Journal of General Physiology最新文献

Gupta et al. (https://doi.org/10.1085/jgp.202413676) reconcile a disconnect between structural and functional data regarding stoichiometry of PANX1 channels and provide new insights about channel activation.

The mechanisms underlying cooperative activation and inactivation of myocardial force extend from local, near-neighbor interactions involving troponin-tropomyosin regulatory units (RU) and crossbridges (XB) to more global interactions across the sarcomere. To better understand these mechanisms in the hearts of small and large mammals, we undertook a simplified mathematical approach to assess the contribution of three types of near-neighbor cooperative interactions, i.e., RU-induced, RU-activation (RU-RU), crossbridge-induced, crossbridge-binding (XB-XB), and XB-induced, RU-activation (XB-RU). We measured the Ca2+ and activation dependence of the rate constant of force redevelopment in murine- and porcine-permeabilized ventricular myocardium. Mathematical modeling of these three near-neighbor interactions yielded nonlinear expressions for the RU-RU and XB-RU rate coefficients (kon and koff) and XB-XB rate coefficients describing the attachment of force-generating crossbridges (f and f'). The derivation of single cooperative coefficient parameters (u = RU-RU, w = XB-RU, and v = XB-XB) permitted an initial assessment of the strength of each near-neighbor interaction. The parameter sets describing the effects of discrete XB-XB or XB-RU interactions failed to adequately fit the in vitro contractility data in either murine or porcine myocardium. However, the Ca2+ dependence of ktr in murine and porcine ventricular myocardium was well fit by parameter sets incorporating the RU-RU cooperative interaction. Our results indicate that a significantly stronger RU-RU interaction is present in porcine ventricular myocardium compared with murine ventricular myocardium and that the relative strength of the near-neighbor RU-RU interaction contributes to species-specific myocardial contractile dynamics in small and large mammals.

Vision is initiated by the reception of light by photoreceptors and subsequent processing via downstream retinal neurons. Proper circuit organization depends on the multifunctional tissue polarity protein FAT3, which is required for amacrine cell connectivity and retinal lamination. Here, we investigated the retinal function of Fat3 mutant mice and found decreases in both electroretinography and perceptual responses to high-frequency flashes. These defects did not correlate with abnormal amacrine cell wiring, pointing instead to a role in bipolar cell subtypes that also express FAT3. The role of FAT3 in the response to high temporal frequency flashes depends upon its ability to transduce an intracellular signal. Mechanistically, FAT3 binds to the synaptic protein PTPσ intracellularly and is required to localize GRIK1 to OFF-cone bipolar cell synapses with cone photoreceptors. These findings expand the repertoire of FAT3's functions and reveal its importance in bipolar cells for high-frequency light response.

Small molecule inhibitors of the sodium channel are common pharmacological agents used to treat a variety of cardiac and nervous system pathologies. They act on the channel via binding within the pore to directly block the sodium conduction pathway and/or modulate the channel to favor a non-conductive state. Despite their abundant clinical use, we lack specific knowledge of their protein-drug interactions and the subtle variations between different compound structures. This study investigates the binding and accessibility of nine different compounds in the pore cavity of the Nav1.5 sodium channel using enhanced sampling simulations. We find that most compounds share a common location of pore binding-near the mouth of the DII-III fenestration-associated with the high number of aromatic residues in this region. In contrast, some other compounds prefer binding within the lateral fenestrations where they compete with lipids, rather than binding in the central cavity. Overall, our simulation results suggest that the drug binding within the pore is highly promiscuous, with most drugs having multiple low-affinity binding sites. Access to the pore interior via two out of four of the hydrophobic fenestrations is favorable for the majority of compounds. Our results indicate that the polyspecific and diffuse binding of inhibitors in the pore contributes to the varied nature of their inhibitory effects and can be exploited for future drug discovery and optimization.

Pannexin 1 (PANX1) is a member of a topologically related and stoichiometrically diverse family of large pore membrane ion channels that support the flux of signaling metabolites (e.g., ATP) and fluorescent dyes. High-resolution structural analyses have identified PANX1 as a heptamer despite early evidence suggesting that it might be a hexamer. To determine if PANX1 channel activity is supported in both hexameric and heptameric conformations, we examined properties of concatenated PANX1 constructs comprising either six or seven subunits with intact or truncated C-termini (the latter to mimic caspase-cleavage activation). In whole-cell recordings from PANX1-deleted cells, the C-tail-truncated hexameric and heptameric concatemers generated outwardly rectifying PANX1-like currents only after severing the intersubunit linkers. Surprisingly, α1D adrenoceptor stimulation activated constructs with intact or truncated C-tails, even without linker cleavage. In inside-out patches from PANX1-deleted cells, linker cleavage activated C-tail truncated channels derived from either hexameric or heptameric concatemers. The heptamers presented peak unitary conductance and mean open time that was similar to channels assembled from the expression of unlinked single PANX1 subunits and greater than from the hexamers. In addition, the linker-cleaved heptameric concatemers supported greater PANX1-dependent ATP release and TO-PRO-3 uptake than the corresponding hexamers. These data indicate that functional PANX1 channels can be obtained in either hexameric or heptameric conformations and suggest that the distinct unitary properties of heptameric channels are more conducive to large molecule permeation by PANX1; they also suggest that there are distinct structural requirements for C-tail cleavage and receptor-mediated PANX1 activation mechanisms.

JGP study (Liu and Bezanilla. https://doi.org/10.1085/jgp.202413667) reveals that a sodium channel mutant blocks fast inactivation downstream of inactivation particle binding, diverting the channel into an alternative open state.

NMDA receptors (NMDAR) convert the major excitatory neurotransmitter glutamate into a synaptic signal. A key question is how efficiently the ion channel opens in response to the rapid exposure to presynaptic glutamate release. Here, we applied glutamate to single channel outside-out patches and measured the successes of channel openings and the latency to first opening to assay the activation efficiency of NMDARs under different physiological conditions and with different human subunit compositions. For GluN1/GluN2A receptors, we find that various factors, including intracellular ATP and GTP, can enhance the efficiency of activation presumably via the intracellular C-terminal domain. Notably, an energy-based internal solution or increasing the time between applications to increase recovery time improved efficiency. However, even under these optimized conditions and with a 1-s glutamate application, there remained around 10-15% inefficiency. Channel activation became more inefficient with brief synaptic-like pulses of glutamate at 2 ms. Of the different NMDAR subunit compositions, GluN2B-containing NMDARs showed the lowest success rate and longest latency to first openings, highlighting that they display the most distinct activation mechanism. In contrast, putative triheteromeric GluN1/GluN2A/GluN2B receptors showed high activation efficiency. Despite the low open probability, NMDARs containing either GluN2C or GluN2D subunits displayed high activation efficiency, nearly comparable with that for GluN2A-containing receptors. These results highlight that activation efficiency in NMDARs can be regulated by environmental surroundings and varies across different subunits.

Titin is the third contractile filament in the sarcomere, and it plays a critical role in sarcomere integrity and both passive and active tension. Unlike the thick and thin filaments, which are polymers of myosin and actin, respectively, titin is a single protein that spans from Z-disk to M-line. The N2A region within titin has been identified as a signaling hub for the muscle and is shown to be involved in multiple interactions. The insertion sequence (UN2A) within the N2A region was predicted as a potential binding site for the Ca2+-binding protein, S100A1. We demonstrate using a combination of size exclusion chromatography, surface plasmon resonance, and fluorescence resonance energy transfer that S100A1 can bind to the UN2A region. We further demonstrate that this interaction occurs under conditions where calcium is bound to S100A1, suggesting that the conformational shift in S100A1 when calcium binds is important. We also observed a conformational change in UN2A induced by shifts in pH, suggesting that conformational flexibility in UN2A plays a critical role in the interaction with S100A1. These results lead us to propose that the interaction of S100A1 and UN2A might act as a sensor to regulate titin's function in response to physiological changes in the muscle.

Voltage-gated sodium channel α-subunits (NaV1.1-1.9) initiate and propagate action potentials in neurons and myocytes. The NaV β-subunits (β1-4) have been shown to modulate α-subunit properties. Homo-oligomerization of β-subunits on neighboring or opposing plasma membranes has been suggested to facilitate cis or trans interactions, respectively. The interactions between several NaV channel isoforms and β-subunits have been determined using cryogenic electron microscopy (cryo-EM). Interestingly, the NaV cryo-EM structures reveal the presence of N-linked glycosylation sites. However, only the first glycan moieties are typically resolved at each site due to the flexibility of mature glycan trees. Thus, existing cryo-EM structures may risk de-emphasizing the structural implications of glycans on the NaV channels. Herein, molecular modeling and all-atom molecular dynamics simulations were applied to investigate the conformational landscape of N-linked glycans on NaV channel surfaces. The simulations revealed that negatively charged sialic acid residues of two glycan sites may interact with voltage-sensing domains. Notably, two NaV1.5 isoform-specific glycans extensively cover the α-subunit region that, in other NaV channel α-subunit isoforms, corresponds to the binding site for the β1- (and likely β3-) subunit immunoglobulin (Ig) domain. NaV1.8 contains a unique N-linked glycosylation site that likely prevents its interaction with the β2 and β4-subunit Ig-domain. These isoform-specific glycans may have evolved to facilitate specific functional interactions, for example, by redirecting β-subunit Ig-domains outward to permit cis or trans supraclustering within specialized cellular compartments such as the cardiomyocyte perinexal space. Further experimental work is necessary to validate these predictions.