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.

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.

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.

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.

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.

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.

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.

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.

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.

Cardiac contractility is driven by shortening of ∼2-μm-long, macromolecular assemblies known as sarcomeres. During contraction, the motor protein myosin binds to, and exerts force upon actin filaments, utilizing energy from the hydrolysis of ATP. When not actively contracting, myosin partition into two subpopulations, distinguished by their basal rates of ATP hydrolysis, known as the "Disordered Relaxed" (DRX) and "Super Relaxed" (SRX) states. Additionally, the slower hydrolyzing SRX state has been proposed as a sequestered or "reserve pool" of myosin that do not contribute to contraction but can be recruited for enhanced contractility in response to external stimuli. Thus, the fraction of myosin in the SRX state is thought to reflect the overall regulatory state of the myosin population. In this volume of the Journal of General Physiology, a study by Pilagov et al. explores how the SRX state is regulated by phosphorylation or haploinsufficiency of a key regulatory protein, Myosin Binding Protein-C (MyBP-C). Surprisingly, they found that perturbations of MyBP-C led to a negligible change in the overall abundance of SRX. Instead, they found a rearrangement of SRX myosin throughout the sarcomere - specifically a decrease in SRX in regions of the sarcomere that contain MyBP-C and a compensatory increase in SRX in regions lacking MyBP-C. Their findings suggest that the influence of MyBP-C extends beyond its immediate vicinity and can simultaneously exert both positive and negative effects in a location-specific manner.

The cardiac voltage-gated sodium channel, Nav1.5, initiates the cardiac action potential. Its dysfunction can lead to dangerous arrhythmias, sudden cardiac arrest, and death. The functional Nav1.5 core consists of four homologous repeats (I, II, III, and IV), each formed from a voltage sensing and a pore domain. The channel also contains three cytoplasmic linkers (I-II, II-III, and III-IV). While Nav1.5 structures have been published, the I-II and II-III linkers have remained absent, are predicted to be disordered, and their functional role is not well understood. We divided the I-II linker into eight regions ranging in size from 32 to 52 residues, chosen based on their distinct properties. Since these regions had unique sequence properties, we hypothesized that they may have distinct effects on channel function. We tested this hypothesis with experiments with individual Nav1.5 constructs with each region deleted. These deletions had small effects on channel gating, though two (430-457del and 556-607del) reduced peak current. Phylogenetic analysis of the I-II linker revealed five prolines (P627, P628, P637, P640, and P648) that were conserved in mammals but absent from the Xenopus sequence. We created mutant channels, where these were replaced with their Xenopus counterparts. The only mutation that had a significant effect on channel gating was P627S, which depolarized channel activation (10.13 ± 2.28 mV). Neither a phosphosilent (P627A) nor a phosphomimetic (P627E) mutation had a significant effect, suggesting that either phosphorylation or another specific serine property is required. Since deletion of large regions had little effect on channel gating while a point mutation had a conspicuous impact, the I-II linker role may be to facilitate interactions with other proteins. Variants may have a larger impact if they create or disrupt these interactions, which may be key in evaluating the pathogenicity of variants.

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.

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.

The sodium leak channel NALCN, a key regulator of neuronal excitability, associates with three ancillary subunits that are critical for its function: a subunit called FAM155, which interacts with the extracellular regions of the channel, and two cytoplasmic subunits called UNC79 and UNC80. Interestingly, NALCN and FAM155 have orthologous phylogenetic relationships with the fungal calcium channel Cch1 and its subunit Mid1; however, UNC79 and UNC80 have not been reported outside of animals. In this study, we leveraged expanded gene sequence data available for eukaryotes to reexamine the evolutionary origins of NALCN and Cch1 channel subunits. Our analysis corroborates the direct phylogenetic relationship between NALCN and Cch1 and identifies a larger clade of related channels in additional eukaryotic taxa. We also identify homologues of FAM155/Mid1 in Cryptista algae and UNC79 and UNC80 homologues in numerous non-metazoan eukaryotes, including basidiomycete and mucoromycete fungi and the microbial eukaryotic taxa Apusomonadida, Malawimonadida, and Discoba. Furthermore, we find that most major animal lineages, except ctenophores, possess a full complement of NALCN subunits. Comparing structural predictions with the solved structure of the human NALCN complex supports orthologous relationships between metazoan and non-metazoan FAM155/Mid1, UNC79, and UNC80 homologues. Together, our analyses reveal unexpected diversity and ancient eukaryotic origins of NALCN/Cch1 channelosome subunits and raise interesting questions about the functional nature of this channel complex within a broad, eukaryotic context.

Cortical spreading depression (CSD) is a transient wave of neuronal and glial depolarization that propagates slowly through the cerebral cortex and is implicated in neurological events such as migraine aura. While glutamate, GABA, and serotonin have established roles in CSD modulation, the contribution of dopaminergic signaling, particularly via D2 receptors (D2Rs), remains unclear. In this study, we examined whether topical cortical application of D2R-targeting agents alters CSD propagation and neuronal activation in vivo. Using a KCl-induced CSD model in anesthetized male Wistar rats, we applied metoclopramide (MCP), raclopride (RCP), and quinpirole (QNP) directly onto the cortex. MCP completely blocked CSD propagation at all time points. RCP and QNP produced opposing, time-dependent effects: RCP initially reduced CSD speed, followed by an increase after prolonged exposure, whereas QNP transiently accelerated propagation at 5 min but suppressed it with longer exposure. These changes were accompanied by alterations in waveform morphology, particularly in the secondary negative deflection. c-Fos immunoreactivity revealed reduced neuronal activation in MCP- and QNP-treated animals, mainly in superficial cortical layers, while RCP showed no significant effect. To support these findings, a reaction-diffusion computational model incorporating drug diffusion, receptor binding kinetics, and excitability parameters successfully reproduced the experimental CSD propagation profiles. Together, these results demonstrate that cortical D2R ligands modulate CSD dynamics and neuronal activation in a ligand-specific and time-dependent manner. This study provides mechanistic insight into how dopaminergic signaling influences cortical excitability and CSD propagation, advancing our understanding of dopamine's role in fundamental neurophysiological processes.

Cantu syndrome (CS) is a rare disease caused by gain-of-function (GOF) mutations of Kir6.1 or SUR2 subunits of ATP-sensitive potassium (KATP) channels. CS patients with SUR2 and Kir6.1 variants display a similar constellation of symptoms, including muscle weakness and fatigue. The effects of CS mutations on skeletal muscle KATP channels, and any consequent direct effects on contractility, are currently unclear. Here, we used two knock-in mouse models of CS, respectively, carrying GOF mutations Kir6.1[V65M] or SUR2[A478V], to assess KATP channel properties and contractility in isolated fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles. Electrophysiological recordings in isolated myofibers showed normal resting potentials, and excised patch-clamp recordings showed normal KATP channel density in both genotypes, but enhanced Mg-nucleotide activation only in SUR2[A478V] fibers, consistent with muscle KATP channels being formed predominantly as complexes of SUR2A and Kir6.2 subunits. Ex vivo testing of isolated SUR2[A478V], but not Kir6.1[V65M], muscles showed an earlier onset of fatigue and a marked intra-tetanic decline of force compared with littermate controls. Importantly, normal contractile behavior was restored ex vivo and in vivo in SUR2[A478V] muscles in the presence of the FDA-approved KATP channel inhibitor glibenclamide, indicating that the increased fatigue of isolated muscles is a direct consequence of overactive sarcolemmal KATP channels. These results shed light on the pathophysiologic relevance of SUR2-dependent KATP channel subunits in skeletal muscle and highlight their role in fatiguing conditions, as well as identifying potential therapeutic benefit of skeletal muscle KATP inhibition in CS.

In this special issue of the Journal of General Physiology (JGP), we bring together a collection of studies that exemplify the multidimensional progress in physiology, pharmacology, and structure-function analysis of voltage-gated sodium (NaV) channels. From computational studies and single-residue mutagenesis to insights into drug interactions and electrophysiological variability, the assembled papers illustrate the richness and continuing momentum of this field.

Precise control of Kir2.1 channel gating is essential for maintaining membrane potential and enabling repolarization in excitable cells. Disruption of Kir2.1 function can cause Andersen-Tawil syndrome type 1 (ATS1), a multisystem channelopathy that predisposes patients to ventricular dysrhythmias and increases the risk of sudden cardiac death. Kir2.1 activity depends on interactions with the membrane phospholipid PIP2, and these interactions can be weakened by genetic mutations or posttranslational modifications. Here, we identify a shared mechanism by which hypoxia-induced SUMOylation and a heterozygous ATS1-linked variant, R67Q, independently and cooperatively suppress Kir2.1 function. We found that SUMOylation reduces Kir2.1 current in a stoichiometric manner, with up to two SUMO proteins per channel tetramer diminishing current by ∼24% each. Channels containing heterozygous R67Q subunits are disproportionately sensitive to hypoxic suppression. Inhibiting the SUMO pathway with TAK-981 prevents this suppression and enhances current in both WT and R67Q-containing channels. Further analysis revealed that both SUMOylation and the R67Q mutation reduce the stability of Kir2.1-PIP2 interactions, indicating a convergent gating defect. These findings support a two-hit model of channel dysfunction, in which a genetic variant and an environmental stressor act through a common structural mechanism to impair Kir2.1 gating. By identifying PIP2 destabilization as the point of convergence, this work provides new insight into how stress-sensitive channelopathies arise and suggests that SUMO pathway inhibition may offer a strategy to restore function under adverse physiological conditions.

TRPM1 channels, regulated by mGluR6 at the dendrites of retinal ON bipolar cells (BCs), play a crucial role in visual signal transduction. Both Trpm1 knockout (KO) and mGluR6 KO mice are models of congenital stationary night blindness with grossly normal morphology. However, robust pathological spontaneous oscillations in retinal ganglion cells (RGCs) have been observed in Trpm1 KO retinas but not in mGluR6 KO retinas. We investigated the mechanism underlying these oscillations in the Trpm1 KO retina using whole-cell clamp techniques. We found that inhibitory and excitatory synaptic inputs produced anti-phase oscillations in OFF and ON RGCs, respectively, and that oscillations could be suppressed by blockers targeting the AII amacrine cell (AC) pathway. The rd1 retina, a model for retinitis pigmentosa with severe photoreceptor degeneration, displays similar oscillations to the Trpm1 KO retina. Morphological and immunohistochemical analyses revealed similar alterations in the Trpm1 KO and rd1 retinas when compared to the mGluR6 KO and wild-type retinas: namely, rod BCs (RBCs) in both Trpm1 KO and rd1 retinas showed reduced dendritic TRPM1 labeling and smaller axon terminals. Furthermore, RBCs in the Trpm1 KO retina were significantly hyperpolarized. In silico simulation of the BC-AII AC-RGC network suggests that the reduction of RBC and ON cone BC outputs to AII ACs contributes to RGC oscillations. Our findings suggest that TRPM1 deficiency in ON BCs produces RGC oscillations in association with RBC axon remodeling and reduced ON BC outputs, and may represent a shared circuit mechanism underlying pathological oscillations across different causes of outer retinal diseases.

JGP study (Horie et al. https://doi.org/10.1085/jgp.202413749) explains why mice lacking TRPM1 exhibit oscillatory firing of their retinal ganglion cells, and suggests that the same mechanism causes similar oscillations in other outer retinal diseases.