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Key Points: K V 1.2 channels, encoded by the KCNA2 gene, regulate neuronal excitability by conducting K + upon depolarization. A new KCNA2 missense variant was discovered in a patient with epilepsy, causing amino acid substitution F302L at helix S4, in the K V 1.2 voltage-sensing domain. Immunocytochemistry and flow cytometry showed that F302L does not impair KCNA2 subunit surface trafficking. Molecular dynamics simulations indicated that F302L alters the exposure of S4 residues to membrane lipids. Voltage clamp fluorometry revealed that the voltage-sensing domain of K V 1.2-F302L channels is more sensitive to depolarization. Accordingly, K V 1.2-F302L channels opened faster and at more negative potentials; however, they also exhibited enhanced inactivation: that is, F302L causes both gain- and loss-of-function effects. Coexpression of KCNA2-WT and -F302L did not fully rescue these effects. The proband's symptoms are more characteristic of patients with loss of KCNA2 function. Enhanced K V 1.2 inactivation could lead to increased synaptic release in excitatory neurons, steering neuronal circuits towards epilepsy., Abstract: An exome-based diagnostic panel in an infant with epilepsy revealed a previously unreported de novo missense variant in KCNA2, which encodes voltage-gated K + channel K V 1.2. This variant causes substitution F302L, in helix S4 of the K V 1.2 voltage-sensing domain (VSD). F302L does not affect KCNA2 subunit membrane trafficking. However, it does alter channel functional properties, accelerating channel opening at more hyperpolarized membrane potentials, indicating gain of function. F302L also caused loss of K V 1.2 function via accelerated inactivation onset, decelerated recovery and shifted inactivation voltage dependence to more negative potentials. These effects, which are not fully rescued by coexpression of wild-type and mutant KCNA2 subunits, probably result from the enhancement of VSD function, as demonstrated by optically tracking VSD depolarization-evoked conformational rearrangements. In turn, molecular dynamics simulations suggest altered VSD exposure to membrane lipids. Compared to other encephalopathy patients with KCNA2 mutations, the proband exhibits mild neurological impairment, more characteristic of patients with KCNA2 loss of function. Based on this information, we propose a mechanism of epileptogenesis based on enhanced K V 1.2 inactivation leading to increased synaptic release preferentially in excitatory neurons, and hence the perturbation of the excitatory/inhibitory balance of neuronal circuits., (© 2020 The Authors. The Journal of Physiology © 2020 The Physiological Society.)

Key Points: Association of plasma membrane BK Ca channels with BK-β subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BK Ca channel (mitoBK Ca ) by BK-β subunits is not established. MitoBK Ca -α and the regulatory BK-β1 subunit associate in mouse cardiac mitochondria. A large fraction of mitoBK Ca display properties similar to that of plasma membrane BK Ca when associated with BK-β1 (left-shifted voltage dependence of activation, V 1/2  = -55 mV, 12 µm matrix Ca 2+ ). In BK-β1 knockout mice, cardiac mitoBK Ca displayed a low P o and a depolarized V 1/2 of activation (+47 mV at 12 µm matrix Ca 2+ ) Co-expression of BK Ca with the BK-β1 subunit in HeLa cells doubled the density of BK Ca in mitochondria. The present study supports the view that the cardiac mitoBK Ca channel is functionally modulated by the BK-β1 subunit; proper targeting and activation of mitoBK Ca shapes mitochondrial Ca 2+ handling., Abstract: Association of the plasma membrane BK Ca channel with auxiliary BK-β1-4 subunits profoundly affects the regulatory mechanisms and physiological processes in which this channel participates. However, functional association of mitochondrial BK (mitoBK Ca ) with regulatory subunits is unknown. We report that mitoBK Ca functionally associates with its regulatory subunit BK-β1 in adult rodent cardiomyocytes. Cardiac mitoBK Ca is a calcium- and voltage-activated channel that is sensitive to paxilline with a large conductance for K + of 300 pS. Additionally, mitoBK Ca displays a high open probability (P o ) and voltage half-activation (V 1/2  = -55 mV, n = 7) resembling that of plasma membrane BK Ca when associated with its regulatory BK-β1 subunit. Immunochemistry assays demonstrated an interaction between mitochondrial BK Ca -α and its BK-β1 subunit. Mitochondria from the BK-β1 knockout (KO) mice showed sparse mitoBK Ca currents (five patches with mitoBK Ca activity out of 28 total patches from n = 5 different hearts), displaying a depolarized V 1/2 of activation (+47 mV in 12 µm matrix Ca 2+ ). The reduced activity of mitoBK Ca was accompanied by a high expression of BK Ca transcript in the BK-β1 KO, suggesting a lower abundance of mitoBK Ca channels in this genotype. Accordingly, BK-β1subunit increased the localization of BKDEC (i.e. the splice variant of BK Ca that specifically targets mitochondria) into mitochondria by two-fold. Importantly, both paxilline-treated and BK-β1 KO mitochondria displayed a more rapid Ca 2+ overload, featuring an early opening of the mitochondrial transition pore. We provide strong evidence that mitoBK Ca associates with its regulatory BK-β1 subunit in cardiac mitochondria, ensuring proper targeting and activation of the mitoBK Ca channel that helps to maintain mitochondrial Ca 2+ homeostasis., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Key Points: Beat-to-beat alternation (alternans) of the cardiac action potential duration is known to precipitate life-threatening arrhythmias and can be driven by the kinetics of voltage-gated membrane currents or by instabilities in intracellular calcium fluxes. To prevent alternans and associated arrhythmias, suitable markers must be developed to quantify the susceptibility to alternans; previous theoretical studies showed that the eigenvalue of the alternating eigenmode represents an ideal marker of alternans. Using rabbit ventricular myocytes, we show that this eigenvalue can be estimated in practice by pacing these cells at intervals varying stochastically. We also show that stochastic pacing permits the estimation of further markers distinguishing between voltage-driven and calcium-driven alternans. Our study opens the perspective to use stochastic pacing during clinical investigations and in patients with implanted pacing devices to determine the susceptibility to, and the type of alternans, which are both important to guide preventive or therapeutic measures., Abstract: Alternans of the cardiac action potential (AP) duration (APD) is a well-known arrhythmogenic mechanism. APD depends on several preceding diastolic intervals (DIs) and APDs, which complicates the prediction of alternans. Previous theoretical studies pinpointed a marker called λalt that directly quantifies how an alternating perturbation persists over successive APs. When the propensity to alternans increases, λalt decreases from 0 to -1. Our aim was to quantify λalt experimentally using stochastic pacing and to examine whether stochastic pacing allows discriminating between voltage-driven and Ca(2+) -driven alternans. APs were recorded in rabbit ventricular myocytes paced at cycle lengths (CLs) decreasing progressively and incorporating stochastic variations. Fitting APD with a function of two previous APDs and CLs permitted us to estimate λalt along with additional markers characterizing whether the dependence of APD on previous DIs or CLs is strong (typical for voltage-driven alternans) or weak (Ca(2+) -driven alternans). During the recordings, λalt gradually decreased from around 0 towards -1. Intermittent alternans appeared when λalt reached -0.8 and was followed by sustained alternans. The additional markers detected that alternans was Ca(2+) driven in control experiments and voltage driven in the presence of ryanodine. This distinction could be made even before alternans was manifest (specificity/sensitivity >80% for -0.4 > λalt  > -0.5). These observations were confirmed in a mathematical model of a rabbit ventricular myocyte. In conclusion, stochastic pacing allows the practical estimation of λalt to reveal the onset of alternans and distinguishes between voltage-driven and Ca(2+) -driven mechanisms, which is important since these two mechanisms may precipitate arrhythmias in different manners., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

Sudden cardiac death (SCD) due to ventricular fibrillation (VF) is a major world-wide health problem. A common trigger of VF involves abnormal repolarization of the cardiac action potential causing early afterdepolarizations (EADs). Here we used a hybrid biological-computational approach to investigate the dependence of EADs on the biophysical properties of the L-type Ca(2+) current (I(Ca,L)) and to explore how modifications of these properties could be designed to suppress EADs. EADs were induced in isolated rabbit ventricular myocytes by exposure to 600 μmol l(-1) H(2)O(2) (oxidative stress) or lowering the external [K(+)] from 5.4 to 2.0-2.7 mmol l(-1) (hypokalaemia). The role of I(Ca,L) in EAD formation was directly assessed using the dynamic clamp technique: the paced myocyte's V(m) was input to a myocyte model with tunable biophysical parameters, which computed a virtual I(Ca,L), which was injected into the myocyte in real time. This virtual current replaced the endogenous I(Ca,L), which was suppressed with nifedipine. Injecting a current with the biophysical properties of the native I(Ca,L) restored EAD occurrence in myocytes challenged by H(2)O(2) or hypokalaemia. A mere 5 mV depolarizing shift in the voltage dependence of activation or a hyperpolarizing shift in the steady-state inactivation curve completely abolished EADs in myocytes while maintaining a normal Ca(i) transient. We propose that modifying the biophysical properties of I(Ca,L) has potential as a powerful therapeutic strategy for suppressing EADs and EAD-mediated arrhythmias.

1. Neuronal alpha 1E Ca2+ channels were expressed alone and in combination with the beta 2a subunit in Xenopus laevis oocytes. 2. The properties of ionic and gating currents of alpha 1E were investigated: ionic currents were measured in 10 mM external Ba2+; gating currents were isolated in 2 mM external Co2+. 3. Charge movement preceded channel opening. The charge movement voltage curve (Q(V)) preceded the ionic conductance voltage dependence (G(V)) by approximately 20 mV. 4. Coexpression of alpha 1E with the beta 2a subunit did not modify the voltage dependence of charge movement but shifted the G(V) curve to more negative potentials. The voltage gap between Q(V) and G(V) curves was reduced by the beta 2a subunit and both curves overlapped at potentials near 0 mV. 5. The coupling efficiency between the charge movement and pore opening was estimated by the ration between limiting conductance and maximum charge movement (Gmax/Qmax). Coexpression of the beta 2a subunit increased the Gmax/Qmax ratio from 9.2 x 10(5) +/- 1.4 x 10(5) to 21.9 x 10(5) +/- 2.8 X 10(5) S C-1 for alpha 1E and alpha 1E + beta 2a, respectively. 6. We conclude that in the neuronal alpha 1E the charge movement is tightly coupled with the pore opening and that the beta 2a subunit coexpression further improves this coupling.