Journal of General Physiology最新文献

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.

Polyphosphoinositides (PPIns) are essential components of membrane lipids and play crucial roles in cell signaling in eukaryotes. Phosphatidylinositol4,5-bisphosphate (PI(4,5)P2) is a species of PPIns enriched in the plasma membrane and regulates numerous membrane proteins, including ion channels, transporters, and receptors, primarily through direct binding to positively charged residues such as lysine and arginine. Despite recent advances in structural biology and biophysics, the specific contributions of individual amino acid residues to PI(4,5)P2 binding in membrane proteins remain unclear. These questions have been explored by functional characterization of mutant proteins with site-specific amino acid replacement and their comparison with the WT proteins. Here, we apply genetic code expansion to investigate the role of lysine residues in the PI(4,5)P2 sensitivity of ion channels. A caged lysine compound, hydroxycoumarin-lysine (HCK), was incorporated at several key lysine residues critical for PI(4,5)P2 sensitivity in the mouse inward-rectifier potassium channel Kir2.1, expressed in Xenopus oocytes. Caging of lysine by introducing HCK at K182 or K187 completely silenced Kir2.1 currents, but light-induced uncaging restored current activity. Voltage-sensing phosphatase assays revealed that this current increase was accompanied by enhanced PI(4,5)P2 sensitivity. On the other hand, introducing HCK at K219, which forms a secondary PI(4,5)P2-binding region, did not fully eliminate Kir2.1 currents, and uncaging resulted in an approximately twofold increase in current. Analysis of uncaging and PI(4,5)P2 sensitivity in Kir2.1-K219HCK revealed that the region C-terminal to residue K219 is dispensable when assembled with the full-length protein. Genetic code expansion using caged lysine provides a valuable tool for studying the mechanisms of PI(4,5)P2 regulation in ion channels, complementing existing approaches.

G protein-gated inwardly rectifying potassium (GIRK) channels mediate membrane hyperpolarization in response to G protein-coupled receptor activation and are critical for regulating neuronal excitability. The membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) is essential for regulating the large family of inward rectifiers, and disruptions in PIP2 interactions contribute to some neurological diseases. Structural analyses have identified arginine-92 (R92) in GIRK2 as a key amino acid interacting with PIP2 as well as the potentiator cholesteryl hemisuccinate (CHS). Using electrophysiological assays and fluorescent K+ flux measurements, we show that substitutions at R92 (F, Y, or Q) disrupt PIP2 regulation, as well as G protein and alcohol activation. Cryo-EM structures of R92F and R92Q show an unexpected change in the orientation of the slide helix that leads to a "domain swap" between adjacent subunits in the cytoplasmic domain, producing a unique arrangement of the alcohol-binding pocket and G protein-interacting domain. These findings indicate that R92 plays a crucial role in how GIRK2 channel subunits assemble for physiological gating, and likely extend to gating of most inward rectifiers due to the high conservation of arginine in that location.

Cyclic nucleotide-binding domain (CNBD) channels are critical components of numerous bioelectrical processes, including cardiac pacemaking, neuronal signaling, phototransduction in the eye, and stomatal regulation in plants. While members of this channel family share a conserved overall structure, they exhibit striking differences in voltage sensitivity. Hyperpolarization-activated cyclic nucleotide-gated channels are activated by membrane hyperpolarization, whereas ether-à-go-go channels open upon depolarization. Mutagenesis and chimeragenesis studies have revealed that some mutants display bipolar gating behavior-remaining closed at intermediate membrane potentials but capable of opening in response to both hyperpolarization and depolarization. Remarkably, in certain cases, just a few mutations are sufficient to reverse the intrinsic gating polarity of the channel. This degree of diversity and plasticity in voltage-dependent gating appears to be unique to the CNBD clade and is not adequately explained by existing models. In this study, we systematically evaluate current models and propose a revised framework that better accounts for the full range of voltage-gating behaviors observed in CNBD channels.

Heart function depends on cardiomyocyte contractile apparatus and proper sarcomere protein expression. Variants in sarcomere genes cause inherited forms of cardiomyopathy and arrhythmias, including atrial fibrillation. Recently, a sarcomere component, myosin-binding protein-H like (MyBP-HL), was identified. MyBP-HL is mainly expressed in cardiac atria and is homologous to the last three C-terminal domains of cardiac myosin-binding protein-C (cMyBP-C). The MYBPHL R255X nonsense variant has been linked to atrial enlargement, dilated cardiomyopathy, and arrhythmias. Similar nonsense mutations in MYBPC3 are linked to hypertrophic cardiomyopathy, with these mutations preventing myofilament incorporation and the degradation of the truncated protein. However, the allele frequency of the MYBPHL R255X variant is too high in the human population to be pathogenic. We sought to determine whether MYBPHL nonsense variants impact on MyBP-HL sarcomere integration and degradation of the truncated protein, and whether the MyBPHL nonsense variants lead to changes in cardiomyocyte calcium dynamics and contractility. We mimicked human MYBPHL nonsense variants in the mouse Mybphl cDNA sequence and tested their sarcomere incorporation. We demonstrated that full-length MyBP-HL overexpression showed the expected C-zone sarcomere incorporation. Nonsense variants showed defective sarcomere incorporation. We demonstrated that full-length MyBP-HL and MyBP-HL nonsense variants were degraded by both proteasome and calpain mechanisms. We did not observe changes in calcium transients. In addition, we observed changes in contraction kinetics, including sarcomere shortening. Together, these data support the hypothesis that MYBPHL nonsense variants are functionally similar.

This study reveals an examination of the phenomenon of coupled gating between ryanodine receptors, the Ca2+ channels of the sarcoplasmic reticulum of skeletal and cardiac muscle, essential for the execution of contraction upon electrical excitation. It asks whether the phenomenon-pairs of channels or larger groups, reconstituted in bilayers, opening and closing together-reflects allosteric interactions that require contact between channels, and whether the phenomenon occurs in vivo with sufficient prevalence to be relevant to physiology and pathophysiology. The examination covers definitions, observations of coupled currents, structural studies of channels, in purified or in native membranes, and quantitative modeling of the phenomena. It concludes with a negative answer to the question whether a physiological role is proven, but a hopeful perspective on further research.

High voltage-activated (HVA) calcium channels (CaV) have four homologous but nonidentical repeats encompassing a voltage-sensing domain (VSD) and a quarter of the pore domain (PD). HVA can be modulated by at least two accessory subunits α2δ and CaVβ. A long-standing issue is how cytoplasmic CaVβ can shift the voltage dependence of channel opening without altering gating currents. Tracking the movement of individual VSDs by voltage-clamp fluorometry in human CaV1.2 revealed that only the VSD from the second repeat (VSD II) is perturbed by CaVβ3 in a construct combining a fluorophore-tagged VSD II (S1623C) with a quenching tryptophan within 11 Å in the PD of repeat III (E1141W). The final construct, S612C_E1141W, exhibited a biphasic voltage-dependent fluorescence whose negative phase was enhanced by CaVβ3. This behavior was well described by a kinetic model that includes three states for VSD II of which the intermediate state contributes the most to pore opening in a CaVβ-dependent manner, and that open channels with VSD II in the intermediate state would yield the lowest fluorescence emissions. Molecular dynamics simulation correlates a structure with two translocated arginines with frequent fluorophore-W contact between VSD II and the pore of open channels.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is recognized as an essential modulator of transient receptor potential (TRP) channels. Specifically, it influences the vanilloid receptor I (TRPV1), a pain receptor activated by a wide range of stimuli, including the binding of phospholipids, such as PIP2. The primary PIP2-binding site in TRPV1 has been identified through advanced techniques, revealing that the PIP2 binds to a specific pocket composed of positively charged residues located predominantly within the proximal C-terminus region. Additionally, a conserved segment with positively charged amino acids, K431 and R432, situated at the beginning of TRPV1's S1 transmembrane domain, has attracted considerable attention from the TRP research community. To date, our knowledge of this site's function and the subsequent effects following PIP2 binding is still emerging. In this work, MD simulations were conducted using coarse-grained models to investigate the binding dynamics of PIP2 on both WT and various mutated forms of TRPV1 channels. Our findings indicate that the K431A and R432A mutations significantly reduce the frequency of PIP2 contacts, suggesting that these mutated residues are part of a "peripheral binding pocket." This pocket seems to play a crucial role in facilitating the entry of PIP2 to the TRPV1 channel's primary binding site. Furthermore, our research has shown that these highly conserved residues within the TRPV subfamily are also structurally conserved across other TRP subfamilies, such as TRPM and TRPC, a detail not evident from sequence alignment alone. Consequently, we propose the existence of a structurally conserved peripheral PIP2-binding site shared among the diverse members of the TRP family, which can be categorized into distinct subfamilies.

Inwardly rectifying potassium (Kir) channel activity is important in the control of membrane potentials in both excitable and non-excitable cells and is regulated through various ligands, including specific membrane lipids. Phosphatidyl-4,5-bisphosphate (PIP2) is required for activity of all Kir channels, binding to the cytoplasmic domain in a compact conformation tightly tethered to the transmembrane domain. Most Kir2 channel structures determined in complex with PIP2 molecules are nevertheless in a closed state, requiring additional conformational changes for channel opening. We have carried out full atomistic MD simulations, which indicate PIP2-dependent conformational changes that are coupled to opening and closing of the channel. In the presence of bound PIP2, the cytoplasmic domain performs clockwise twisting motions, with a pivot residing near the C-linker in each subunit. These motions are reduced when PIP2 is removed, leading to narrowing of the critical gate at the M2 helix bundle crossing (HBC), but expansion at the region G-loop, as well as reduced overall fourfold symmetry, in turn coupled to cessation of ion permeation.

Pain perception is a complex experience, the initiation of which is mediated, among others, by voltage-gated sodium channels. Pathogenic variants in the sodium channel gene SCN11A encoding for NaV1.9 have been associated with various pain loss and neuropathic pain conditions. We herein describe the novel heterozygous SCN11A variant c.197A>C; p.(Tyr66Ser) that is absent in controls and cosegregates with small fiber neuropathy in a mother-and-son duo. To a variable degree, but progressively over time, both patients developed positive and negative sensory symptoms and milder autonomic signs. Upon quantitative sensory testing, we found significant thermal hypoesthesia and pinprick hyperalgesia in both individuals. Rectangle, half-sine-, and sine-wave stimulation applied to hands and feet in both individuals revealed signs of axonal on/off-like hyperexcitability, possibly due to continuous activation of CMi-fibers that are insensitive to mechanical stimulation (also known as sleeping nociceptors). Nerve conduction studies were unremarkable, whereas pain-related evoked potentials showed pathological responses in both individuals. The intraepidermal nerve fiber density was reduced at the index patient's distal leg. Patch-clamp analyses revealed that p.(Tyr66Ser) shifted both the voltage dependence of activation and steady-state inactivation of NaV1.9 to more depolarized potentials, accompanied by accelerated deactivation and a slowdown of the channel's inactivation kinetics. In addition, overexpression of the variant in mouse sensory neurons shortened the duration of individual action potentials and enhanced action potentials after hyperpolarization. In this translational n-of-two study, we present longitudinal data on disease progression and provide functional evidence that the SCN11A variant p.(Tyr66Ser) is a strong candidate to contribute to the patients' phenotype.