Journal of General Physiology最新文献

Voltage-gated sodium (NaV) channels are vital regulators of electrical activity in excitable cells. Given their importance in physiology, NaV channels are key therapeutic targets for treating numerous conditions, yet developing subtype-selective drugs remains challenging due to the high sequence and structural conservation among NaV subtypes. Recent advances in cryo-electron microscopy have resolved most human NaV channels, providing valuable insights into their structure and function. However, limitations persist in fully capturing the complex conformational states that underlie NaV channel gating and modulation. This study explores the capability of AlphaFold2 to sample multiple NaV channel conformations and assess AlphaFold Multimer's accuracy in modeling interactions between the NaV α-subunit and its protein partners, including auxiliary β-subunits and calmodulin. We enhance conformational sampling to explore NaV channel conformations using a subsampled multiple sequence alignment approach and varying the number of recycles. Our results demonstrate that AlphaFold2 models multiple NaV channel conformations, including those observed in experimental structures, states that have not been described experimentally, and potential intermediate states. Correlation and clustering analyses uncover coordinated domain behavior and recurrent state ensembles. Furthermore, AlphaFold Multimer models NaV complexes with auxiliary β-subunits and calmodulin with high accuracy, and the presence of protein partners significantly alters both the modeled conformational landscape of the NaV α-subunit and the coupling between its functional states. These findings highlight the potential of deep learning-based methods to expand our understanding of NaV channel structure, gating, and modulation, while also underscoring the limitations of predicted models that remain hypotheses until validated by experimental data.

The presence of significant amounts of the transition metal, manganese, is essential for living cells where it is bound to some intracellular enzymes. The free (i.e., unbound) Mn2+ concentration in both extracellular and intracellular space is tightly regulated and thought to be considerably lower than the free calcium ion (Ca2+) concentration. Mn2+ can pass through plasmalemma Ca2+ ion channels, but under normal circumstances due to channel selectivity and relative concentrations, this event is rare. But when extracellular Mn2+ is increased to mM levels, significant Mn2+ influx occurs through Ca2+ channels in the plasma membrane and intracellular manganese levels increase above normal physiological levels. Mn2+ ions also have the property of binding to and quenching the fluorescence of fluorophores. This property can be used to detect Mn2+ influx and is the basis of the use of raised extracellular Mn2+ in experiments designed to detect pathways for Ca2+ influx. This commentary features the manganese quench technique as used in a recently published article and discusses in detail the potential consequences for the intracellular Ca2+ handling when intracellular Mn2+ is increased, as it now competes to a greater extent than normal with Ca2+ for intracellular buffers.

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.

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.

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.

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.

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.