Modulation of KV4.3-KChIP2 Channels by IQM-266: Role of DPP6 and KCNE2

The transient outward potassium current (Itof) is generated by the activation of KV4 chan- nels assembled with KChIP2 and other accessory subunits (DPP6 and KCNE2). To test the hypothesis that these subunits modify the channel pharmacology, we analyzed the electrophysiological effects of (3-(2-(3-phenoxyphenyl)acetamido)-2-naphthoic acid) (IQM-266), a new KChIP2 ligand, on the currents generated by KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 channels. CHO cells were transiently transfected with cDNAs codifying for different proteins (KV4.3/KChIP2, KV4.3/KChIP2/DPP6 or KV4.3/KChIP2/KCNE2), and the potassium currents were recorded using the whole-cell patch-clamp technique. IQM-266 decreased the maximum peak of KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 currents, slowing their time course of inactivation in a concentration-, voltage-, time- and use-dependent manner. IQM-266 produced an increase in the charge in KV4.3/KChIP2 channels that was intensified when DPP6 was present and abolished in the presence of KCNE2. IQM-266 induced an activation unblocking effect during the applica- tion of trains of pulses to cells expressing KV4.3/KChIP2 and KV4.3/KChIP2/KCNE2, but not in KV4.3/KChIP2/DPP6 channels. Overall, all these results are consistent with a preferential IQM-266 binding to an active closed state of Kv4.3/KChIP2 and Kv4.3/KChIP2/KCNE2 channels, whereas in the presence of DPP6, IQM-266 binds preferentially to an inactivated state. In conclusion, DPP6 and KCNE2 modify the pharmacological response of KV4.3/KChIP2 channels to IQM-266.This publication is the results of the: Grants SAF2016-75021-R (to C.V.), RTI2018-097189-B- C22 (to M.M.-M.) and BIO2017-89523-R (to A.A.) funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”; Grants PID2019-104366RB-C21 (to C.V.), PID2019-104366RB- C22 (to M.G.-R.), PID2020-114256RB-I00 (to A.O. and J.A.G.-V.), PID2020-119805RB-I00 (to A.A.) funded by MCIN/AEI/10.13039/501100011033; Grant A-FQM-386-UGR20 funded by FEDER/Junta de Andalucía-Consejería de Transformación Económica, Industria, Conocimiento (to J.A.G.-V.); Grant CB/11/00222 funded by Instituto de Salud Carlos III CIBERCV (to C.V.); Grants PIE202180E073 (to M.M.-M. and M.G.-R.), PIE201820E104 and 2019AEP148 (to C.V.) funded by Consejo Superior de Investigaciones Científicas. Grants BES-2017-080184 (to A.d.B.-B.), BES-2010-036573 (to P.C.), PRE2018- 083280 (to M.D.-M.) and RYC2018-023837-I (to A.P.-L.) funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”; Grant FPU17/02731 (to P.G.S.) funded by Ministerio de Ciencia e Innovación.Peer reviewe

Identification of IQM-266, a Novel DREAM Ligand That Modulates KV4 Currents

Downstream Regulatory Element Antagonist Modulator (DREAM)/KChIP3/calsenilin is a neuronal calcium sensor (NCS) with multiple functions, including the regulation of A-type outward potassium currents (IA). This effect is mediated by the interaction between DREAM and KV4 potassium channels and it has been shown that small molecules that bind to DREAM modify channel function. A-type outward potassium current (IA) is responsible of the fast repolarization of neuron action potentials and frequency of firing. Using surface plasmon resonance (SPR) assays and electrophysiological recordings of KV4.3/DREAM channels, we have identified IQM-266 as a DREAM ligand. IQM-266 inhibited the KV4.3/DREAM current in a concentration-, voltage-, and time-dependent-manner. By decreasing the peak current and slowing the inactivation kinetics, IQM-266 led to an increase in the transmembrane charge (QKV4.3/DREAM) at a certain range of concentrations. The slowing of the recovery process and the increase of the inactivation from the closed-state inactivation degree are consistent with a preferential binding of IQM-266 to a pre-activated closed state of KV4.3/DREAM channels. Finally, in rat dorsal root ganglion neurons, IQM-266 inhibited the peak amplitude and slowed the inactivation of IA. Overall, the results presented here identify IQM-266 as a new chemical tool that might allow a better understanding of DREAM physiological role as well as modulation of neuronal IA in pathological processes

Chemical tolls to modulate kchip3 signaling

Trabajo presentado en el 19th Meeting SEQT, celebrado en Vitoria-Gasteiz (España) del 8 al 11 de julio de 2019.DREAM (Downstream Regulatory Element Antagonist Modulator), also known as KChIP-3 or calsenilin, is a multifunctional calcium binding protein that controls the expression level and/or the activity of several proteins related to calcium homeostasis, neuronal excitability and neuronal survival. As an auxiliary protein in the plasma membrane, DREAM interacts with KV4 potassium channels, L- and T-type voltage-dependent calcium channels, NMDA receptors, presenilins and the transcriptor factor ATF6.1 The interaction between DREAM and KV4 potassium channels regulates of A-type outward potassium currents (IA) that is responsible of the fast repolarization of neuron action potentials and frequency of firing.2 Using a multidisciplinary approach that involves drug design, organic chemistry, surface plasmon resonance assays, electrophysiological recordings of KV4.3/DREAM channels, and IA recordings in rat dorsal root ganglion neurons, we have identified IQM-266.3 IQM-266 slows the inactivation kinetics, and this effect may explain why at concentrations lower than the IC50, IQM-266 augments the efflux of potassium ions resulting in an increase of the charge (activating effect). This effect could be the basis of a promising therapeutic strategy for the treatment of certain neuronal pathologies (epilepsy, Alzheimer disease or ataxia), in which a downregulation of KV4.3 or DREAM has been demonstrated.4 IQM-266 also modulated IA from rat DRG neurons. Overall, IQM-266 constitutes a novel small chemical tool suitable to modulate KV4.3 channels in native systems, that might allow a better understanding of DREAM physiological roles, as well as modulation of neuronal IA current in pathological processes

Identification of IQM-266, a novel DREAM ligand that modulates KV4 currents

Downstream Regulatory Element Antagonist Modulator (DREAM)/KChIP3/calsenilin is a neuronal calcium sensor (NCS) with multiple functions, including the regulation of A-type outward potassium currents (I A). This effect is mediated by the interaction between DREAM and KV4 potassium channels and it has been shown that small molecules that bind to DREAM modify channel function. A-type outward potassium current (I A) is responsible of the fast repolarization of neuron action potentials and frequency of firing. Using surface plasmon resonance (SPR) assays and electrophysiological recordings of KV4.3/DREAM channels, we have identified IQM-266 as a DREAM ligand. IQM-266 inhibited the KV4.3/DREAM current in a concentration-, voltage-, and time-dependent-manner. By decreasing the peak current and slowing the inactivation kinetics, IQM-266 led to an increase in the transmembrane charge ( QKV4.3/DREAM ) at a certain range of concentrations. The slowing of the recovery process and the increase of the inactivation from the closed-state inactivation degree are consistent with a preferential binding of IQM-266 to a pre-activated closed state of KV4.3/DREAM channels. Finally, in rat dorsal root ganglion neurons, IQM-266 inhibited the peak amplitude and slowed the inactivation of I A. Overall, the results presented here identify IQM-266 as a new chemical tool that might allow a better understanding of DREAM physiological role as well as modulation of neuronal I A in pathological processes.PC was the recipient of a postgraduate FPI fellowship from the Spanish Ministry of Economy, Industry and Competitivity (MINECO). This work was funded by the Spanish Ministry of Economy, Industry and Competitivity (Ministerio de Economía y Competitividad; AEI-FEDER, EU grants): SAF2012-32209 and BFU2015-67284-R (to MG-R), SAF2014-53412-R and SAF2017- 89554-R (to JN), SAF2013-45800-R, SAF2016-75021-R (to CV) and SAF2015-66275-C2-2-R (to MM-M); Universidad Complutense de Madrid (UCM) grant: PR75/18-21593 (to AA); the Instituto de Salud Carlos III CIBERNED and CIBERCV programs (to JN and to CV, respectively) and the Madrid regional government/Neurodegmodels (to JN); Consejo Superior de Investigaciones Científicas (CSIC) grants: PIE 201820E104 (to CV) and 201880E109 (to MG-R and MM-M).We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Identification of IQM-266, a novel DREAM ligand that modulates Kv4 currents

Resumen del trabajo presentado al VII Congreso Red Española Canales Iónicos: Symposium 6: Drug Discovery and ion channel Pharmacology, celebrado en Cáceres del 15 al 17 de mayo de 2019.Funded by BFU2015-67284-R (to MG-R), SAF2014-53412-R and SAF2017-89554-R (to JN), SAF2013-45800-R, SAF2016-75021-R (to CV), SAF2015-66275-C2-2-R (to MM-M); UCM grant: PR75/18-21593 (to AA); and CSIC grants: PIE 201820E104 (to CV) and 201880E109 (to MG-R and MM-M).Peer Reviewe