Low-voltage-activated (LVA, T-type, or CaV3), calcium-selective channels open in response to modest depolarizations, just above the resting membrane potential, supporting neuronal burst-firing patterns and spontaneous firing in cardiac pacemaker cells. How LVA-channels open at low voltages is unclear: traditional gating-current experiments suggest that LVA-channel voltage-sensing domains (VSDs) paradoxically require stronger depolarization to activate than pore opening. Using voltage-clamp fluorometry, we find that the activation of all four VSDs in human CaV3.1-channels precedes opening in voltage, solving the longstanding conundrum. We also uncover confounding effects of La3+ (used for gating-current measurements) on VSD function and clarify the role of distinct LVA-channel structure S6Cyto. CaV3.1-VSDs operate within a narrow voltage-range, resembling the VSDs of related NaV-channels more than those of other CaV-channels. Likely, NaV-like VSDs emerge before sodium selectivity.

For more than two centuries, digoxin has been used to treat heart failure by increasing the strength of cardiac contraction and, more recently, is used for heart rate control. The proposed, yet unproven, mechanism underlying digoxin’s positive inotropic effect is as follows: By inhibiting the Na + -K + ATPase (NKA), digoxin partially dissipates the transmembrane Na + gradient, which is used by the Na + -Ca 2+ exchanger (NCX1) to extrude Ca 2+ from myocytes, thus causing accumulation of cytosolic Ca 2+ and therefore increased cardiac contractility. Here, we demonstrate that digoxin critically relies on a specific allosteric regulation of NCX1, known as Na + -dependent inactivation, to exert its positive inotropic effect, establishing the precise mechanism of action of this historic drug. These findings identify a distinct molecular target for the development of positive inotropes that avoid the undesirable effects associated with the blockade of NKA. As the structural information for the region involved with NCX1 Na + -dependent inhibition is well resolved, we provide the mechanistic foundation for drug development.

No targeted interventions exist that improve the outcomes of patients with acute lung injury/ARDS. A few studies investigated Na + and Ca 2+ channels/transporters for potential therapeutic intervention but with limited translational success. This study highlights the regulatory role of TREK-1 K + channels during HO+stretch/mechanical ventilation-induced lung injury in ROS production, caspase activation, cytokine secretion, and explores the underlying TREK-1-mediated signaling mechanisms. These preclinical findings lay the groundwork for future rational drug design targeting TREK-1 channels.

Depolarization-evoked opening of CaV2.1 (P/Q-type) Ca2+-channels triggers neurotransmitter release, while voltage-dependent inactivation (VDI) limits channel availability to open, contributing to synaptic plasticity. The mechanism of CaV2.1 response to voltage is unclear. Using voltage-clamp fluorometry and kinetic modeling, we optically track and physically characterize the structural dynamics of the four CaV2.1 voltage-sensor domains (VSDs). The VSDs are differentially sensitive to voltage changes, both brief and long-lived. VSD-I seems to directly drive opening and convert between two modes of function, associated with VDI. VSD-II is apparently voltage-insensitive. VSD-III and VSD-IV sense more negative voltages and undergo voltage-dependent conversion uncorrelated with VDI. Auxiliary β-subunits regulate VSD-I-to-pore coupling and VSD conversion kinetics. Hence, the central role of CaV2.1 channels in synaptic release, and their contribution to plasticity, memory formation and learning, can arise from the voltage-dependent conformational changes of VSD-I.

What is the molecular origin of voltage dependence in skeletal muscle excitation-contraction? Cholinergic transmission to the muscle fiber triggers action potentials, which are sensed by voltage-gated L-type calcium channels (Ca V 1.1). In turn, the conformational changes in Ca V 1.1 propagate to and activate intracellular ryanodine receptors (RyR1), causing Ca 2+ release and contraction. The Ca V 1.1 channel has four voltage-sensing domains (VSD-I to -IV) with diverse voltage-sensing properties, so the identity of VSD(s) responsible for conferring voltage dependence to RyR1 opening, is unknown. Using voltage-clamp fluorometry, we show that only VSD-III possesses kinetic, voltage-dependent and pharmacological properties consistent with skeletal-muscle excitability and Ca 2+ release. We propose that the earliest voltage-dependent event in the excitation-contraction process is the structural rearrangement of VSD-III that propagates to RyR1 to initiate Ca 2+ release and contraction.

T-type calcium-selective (CaV3) channels control calcium entry in various cell types, regulating essential processes such as neuronal excitability and cardiac pacemaker activity. CaV3 display unique functional and structural features that set them apart from other CaV channels as they activate in response to small depolarizations (<−60 mV). Recent work in CaV3.3 has resolved a long S6 helix of Domain-III that incorporates a positively charged region (S6Cyto) protruding into the cytosol (He et al., Nat Commun. 2022). This region (RRREEKRLRRLEKKRR), conserved among CaV3 channels, is thought to be critical for the T-type-channel low-voltage activation (LVA). We probed the mechanism by which this region shapes CaV3.1 voltage-dependent activation, testing the hypothesis that the S6Cyto positive charge modulates voltage-sensing domain(s) (VSDs). Using cut-open-oocyte voltage-clamp fluorometry, we found that neutralizing 11 positively charged amino-acids with glutamine (CaV3.1-11Q) caused an overall +5 mV shift of Vhalf activation (GV-WT: Vhalf1 = −37±1 mV (55%), Vhalf2 = −4±1 mV; GV-11Q: Vhalf1 = −31±1 mV (55%), Vhalf2 = 0±1 mV). This partial loss of "low-voltage activation" was associated with a large perturbation of VSD-IV activation revealed by a new component in the fluorescence voltage dependence at negative membrane potential (<−90 mV), possibly revealing a new resting state of VSD-IV. To further probe the contribution of VSD-IV to voltage-dependent activation, we introduced an ataxia-causing mutation in S4 VSD-IV (R1715H) (Coutelier et al., Am J Hum Genet. 2015). This charge neutralization also caused a ∼+5 mV shift in CaV3.1 activation, associated with a positive shift of VSD-IV activation (WT: −46±1 mV; R1715H: −29±1 mV). All other VSDs were not significantly affected by R1715H. In summary, this study establishes the significant contribution of VSD-IV to CaV3.1 voltage-dependent activation and reveals the mechanism by which an ataxia-causing mutation impairs channel activation. S6Cyto synergizes with VSD-IV to contribute ∼5 mV to "low-voltage activation."

Postoperative atrial fibrillation (POAF) is the most common complication after cardiac surgery and a significant cause of increased morbidity and mortality. The development of novel POAF therapeutics has been limited by an insufficient understanding of molecular mechanisms promoting atrial fibrillation. In this observational cohort study, we enrolled 28 patients without a history of atrial fibrillation that underwent mitral valve surgery for degenerative mitral regurgitation and obtained left atrial tissue samples along the standard atriotomy incision in proximity to the right pulmonary veins. We isolated cardiomyocytes and performed transcriptome analyses demonstrating 13 differentially expressed genes associated with new-onset POAF. Notably, decreased expression of fibroblast growth factor 13 (FGF13), a fibroblast growth factor homologous factor known to modulate voltage-gated sodium channel Na V 1.5 inactivation, had the most significant association with POAF. To assess the functional significance of decreased FGF13 expression in atrial myocytes, we performed patch clamp experiments on neonatal rat atrial myocytes after siRNA-mediated FGF13 knockdown, demonstrating action potential prolongation. These critical findings indicate that decreased FGF13 expression promotes vulnerability to POAF.

How G-proteins inhibit N-type, voltage-gated, calcium-selective channels (Ca V 2.2) during presynaptic inhibition is a decades-old question. G-proteins Gβγ bind to intracellular Ca V 2.2 regions, but the inhibition is voltage-dependent. Using the hybrid electrophysiological and optical approach voltage-clamp fluorometry, we show that Gβγ acts by selectively inhibiting a subset of the four different Ca V 2.2 voltage-sensor domains (VSDs I-IV). During regular "willing" gating, VSDs I and IV activation resemble pore opening, VSD III activation is hyperpolarized, and VSD II appears unresponsive to depolarization. In the presence of Gβγ, Ca V 2.2 gating is "reluctant": pore opening and VSD-I activation are strongly and proportionally inhibited, VSD IV is modestly inhibited while VSD III is not. We propose that Gβγ inhibition of VSD-I and -IV underlies reluctant Ca V 2.2 gating and subsequent presynaptic inhibition.

The Na+-Ca2+ exchanger (NCX1) is the dominant Ca2+ extrusion mechanism in cardiac myocytes. NCX1 activity is inhibited by intracellular Na+ via a process known as Na+-dependent inactivation. A central question is whether this inactivation plays a physiological role in heart function. Using CRISPR/Cas9, we inserted the K229Q mutation in the gene (Slc8a1) encoding for NCX1. This mutation removes the Na+-dependent inactivation while preserving transport properties and other allosteric regulations. NCX1 mRNA levels, protein expression, and protein localization are unchanged in K229Q male mice. However, they exhibit reduced left ventricular ejection fraction and fractional shortening, while displaying a prolonged QT interval. K229Q ventricular myocytes show enhanced NCX1 activity, resulting in action potential prolongation, higher incidence of aberrant action potentials, a faster decline of Ca2+ transients, and depressed cell shortening. The results demonstrate that NCX1 Na+-dependent inactivation plays an essential role in heart function by affecting both cardiac excitability and contractility. The sodium-calcium exchanger (NCX1) is the primary calcium extrusion mechanism of cardiac myocytes. Here, the authors show that removal of a long questioned allosteric regulation of NCX1 by intracellular sodium alters cardiac excitation-contraction coupling.

T-type calcium channels (CaV3) are critical regulators of a variety of physiological processes and are considered attractive pharmacological targets for the treatment of diverse neurological disorders. Z944 is a highly selective piperazine-derivative T-type-acting compound that holds promise for treating seizures, neuropathic pain and tremor, and is currently being evaluated in clinical trials. Z944 binds to the intracellular pore at the fenestration between Repeat-II and -III of CaV3.1, and is thought to block ionic conduction in a state-dependent manner (Zhao et al.

How G proteins inhibit N-type, voltage-gated, calcium-selective channels (Ca V 2.2) during presynaptic inhibition is a decades-old question. G proteins Gβγ bind to intracellular Ca V 2.2 regions, but the inhibition is voltage dependent. Using the hybrid electrophysiological and optical approach voltage-clamp fluorometry, we show that Gβγ acts by selectively inhibiting a subset of the four different Ca V 2.2 voltage-sensor domains (VSDs I to IV). During regular “willing” gating, VSD-I and -IV activations resemble pore opening, VSD III activation is hyperpolarized, and VSD II appears unresponsive to depolarization. In the presence of Gβγ, Ca V 2.2 gating is “reluctant”: pore opening and VSD I activation are strongly and proportionally inhibited, VSD IV is modestly inhibited, while VSD III is not. We propose that Gβγ inhibition of VSDs I and IV underlies reluctant Ca V 2.2 gating and subsequent presynaptic inhibition.

The electrogenic Na+-Ca2+ exchanger (NCX1) transports three Na+ into the cell while extruding one Ca2+, thus regulating both cardiac excitability and contractility. In addition to being transported, intracellular Na+ inhibits NCX1 via a mechanism termed Na+-dependent inactivation. The physiological role of this regulation remains unknown. We created a novel mouse line in which the native NCX1 carries mutation K229Q. This modification removes NCX1 Na+-dependent inactivation without affecting other regulatory properties (Matsuoka et al., JPG 109:273-86, 1997; John et al., JGP 150:245-257, 2018). K229Q mice show a prolonged QT interval and action potential (Steccanella et al., Biophys. J. 118, 100a, 2020). To determine the mechanism underlying these altered electrical properties, we recorded NCX1 currents from patch clamped myocytes. Currents were elicited by a voltage ramp protocol and acquired in the absence and presence of intracellular Na+. In the absence of cytosolic Na+ both WT and K229Q exchangers do not undergo Na+-dependent inactivation and thus generated similar current amplitudes (WT = −0.13±0.04 pA/pF, n = 7/5 cells/animals; K229Q = −0.12±0.04, n = 7/3; p = 0.87, measured at −70 mV, mean±SEM). In contrast, in the presence of 25mM Na+, NCX1 currents recorded from K229Q cardiomyocytes were significantly larger than those measured from WT cells (WT = 0.72±0.07 pA/pF, n = 8/4; K229Q = 1.07±0.10, n = 10/6; p = 0.009, measured at 50 mV). This is consistent with increased activity of NCX1-K229Q which lacks Na+-dependent inactivation. L-type Ca2+ current amplitudes (WT = −12.26±1.32 pA/pF, n = 7/5; K229Q = −14.82±2.29, n = 6/3; p = 0.36, measured at 10 mV) and transcript levels of CaV1.2, KV2.1, KV4.2, and Kir2.1 channels were not significantly different between WT and K229Q mice. These findings suggest that action potential prolongation observed in K229Q cardiomyocytes is primarily mediated by NCX1 current enhanced by the lack of Na+-dependent inactivation and that NCX1 Na+-dependent inactivation is an important modulator of cardiac electrical activity.

Low-voltage-activated CaV3.1 (T-type) Ca2+ channels contribute to neuronal burst-mode firing and heart pacemaker activity. Similar to other voltage-gated channels, the four Voltage-Sensing Domains (VSDs) are expected to activate before the pore opens. However, previous studies attempting to characterize the CaV3.1 voltage-sensing apparatus in channels blocked by La3+ found that most of the gating charge moves after the channel has opened. To shed light on the molecular mechanisms underlying the unique voltage-dependent gating of CaV3.1, we expressed the human CaV3.1 pore-forming subunit α1G in Xenopus oocytes and used voltage-clamp fluorometry (VCF) to optically track the structural rearrangements of the four VSDs.

Myocardial fibrosis and calcification associate with adverse outcomes in nonischemic heart failure. Cardiac fibroblasts (CF) transition into myofibroblasts (MF) and osteogenic fibroblasts (OF) to promote myocardial fibrosis and calcification. However, common upstream mechanisms regulating both CF-to-MF transition and CF-to-OF transition remain unknown. microRNAs are promising targets to modulate CF plasticity. Our bioinformatics revealed downregulation of miR–129-5p and upregulation of its targets small leucine–rich proteoglycan Asporin (ASPN) and transcription factor SOX9 as common in mouse and human heart failure (HF). We experimentally confirmed decreased miR–129-5p and enhanced SOX9 and ASPN expression in CF in human hearts with myocardial fibrosis and calcification. miR–129-5p repressed both CF-to-MF and CF-to-OF transition in primary CF, as did knockdown of SOX9 and ASPN. Sox9 and Aspn are direct targets of miR–129-5p that inhibit downstream β-catenin expression. Chronic Angiotensin II infusion downregulated miR–129-5p in CF in WT and TCF21-lineage CF reporter mice, and it was restored by miR–129-5p mimic. Importantly, miR–129-5p mimic not only attenuated progression of myocardial fibrosis, calcification marker expression, and SOX9 and ASPN expression in CF but also restored diastolic and systolic function. Together, we demonstrate miR–129-5p/ASPN and miR–129-5p/SOX9 as potentially novel dysregulated axes in CF-to-MF and CF-to-OF transition in myocardial fibrosis and calcification and the therapeutic relevance of miR–129-5p.

Elevated TNF-α levels in serum and broncho-alveolar lavage fluid of acute lung injury patients correlate with mortality rates. We hypothesized that pharmacological plasma membrane potential (Em) hyperpolarization protects against TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells through inhibition of inflammatory Ca2+-dependent MAPK pathways. Since the role of Ca2+ influx in TNF-α-mediated inflammation remains poorly understood, we explored the role of L-type voltage-gated Ca2+ (CaV) channels in TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells. The CaV channel blocker, Nifedipine, decreased both CCL-2 and IL-6 secretion, suggesting that a fraction of CaV channels is open at the significantly depolarized resting Em of human microvascular pulmonary endothelial cells (−6 ± 1.9 mV), as shown by whole-cell patch-clamp measurements. To further explore the role of CaV channels in cytokine secretion, we demonstrated that the beneficial effects of Nifedipine could also be achieved by Em hyperpolarization via the pharmacological activation of large conductance K+ (BK) channels with NS1619, which elicited a similar decrease in CCL-2 but not IL-6 secretion. Using functional gene enrichment analysis tools, we predicted and validated that known Ca2+-dependent kinases, JNK-1/2 and p38, are the most likely pathways to mediate the decrease in CCL-2 secretion.