r 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. r Immunocytochemistry and flow cytometry showed that F302L does not impair KCNA2 subunit surface trafficking. r Molecular dynamics simulations indicated that F302L alters the exposure of S4 residues to membrane lipids. r 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. r 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. Antonios Pantazis: This project was Antonios' last at his previous position (UCLA; PI: Riccardo Olcese) and first at his first fully independent appointment, as Assistant Professor at Linköping University. He is particularly fond of this work because it combines highly diverse contributions from a truly international and multidisciplinary team of investigators, to address a case straight from the clinic. Antonios' research focus is on ion channel function and dysfunction. His team at LiU study how the intricate and dynamic structure of ion channels, their biophysical properties and their regulation relate to cellular excitability in health and, as in this article, disease.

Initiation of skeletal muscle contraction is triggered by rapid activation of RYR1 channels in response to sarcolemmal depolarization. RYR1 is intracellular and has no voltage-sensing structures, but it is coupled with the voltage-sensing apparatus of CaV1.1 channels to inherit voltage sensitivity. Using an opto-electrophysiological approach, we resolved the excitation-driven molecular events controlling both CaV1.1 and RYR1 activations, reported as fluorescence changes. We discovered that each of the four human CaV1.1 voltage-sensing domains (VSDs) exhibits unique biophysical properties: VSD-I time-dependent properties were similar to ionic current activation kinetics, suggesting a critical role of this voltage sensor in CaV1.1 activation; VSD-II, VSD-III, and VSD-IV displayed faster activation, compatible with kinetics of sarcoplasmic reticulum Ca2+ release. The prominent role of VSD-I in governing CaV1.1 activation was also confirmed using a naturally occurring, charge-neutralizing mutation in VSD-I (R174W). This mutation abolished CaV1.1 current at physiological membrane potentials by impairing VSD-I activation without affecting the other VSDs. Using a structurally relevant allosteric model of CaV activation, which accounted for both time- and voltage-dependent properties of CaV1.1, to predict VSD-pore coupling energies, we found that VSD-I contributed the most energy (~75 meV or ∼3 kT) toward the stabilization of the open states of the channel, with smaller (VSD-IV) or negligible (VSDs II and III) energetic contribution from the other voltage sensors (<25 meV or ∼1 kT). This study settles the longstanding question of how CaV1.1, a slowly activating channel, can trigger RYR1 rapid activation, and reveals a new mechanism for voltage-dependent activation in ion channels, whereby pore opening of human CaV1.1 channels is primarily driven by the activation of one voltage sensor, a mechanism distinct from that of all other voltage-gated channels.

Significance A child with epilepsy has a previously unreported, heterozygous mutation in KCNA2 , the gene encoding K V 1.2 proteins. Four K V 1.2 assemble into a potassium-selective channel, a protein complex at the neuronal cell surface regulating electrical signaling. K V 1.2 subunits assemble with other K V 1-family members to form heterotetrameric channels, contributing to neuronal potassium-channel diversity. The most striking consequence of this mutation is preventing K V 1.2-subunit trafficking, i.e., their ability to reach the cell surface. Moreover, the mutation is dominant negative, as mutant subunits can assemble with wild-type K V 1.2 and K V 1.4, trapping them into nontrafficking heterotetramers and decreasing their functional expression. Thus, K V 1-family genes’ ability to form heterotetrameric channels is a double-edged sword, rendering K V 1-family members vulnerable to dominant-negative mutations in a single member gene.

Chorionic stem cells represent a promising opportunity for regenerative medicine. A deeper understanding of the stimuli that regulate their physiology, could lead to innovative clinical approaches. We revealed the presence of multiple sphingosine-1-phosphate (S1P) receptor isoforms in chorion-derived mesenchymal stem cells (CMSCs). Their activation simultaneously propagated from the plasma membrane through Gi and other heterotrimeric G proteins and further diverged toward extracellular-signal-regulated kinase 1/2 (ERK1/2), p38 and protein kinase D 1. At a functional level, S1P signaling inhibited CMSC migration, while promoting proliferation. Instead, a reduction of cell density was obtained when S1P was combined to treatments that increased cAMP intracellular concentration. Such surprising reduction of cell viability was relatively specific as it was not observed with stromal stem cells from bone marrow. Neither it was observed by activating analogous G proteins with bradykinin nor by inducing cell death via a cAMP-independent pathway. S1P could thus reveal novel keys to improve CMSC differentiation programs acting on cAMP concentration. Furthermore, S1P receptor agonists/antagonists could become instrumental in favoring CMSC engraftment by controlling cell motility.

Ventricular arrhythmias, a leading cause of sudden cardiac death, can be triggered by cardiomyocyte early afterdepolarizations (EADs). EADs can result from an abnormal late activation of L-type Ca2+ channels (LTCCs). Current LTCC blockers (class IV antiarrhythmics), while effective at suppressing EADs, block both early and late components of ICa,L, compromising inotropy. However, computational studies have recently demonstrated that selective reduction of late ICa,L (Ca2+ influx during late phases of the action potential) is sufficient to potently suppress EADs, suggesting that effective antiarrhythmic action can be achieved without blocking the early peak ICa,L, which is essential for proper excitation–contraction coupling. We tested this new strategy using a purine analogue, roscovitine, which reduces late ICa,L with minimal effect on peak current. Scaling our investigation from a human CaV1.2 channel clone to rabbit ventricular myocytes and rat and rabbit perfused hearts, we demonstrate that (1) roscovitine selectively reduces ICa,L noninactivating component in a human CaV1.2 channel clone and in ventricular myocytes native current, (2) the pharmacological reduction of late ICa,L suppresses EADs and EATs (early after Ca2+ transients) induced by oxidative stress and hypokalemia in isolated myocytes, largely preserving cell shortening and normal Ca2+ transient, and (3) late ICa,L reduction prevents/suppresses ventricular tachycardia/fibrillation in ex vivo rabbit and rat hearts subjected to hypokalemia and/or oxidative stress. These results support the value of an antiarrhythmic strategy based on the selective reduction of late ICa,L to suppress EAD-mediated arrhythmias. Antiarrhythmic therapies based on this idea would modify the gating properties of CaV1.2 channels rather than blocking their pore, largely preserving contractility.