Journal of General Physiology最新文献

Among the three classes of voltage-gated Ca2+ channels (CaV1, CaV2, and CaV3), CaV3 T-type channels are drug targets for disorders, including epilepsy and pain. Antagonists such as Z944 and ML218 are highly selective for CaV3 compared with the CaV1.2 L-type channel, but whether they have additional activity on other CaV1 subtypes is unknown. Here, we investigated the effects of Z944 and ML218 on the CaV1.4 channel, which regulates neurotransmitter release from retinal photoreceptors. In HEK293T cells transfected with CaV1.4 and the auxiliary β2x13 and α2δ-4 subunits, Z944 and ML218 inhibited Ca2+ currents with IC50 values of ∼30 and 2 µM, respectively. Structure-based modeling combined with functional studies revealed the importance of a cluster of methionine residues, particularly M1004, within the DHP-binding site for the effects of ML218. Compared with mutation of a conserved threonine (T1007) that is required for DHP sensitivity of CaV1 channels, mutation of M1004 had a 10-fold greater impact in diminishing the potency of ML218. CaV1.2 was significantly less sensitive to ML218 inhibition (IC50 ∼37 µM) than CaV1.4, which could not be attributed to a valine in place of M1004 in CaV1.2. We conclude that ML218 and Z944 are dual CaV1/CaV3 modulators of CaV1.4 and should be used with caution when dissecting the contributions of CaV3 channels in tissues where CaV1.4 is expressed.

Electrical synapses mediated by gap junctions are widespread in the mammalian brain, playing essential roles in neural circuit function. Beyond their role synchronizing neuronal activity, they also support complex computations such as coincidence detection-a circuit mechanism in which differences in input timing are encoded by the firing rates of coupled neurons, enabling preferential responses to synchronous over temporally dispersed inputs. Electrical coupling allows each neuron to act as a current sink for its partner during independent depolarizations, thereby reducing excitability. In contrast, synchronous inputs across the network minimize voltage differences through gap junctions, reducing current shunting and increasing spiking probability. However, the contribution of intrinsic neuronal properties to coincidence detection remains poorly understood. Here, we investigated this issue in the mesencephalic trigeminal (MesV) nucleus of mice, a structure composed of somatically coupled neurons. Using whole-cell recordings and pharmacological tools, we examined the role of the D-type K+ current (ID), finding that it critically shapes both the intrinsic electrophysiological properties of MesV neurons and the dynamics of electrical synaptic transmission. Its fast activation kinetics and subthreshold voltage range of activation make ID a key determinant of transmission strength and timing. Furthermore, the ID, likely mediated by Kv1 subunits, is expressed at the soma and the axon initial segment. Finally, we characterized two key parameters of coincidence detection-precision (time window for effective input summation) and gain (differential response to coincident versus dispersed inputs)-finding that ID enhances precision by accelerating membrane repolarization and reduces the gain by limiting neuronal excitability.

Ca2+- and voltage-activated BK-type K+ channels are influenced profoundly by associated regulatory subunits, including β subunits (Kcnmb1-4; β1-β4). Although overlap in expression of different BK β subunits occurs in native tissues, whether they can coassemble in the same channel complex is not known. We coexpress β2 and β3a subunits together with BK α and, through a combination of macroscopic and single-channel recordings, along with quantitative pull-down of tagged subunits, test whether coassembly can occur. We evaluate two models: (1) random mixing in which β2 and β3a subunits coassemble in the same channels, and (2) segregation in which β2 and β3a are found in separate complexes. Our results support the view that, for β2 and β3a, BK currents arise from the random, independent assembly of both subunits in the same channels. Single-channel recordings directly confirm coassembly of β2 and β3a subunits in the same channels. Quantitative biochemical analysis of coexpression of tagged β2, β3a, and BK α subunits also reveals that β2:β3a:α ternary complexes form.

In mammalian skeletal muscle fibers, transmembrane Ca2+ influx is known to occur at rest and to increase in response to depolarization. In parallel to the well-identified dihydropyridine receptor (DHPR) pathway underlying this depolarization-induced Ca2+ influx, a tubular Ca2+ entry pathway activated by sarcoplasmic reticulum (SR) Ca2+ depletion, named store-operated Ca2+ entry (SOCE), has been identified. The use of the Mn2+ quenching technique has been instrumental for the characterization of these Ca2+ influxes. But, because both should be activated by depolarization, it is difficult to discriminate between these two Ca2+ entry pathways. In that context, the zebrafish muscle fiber is an ideal model to determine whether or not SOCE develops in response to depolarization, because the zebrafish DHPR is not conductive to any divalent cation. Using the technique of Mn2+ quenching of fura-2 fluorescence in voltage-clamped zebrafish fast muscle fibers, we show that depolarization pulses evoke slow transient Mn2+ quenching signals that persist after washout of external Mn2+. The Mn2+ quenching signal displays rate of recovery and voltage dependence correlated to the rate of recovery and voltage dependence of SR Ca2+ release, respectively. Our data suggest that the voltage-evoked Mn2+ quenching signal of zebrafish muscle fibers does not result from a Mn2+ influx provoked by depletion of SR Ca2+ content but from a displacement of Mn2+ accumulated on intracellular Ca2+ buffers by Ca2+ released from the SR. These findings should encourage to consider that increase in Mn2+ quenching can result from changes in intracellular Ca2+ and not from SOCE.

Voltage-gated sodium channels undergo reversible voltage/time-dependent transitions from closed to open and inactivated states. The voltage setpoints and efficiency of cardiac sodium channel Nav1.5 state transitions are crucial for tuning the initiation and conduction of myocardial action potentials. The channel's cytoplasmic carboxyl-terminal domain (CTD) regulates gating by intramolecular interactions and by serving as a hub for the binding of accessory proteins. We have investigated the roles of the CTD in intrinsic and FGF homologous factor (FHF)-modulated Nav1.5 gating through structure-guided CTD subdomain mutagenesis. The EF-hand module within the CTD was found to exert the most profound effects on channel gating, strongly influencing voltage dependence of inactivation and activation, accelerating inactivation from the closed state, decelerating inactivation from the open state, minimizing persistent sodium current, and serving as the binding domain for FHF proteins. Nav1.5D1788K bearing a missense mutation in the EF-hand motif displayed a depolarizing shift in voltage dependence of activation and generated enhanced persistent sodium current without altering the voltage dependence of channel inactivation. Another EF-hand mutant, Nav1.5L1861A, underwent closed-state inactivation at more negative membrane potential and at an accelerated rate but did not display other phenotypes associated with CTD deletion. Missense mutation Nav1.5V1776A in the juxtamembrane region between the EF-hand and the channel pore helices did not alter intrinsic gating properties but impaired FHF modulation of inactivation gating. Our channel physiology studies, together with the prior structural data from others, suggest that the voltage and rate of channel inactivation from the closed state are governed by an intramolecular hydrophobic interaction of the CTD EF-hand with the cytoplasmic inactivation loop helix and the extension of this binding interface upon FHF-induced restructuring of the juxtamembrane region. The CTD also tunes voltage-dependent activation and helps minimize persistent sodium current through distinct, presumed electrostatic mechanisms.

KdpFABC is an ATP-dependent membrane complex that enables prokaryotes to maintain potassium homeostasis under potassium-limited conditions. It features a unique hybrid mechanism combining a channel-like selectivity filter in KdpA with the ATP-driven transport functionality of KdpB. A key unresolved question is whether K+ ions translocate through the inter-subunit tunnel as a queue of ions or individually within a hydrated environment. Using molecular dynamics simulations, metadynamics, anomalous X-ray scattering, and biochemical assays, we demonstrate that the tunnel is predominantly occupied by water molecules rather than multiple K+ ions. Our results identify only one stable intermediate binding site for K+ within the tunnel, apart from the canonical sites in KdpA and KdpB. Free energy calculations reveal a substantial barrier (∼22 kcal/mol) at the KdpA-KdpB interface, making spontaneous K+ translocation unlikely. Furthermore, mutagenesis and functional assays confirm previous findings that Phe232 at this interface plays a key role in coupling ATP hydrolysis to K+ transport. These findings challenge previous models containing a continuous wire of K+ ions through the tunnel and suggest the existence of an as-yet unidentified intermediate state or mechanistic detail that facilitates K+ movement into KdpB.

Ex vivo culture of isolated muscle fibers can serve as an important model for in vitro research on mature skeletal muscle fibers. Nevertheless, this model has limitations for long-term studies due to structural loss and dedifferentiation following prolonged culture periods. This study aimed to investigate how ex vivo culture affects muscle fiber contraction and to improve the culture system to preserve muscle fiber morphology and sarcomere function. Additionally, we sought to determine which culture-induced changes can negatively affect muscle fiber contraction. We cultured isolated flexor digitorum brevis (FDB) muscle fibers in several conditions for up to 7 days and investigated viability, morphology, and the unloaded sarcomere shortening in intact fibers, along with force generation in permeabilized muscle fibers. In addition, we examined changes to the microtubule network. We found a time-dependent decrease in contractility and viability in muscle fibers cultured for 7 days on a laminin-coated culture dish (2D). Conversely, we found that culturing FDB muscle fibers in a low-serum, fibrin/Geltrex hydrogel (3D) reduces markers of muscle fiber dedifferentiation (i.e., sprouting), improves viability, and retains contractility over time. We discovered that the loss of contractility of cultured muscle fibers was not the direct result of reduced sarcomere function but may be related to changes in the microtubule network. Collectively, our findings highlight the importance of providing muscle fibers with a 3D environment during ex vivo culture, particularly when testing pharmacological or genetic interventions to study viability or contractile function.

NMDA-type ionotropic glutamate receptors mediate excitatory neurotransmission and synaptic plasticity, but aberrant signaling by these receptors is also implicated in brain disorders. Here, we present the binding site and the mechanism of action for UCM-101, a novel negative NMDA receptor modulator that produces full inhibition of NMDA receptor-mediated excitatory postsynaptic currents in hippocampal CA pyramidal neurons from juvenile mouse brain slices. UCM-101 has a 59-fold higher binding affinity at GluN1/2A compared with GluN1/2B receptors and inhibits diheteromeric GluN1/2A and triheteromeric GluN1/2A/2B receptors with IC50 values of 110 and 240 nM, respectively, in the presence of 1 µM glycine. The novel binding mode for UCM-101 is revealed in a high-resolution crystal structure of the GluN1/2A agonist binding domain heterodimer. UCM-101 and its analog TCN-213 inhibit NMDA receptors by negatively modulating co-agonist binding to the GluN1 subunit via an allosteric mechanism that is conserved with previously described GluN2A-selective antagonists, TCN-201 and MPX-004. Despite the shared mechanism of action, the structural determinants that mediate subunit selectivity for UCM-101 are distinct from those of TCN-201 and MPX-004. These findings provide detailed insights into the binding site and mechanism of action of a novel NMDA receptor modulator and open new avenues for the development of NMDA receptor ligands with therapeutic potential.

JGP study (De Giorgis et al. https://doi.org/10.1084/jgp.202413739) reveals that the auxiliary Cavβ3 subunit regulates the cardiac calcium channel Cav1.2 by modulating the two-step activation of VSD II.

Subtype-selective modulation of N-methyl-D-aspartate receptors (NMDARs) remains a major goal in neuropharmacology, with the potential to advance basic research and enable targeted therapies for disorders involving dysregulated glutamatergic signalling. In this volume of the Journal of General Physiology, Lotti et al. describe UCM-101, a newly optimized GluN2A-selective allosteric inhibitor derived from the weakly active scaffold TCN-213. Introduction of a single ethyl group resulted in a 7.5-fold increase in potency, yielding an inhibitor with an IC₅₀ of 110 nM at GluN1/2A receptors and up to 118-fold selectivity over other NMDAR subtypes under physiologically relevant conditions. A 1.7 Å crystal structure of the GluN1-2A ligand-binding domain (LBD) revealed that UCM-101 adopts an extended conformation spanning the inter-subunit allosteric pocket, engaging a previously unexploited "UCM-subsite" distinct from those used by TCN- or MPX-class modulators. Despite its novel orientation, UCM-101 stabilizes the inactive, open-clamshell conformation of the GluN1 LBD, thereby reducing glycine affinity and preventing receptor activation. Mutagenesis identified new selectivity determinants (GluN2A V529, M788, and T797) that are not utilized by TCN-201, demonstrating that different scaffolds exploit distinct microenvironments within the same allosteric site. Functionally, UCM-101 produced robust inhibition of NMDAR-mediated synaptic currents in hippocampal slices (89% at 3 μM) and displayed similar potency at triheteromeric GluN1/2A/2B receptors. Together, these findings validate the mechanistic framework for GluN2A-selective inhibition while broadening the structural landscape for ligand engagement. UCM-101 provides both a potent research tool and a promising scaffold for the development of next-generation subtype-selective NMDAR modulators.