Potassium channels are essential for life. They are key players of many vital functions as diverse as those of the heart, brain, muscle, kidney, pancreas or immune system. Members of the Kv7 family of voltage-gated potassium channels play a major role in modulating brain and cardiac excitability. Consistent with their physiological importance, mutations in human Kv7 genes lead to severe inherited cardiovascular and neurological disorders such as cardiac arrhythmia, neonatal epilepsy and deafness. Our research aims at elucidating the structural, biophysical and physiological attributes of normal and diseased Kv7 channel proteins in brain and heart. We apply the state of the art technologies, including voltage- and current-clamp modes of the patch-clamp technique, simultaneous optical and electrophysiological recording (FRET spectral analysis and voltage-clamp), TIRF imaging and various biochemical methods (pull-down, ITC and MST) and in collaboration with Dr. Joel Hirsch, X-ray crystallographic techniques.
Using the powerful combination of electrophysiology, cell biophysics, molecular biology and biochemistry, we study the properties of a family of voltage-gated potassium channel proteins, Kv7 (KCNQ), whose mutations in humans lead to major neurological and cardiovascular disorders like epilepsy, deafness and cardiac arrhythmias (long QT syndrome). Our research aims at elucidating the structural, biophysical and physiological characteristics of neuronal and cardiac Kv7 channels.
1-We triggered a unique form of homeostatic plasticity by targeting M-channels (Kv7.2/3), which regulate neuronal excitability and are prominently localized in the axon initial segment (AIS). Protracted exposure of hippocampal pyramidal neurons to the M-channel blocker XE-991 rapidly moved away from the soma, axonal Na+ channels and Kv7.3 K+ channels but not Ankyrin G. M-channel blockade induced fast adaptive changes in intrinsic excitability with a distal shift in the spike trigger zone as measured by dual patch recording. The rapid homeostatic changes in AIS induced by M-current inhibition were contingent on the crucial AIS component, casein kinase 2. Thus, alterations to M-channel activity rapidly trigger unique homeostatic plasticity features to stabilize network excitability. We are also studying these plasticity mechanisms in dorsal root ganglion neurons.
2-We revealed the competition of PIP2 and the calcified form of the calmodulin N-lobe to a previously unidentified site in helix B of the proximal Kv7.1 C-terminus in the cardiac KV7.1 channel. Notably, this site bears a mutation causing a cardiac arrhythmia called the long QT syndrome. Our results suggest that following receptor-mediated PIP2 depletion and increased cytosolic Ca2+, calcified calmodulin N-lobe interacts with helix B in place of PIP2, to limit excessive IKS current depression.
3-We showed that SK4 K+ channels were expressed in human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) from healthy and CPVT2 patients bearing an arrhythmia mutation in calsequestrin 2 (CASQ2-D307H) and in pacemaker sinoatrial node cells (SAN) from WT and CASQ2-D307H knock-in (KI) mice. TRAM-34, a selective blocker of SK4 channels, prominently reduced delayed-afterdepolarizations and arrhythmic Ca2+ transients observed following application of the b-adrenergic agonist isoproterenol in CPVT2-derived hiPS-CMs and in SAN cells from KI mice. Strikingly, in vivo ECG recording showed that intraperitoneal injection of the SK4 channel blockers, TRAM-34 or clotrimazole, greatly reduced the arrhythmic features of CASQ2-D307H KI and CASQ2 knockout mice at rest and following exercise. Our work demonstrates the critical role of SK4 Ca2+-activated K+ channels in adult pacemaker function, making them promising therapeutic targets for the treatment of cardiac ventricular arrhythmias such as CPVT.
4-We designed novel chemical entities that are powerful openers or blockers of neuronal Kv7.2 channels and act as gating modifiers. Using these new pharmacological tools, we characterized the properties of pre- and post-synaptic Kv7.2/3 channels, which play a crucial in brain excitability. Our results show that the voltage sensor domain of both Kv7 and TRPV channels is the target of these gating-modifier molecules, indicating that this structure is a novel pharmacological target to cure human hyperexcitability diseases, like epilepsy, migraine, or neuropathic pain. In addition, these new ligands provide us with the unique opportunity to explore the gating mechanisms of voltage-dependent cation channels in a way that was never examined before.
Current research projects in the lab:
- Cardiac Kv7.1 potassium channels: structure, gating mechanisms, thermodynamics and implications for diseased cardiac long QT mutations.
- Brain Kv7.2/3 potassium channels: neurophysiology, homeostatic plasticity at the axon initial segment and implications for epileptic encephalopathy mutations.
- Mechanisms underlying the cardiac pacemaker activity in normal and diseased arrhythmic conditions using human induced-pluripotent stem cell-derived cardiomyocytes and mouse animal models.
- Targeting the voltage sensor of Kv7 and TRPV1 channels with gating-modifier molecules.