by the substitution of threonine for Ala315. KCNQ3 subunits require assembly with KCNQ2 to exit this compartment, whereas KCNQ3-A315T is no longer dependent on KCNQ2 to form channels that are efficiently trafficked to the plasma membrane. The presence of this alanine, therefore, plays an important role in regulating the subunit composition of functional M-channels expressed at the surface of neurons

Functional assembly of Kv7.1/Kv7.5 channels with emerging properties on vascular muscle physiology

et al.[Objective]: Voltage-dependent K (Kv) channels from the Kv7 family are expressed in blood vessels and contribute to cardiovascular physiology. Although Kv7 channel blockers trigger muscle contractions, Kv7 activators act as vasorelaxants. Kv7.1 and Kv7.5 are expressed in many vessels. Kv7.1 is under intense investigation because Kv7.1 blockers fail to modulate smooth muscle reactivity. In this study, we analyzed whether Kv7.1 and Kv7.5 may form functional heterotetrameric channels increasing the channel diversity in vascular smooth muscles. [Approach and Results]: Kv7.1 and Kv7.5 currents elicited in arterial myocytes, oocyte, and mammalian expression systems suggest the formation of heterotetrameric complexes. Kv7.1/Kv7.5 heteromers, exhibiting different pharmacological characteristics, participate in the arterial tone. Kv7.1/Kv7.5 associations were confirmed by coimmunoprecipitation, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching experiments. Kv7.1/Kv7.5 heterotetramers were highly retained at the endoplasmic reticulum. Studies in HEK-293 cells, heart, brain, and smooth and skeletal muscles demonstrated that the predominant presence of Kv7.5 stimulates release of Kv7.1/Kv7.5 oligomers out of lipid raft microdomains. Electrophysiological studies supported that KCNE1 and KCNE3 regulatory subunits further increased the channel diversity. Finally, the analysis of rat isolated myocytes and human blood vessels demonstrated that Kv7.1 and Kv7.5 exhibited a differential expression, which may lead to channel diversity. [Conclusions]: Kv7.1 and Kv7.5 form heterotetrameric channels increasing the diversity of structures which fine-tune blood vessel reactivity. Because the lipid raft localization of ion channels is crucial for cardiovascular physiology, Kv7.1/Kv7.5 heteromers provide efficient spatial and temporal regulation of smooth muscle function. Our results shed light on the debate about the contribution of Kv7 channels to vasoconstriction and hypertension.This work was supported by the Ministerio de Ciencia e Innovación (MINECO), Spain (BFU2011-23268 to A. Felipe, and BFU2012-39883 to A. Villarroel; CSD2008-00005 to A. Felipe and A. Villarroel; SAF2010-14916 and RD12/0042/0019 to C. Valenzuela; BFU2010-15674 to C. Soler). L. Solé, A. Oliveras, and A. Prieto are fellows from MINECO. N. Comes and A. de la Cruz are supported by Juan de la Cierva program (MINECO) and CSIC contract, respectively.Peer Reviewe

Kv7.1/Kv7.5 heterotetramers with emerging properties on vascular smooth muscle physiology

Resumen del trabajo presentado al 37th Congress of the Spanish Society of Physiological Sciences (SECF), celebrado del 24 al 26 de Septiembre de 2014 en Granada (España):-- et al.[Introduction]: Voltage-dependent K+ channels from Kv7 (KCNQ) family have well-established physiological roles in cardiovascular and nervous system, although functions in blood vessels remain unclear. Evidence suggests that Kv7.1, Kv7.4 and Kv7.5 control vascular reactivity. However, because controversial pharmacological results Kv7.1 is under intense investigation. In this scenario, the ability of Kv7 channels to form heterotetramers is of physiological relevance. Thus, Kv7.4/Kv7.5 heterotetramers paves the way for novel interaction that could shed light to controversial pharmacological results. We aim whether Kv7.1 and Kv7.5 form heterotetramers increasing the diversity of channel responses in vascular smooth muscle. [Methods and Results]: We demonstrated Kv7.1/Kv7.5 structures in heterologous system by many different approaches, such as electrophysiology, coimmunoprecipitation and FRET. Heteromeric channels are retained at the endoplasmatic reticulum and, unlike Kv7.1 channels, heteromers localize out of lipid rafts. These results are supported by experiments in isolated smooth muscle myocytes. Kv7.1 and Kv7.5 are expressed in aorta, cava and coronary myocytes. Electrophysiological and miography recordings using linopiridine, chromanol 293B and retigabine support Kv7.1/Kv7.5 heterotetramers that co-immunoprecipitation experiments further confirmed. Finally, lipid raft isolation from different tissues corroborated that expression of Kv7.5 releases Kv7.1/Kv7.5 channels out of raft structures. [Discussion]: Kv7.1 and Kv7.5 are differentially expressed in blood vessels where they contribute to control vascular reactivity. We prove that they do heterotetramerize increasing the diversity of their physiological response. These data may help to better understand the scenario of Kv7 channels and vascular physiology.Supported by BFU2011-23268 and CSD2008-00005 to AF (MINECO, Spain)Peer Reviewe

Calmodulin Activation Limits the Rate of KCNQ2 K+ Channel Exit from the Endoplasmic Reticulum*

The potential regulation of protein trafficking by calmodulin (CaM) is a novel concept that remains to be substantiated. We proposed that KCNQ2 K+ channel trafficking is regulated by CaM binding to the C-terminal A and B helices. Here we show that the L339R mutation in helix A, which is linked to human benign neonatal convulsions, perturbs CaM binding to KCNQ2 channels and prevents their correct trafficking to the plasma membrane. We used glutathione S-transferase fused to helices A and B to examine the impact of this and other mutations in helix A (I340A, I340E, A343D, and R353G) on the interaction with CaM. The process appears to require at least two steps; the first involves the transient association of CaM with KCNQ2, and in the second, the complex adopts an “active” conformation that is more stable and is that which confers the capacity to exit the endoplasmic reticulum. Significantly, the mutations that we have analyzed mainly affect the stability of the active configuration of the complex, whereas Ca2+ alone appears to affect the initial binding step. The spectrum of responses from this collection of mutants revealed a strong correlation between adopting the active conformation and channel trafficking in mammalian cells. These data are entirely consistent with the concept that CaM bound to KCNQ2 acts as a Ca2+ sensor, conferring Ca2+ dependence to the trafficking of the channel to the plasma membrane and fully explaining the requirement of CaM binding for KCNQ2 function