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SignificanceOur data challenge the generally accepted view that Kv2.1 proteins regulate arterial smooth muscle function by regulating their membrane potential. Rather, we discovered that Kv2.1 plays both conductive and structural roles with opposing functional consequences on arterial myocytes, with the former predominating in males, the latter in females. Opening of Kv2.1 channels opposes vasoconstriction by inducing membrane hyperpolarization. In addition to this conductive function, Kv2.1 promotes the structural clustering of CaV1.2 channels, thereby enhancing Ca2+ influx and inducing vasoconstriction. These two functions are highlighted by differences in the regulation of membrane potential, intracellular Ca2+, and myogenic tone between males and females. Our data suggest that these disparities derive from sex-specific variations in Kv2.1 expression levels in male versus female myocytes. The accepted role of the protein Kv2.1 in arterial smooth muscle cells is to form K+ channels in the sarcolemma. Opening of Kv2.1 channels causes membrane hyperpolarization, which decreases the activity of L-type CaV1.2 channels, lowering intracellular Ca2+ ([Ca2+]i) and causing smooth muscle relaxation. A limitation of this model is that it is based exclusively on data from male arterial myocytes. Here, we used a combination of electrophysiology as well as imaging approaches to investigate the role of Kv2.1 channels in male and female arterial myocytes. We confirmed that Kv2.1 plays a canonical conductive role but found it also has a structural role in arterial myocytes to enhance clustering of CaV1.2 channels. Less than 1% of Kv2.1 channels are conductive and induce membrane hyperpolarization. Paradoxically, by enhancing the structural clustering and probability of CaV1.2–CaV1.2 interactions within these clusters, Kv2.1 increases Ca2+ influx. These functional impacts of Kv2.1 depend on its level of expression, which varies with sex. In female myocytes, where expression of Kv2.1 protein is higher than in male myocytes, Kv2.1 has conductive and structural roles. Female myocytes have larger CaV1.2 clusters, larger [Ca2+]i, and larger myogenic tone than male myocytes. In contrast, in male myocytes, Kv2.1 channels regulate membrane potential but not CaV1.2 channel clustering. We propose a model in which Kv2.1 function varies with sex: in males, Kv2.1 channels control membrane potential but, in female myocytes, Kv2.1 plays dual electrical and CaV1.2 clustering roles. This contributes to sex-specific regulation of excitability, [Ca2+]i, and myogenic tone in arterial myocytes.

In mammalian brain neurons, membrane depolarization leads to voltage-gated Ca2+ channel-mediated Ca2+ influx that triggers diverse cellular responses, including gene expression, in a process termed excitation–transcription coupling. Neuronal L-type Ca2+ channels, which have prominent populations on the soma and distal dendrites of hippocampal neurons, play a privileged role in excitation–transcription coupling. The voltage-gated K⁺ channel Kv2.1 organizes signaling complexes containing the L-type Ca2+ channel Cav1.2 at somatic endoplasmic reticulum–plasma membrane junctions. This leads to enhanced clustering of Cav1.2 channels, increasing their activity. However, the downstream consequences of the Kv2.1-mediated regulation of Cav1.2 localization and function on excitation–transcription coupling are not known. Here, we have identified a region between residues 478 to 486 of Kv2.1’s C terminus that mediates the Kv2.1-dependent clustering of Cav1.2. By disrupting this Ca2+ channel association domain with either mutations or with a cell-penetrating interfering peptide, we blocked the Kv2.1-mediated clustering of Cav1.2 at endoplasmic reticulum–plasma membrane junctions and the subsequent enhancement of its channel activity and somatic Ca2+ signals without affecting the clustering of Kv2.1. These interventions abolished the depolarization-induced and L-type Ca2+ channel-dependent phosphorylation of the transcription factor CREB and the subsequent expression of c-Fos in hippocampal neurons. Our findings support a model whereby the Kv2.1-Ca2+ channel association domain-mediated clustering of Cav1.2 channels imparts a mechanism to control somatic Ca2+ signals that couple neuronal excitation to gene expression.

Calcium binding to BK channels lowers BK activation threshold, substantiating functional coupling with calcium-permeable channels. This coupling requires close proximity between different channel types, and the formation of BK-Ca V 1.3 hetero-clusters at nanometer distances exemplifies this unique organization. To investigate the structural basis of this interaction, we tested the hypothesis that BK and Ca V 1.3 channels assemble before their insertion into the plasma membrane. Our approach incorporated four strategies: (1) detecting interactions between BK and Ca V 1.3 proteins inside the cell, (2) identifying membrane compartments where intracellular hetero-clusters reside, (3) measuring the proximity of their mRNAs, and (4) assessing protein interactions at the plasma membrane during early translation. These analyses revealed that a subset of BK and Ca V 1.3 transcripts are spatially close in micro-translational complexes, and their newly synthesized proteins associate within the endoplasmic reticulum (ER) and Golgi. Comparisons with other proteins, transcripts, and randomized localization models support the conclusion that BK and Ca V 1.3 hetero-clusters form before their insertion at the plasma membrane.

Junctophilins (JPH) are a class of proteins found at junctions between the plasma membrane and the endoplasmic or sarcoplasmic reticulum, allowing for communications between proteins embedded in different membranes. JPHs have been proposed to interact with lipids as well as several ion channels, allowing for specialized communication between them. The JPH3 isoform is the target for repeats that cause Huntington’s disease-like 2, whereas JPH2 is a hot spot for mutations linked to cardiomyopathy. Here we present crystal structures of two JPH isoforms, which resemble a twisted skeleton with ribs formed by membrane occupation recognition nexus repeats, and a backbone built by a long α-helix. We captured the structure of a complex between JPH2 and a C-terminal binding site in the L-type calcium channel (CaV1.1) and show that this interaction is required for clustering of these channels and for robust muscle excitation–contraction coupling. Over 80 sequence variants linked to cardiomyopathy are found in different structurally important regions of JPH2, most of which affect stabilizing interactions. A subset directly affects the interaction with the L-type calcium channel. In parallel, sequence variants in the L-type calcium channel, linked to cardiac arrhythmia, also affect critical interactions.

Two-pore channel 2 (TPC2) is a lysosomal cation channel involved in calcium and sodium signaling and membrane remodeling. Its dysfunction has been linked to diseases including viral infections, neurodegenerative disorders, and cancer. Here, we identify mucin-type O-linked glycosylation at two conserved luminal residues of TPC2, Ser612 and Ser613, as a structural gating brake. Combining structure-guided mutagenesis, lysosomal patch-clamp recordings, calcium nanodomain imaging, molecular dynamics simulations, and small-molecule modulation, we show that genetic, enzymatic, or pharmacological glycan removal enhances basal and ligand-evoked TPC2 activity. This hyperactivation induces lysosomal tubulation, increases vesicle mobility, and promotes TPC2 clustering. In cancer-related models, glycan-deficient TPC2 also increases cell migration, which can be reversed by inhibitors such as tetrandrine and SC-3. These findings establish luminal glycosylation as a key post-translational regulator of TPC2 gating and reveal a mechanism by which ion channel activity controls lysosomal architecture and disease-relevant cell behavior. Lysosomal ion channels regulate diverse cellular processes, yet their modulation remains poorly understood. Here, authors show that mucin-type O-linked glycosylation restrains TPC2 gating, revealing a mechanism linking glycan modification to lysosomal function.

The cardiac ryanodine receptor (RyR2) constitutes the molecular basis of the process of calcium-induced calcium release where activation of RyR2s can be locally regenerative. Here, we present purely optical data of RyR2 distribution with sub-molecular resolution by applying 3D MINFLUX microscopy. Using single-domain antibodies and DNA-PAINT we determine the location of individual RyR2 subunits with high precision (~3 nm) and resolve the 3D orientations of RyR2s in-situ. We measured labeling efficiencies of ~50%, implying RyR2 tetramer detection probability approaching 95%. In HEK293 cells, RyR2 expression was dense, with some clusters containing several hundred RyR2s. Ventricular myocytes from mice contained large clusters containing many tens of close-packed RyR2s, resolving apparent discrepancies between electron microscopy and previous super-resolution microscopy data. The methodology developed here reveals the full 3D morphological complexity of RyR2 channels and is applicable to other multi-subunit complexes in a variety of cell types. Cardiac ryanodine receptors (RyR2) are critical for heart contraction. Here, the authors use 3D MINFLUX microscopy to image receptor subunits and RyR2 orientation with nanometre resolution, thereby providing a molecular view of the organisation and clustering of these cardiac muscle receptors.

Cardiac dysfunction is a hallmark of aging in humans and mice. Here we report that a two-week treatment to restore youthful Bridging Integrator 1 (BIN1) levels in the hearts of 24-month-old mice rejuvenates cardiac function and substantially reverses the aging phenotype. Our data indicate that age-associated overexpression of BIN1 occurs alongside dysregulated endosomal recycling and disrupted trafficking of cardiac Ca V 1.2 and type 2 ryanodine receptors. These deficiencies affect channel function at rest and their upregulation during acute stress. In vivo echocardiography reveals reduced systolic function in old mice. BIN1 knockdown using an adeno-associated virus serotype 9 packaged shRNA-mBIN1 restores the nanoscale distribution and clustering plasticity of ryanodine receptors and recovers Ca 2+ transient amplitudes and cardiac systolic function toward youthful levels. Enhanced systolic function correlates with increased phosphorylation of the myofilament protein cardiac myosin binding protein-C. These results reveal BIN1 knockdown as a novel therapeutic strategy to rejuvenate the aging myocardium. Cardiac dysfunction is a hallmark of aging in humans and mice. Here, the authors show that by restoring youthful Bridging Integrator 1 (BIN1) protein levels in the hearts of 24-month-old mice in vivo cardiac systolic function is rejuvenated, and the aging phenotype partially reversed within two weeks.

Action potentials trigger neurotransmitter release at the presynaptic active zone with spatiotemporal precision. This is supported by protein machinery that mediates synaptic vesicle priming and clustering of Ca V 2 Ca 2+ channels nearby. One model posits that scaffolding proteins directly tether vesicles to Ca V 2s; however, here we find that at mouse hippocampal synapses, Ca V 2 clustering and vesicle priming are executed by separate machineries. Ca V 2 nanoclusters are positioned at variable distances from those of the priming protein Munc13. The active zone organizer RIM anchors both proteins but distinct interaction motifs independently execute these functions. In transfected cells, Liprin-α and RIM form co-assemblies that are separate from Ca V 2-organizing complexes. At synapses, Liprin-α1–Liprin-α4 knockout impairs vesicle priming but not Ca V 2 clustering. The cell adhesion protein PTPσ recruits Liprin-α, RIM and Munc13 into priming complexes without co-clustering Ca V 2s. We conclude that active zones consist of distinct machineries to organize Ca V 2s and prime vesicles, and Liprin-α and PTPσ specifically support priming site assembly. The active zone primes synaptic vesicles and clusters voltage-gated Ca 2+ channels fast neurotransmitter release. Here the authors dissect the underlying molecular architecture and show that distinct protein machineries execute these functions.

The L-type Ca 2+ channel Ca V 1.2 is essential for arterial myocyte excitability, gene expression and contraction. Elevations in extracellular glucose (hyperglycemia) potentiate vascular L-type Ca 2+ channel via PKA, but the underlying mechanisms are unclear. Here, we find that cAMP synthesis in response to elevated glucose and the selective P2Y 11 agonist NF546 is blocked by disruption of A-kinase anchoring protein 5 (AKAP5) function in arterial myocytes. Glucose and NF546-induced potentiation of L-type Ca 2+ channels, vasoconstriction and decreased blood flow are prevented in AKAP5 null arterial myocytes/arteries. These responses are nucleated via the AKAP5-dependent clustering of P2Y 11 / P2Y 11 -like receptors, AC5, PKA and Ca V 1.2 into nanocomplexes at the plasma membrane of human and mouse arterial myocytes. Hence, data reveal an AKAP5 signaling module that regulates L-type Ca 2+ channel activity and vascular reactivity upon elevated glucose. This AKAP5-anchored nanocomplex may contribute to vascular complications during diabetic hyperglycemia. Molecular mechanisms by which glucose modulates L-type Ca 2+ channel activity and vascular reactivity are unclear. Here the authors report a nanocomplex orchestrated by AKAP5 that facilitates local purinergic stimulation of L-type Ca 2+ channels and vasoconstriction during diabetic hyperglycemia.

Biophysical properties of ligand-gated receptors can be profoundly modified by auxiliary subunits or by the lipid microenvironment of the membrane. Hence, it is sometimes challenging to relate the properties of receptors reconstituted in heterologous expression systems to those of their native counterparts. Here we show that the properties of Caenorhabditis elegans levamisole-sensitive acetylcholine receptors (L-AChRs), the ionotropic acetylcholine receptors targeted by the cholinergic anthelmintic levamisole at neuromuscular junctions, can be profoundly modified by their clustering machinery. We uncovered that L-AChRs exhibit a strong outward rectification in vivo, which was not previously described in heterologous systems. This unusual feature for an ionotropic AChR is abolished by disrupting the interaction of the receptors with the extracellular complex required for their synaptic clustering. When recorded at 260 mV, levamisole-induced currents are similar in the wild type and in L-AChR-clustering– defective mutants, while they are halved in these mutants at more depolarized physiological membrane potentials. Consequently, levamisole causes a strong muscle depolarization in the wild type, which leads to complete inactivation of the voltage-gated calcium channels and to an irreversible flaccid paralysis. In mutants defective for L-AChR clustering, the levamisole-induced depolarization is weaker, allowing voltage-gated calcium channels to remain partially active, which eventually leads to adaptation and survival of the worms. This explains why historical screens for C. elegans mutants resistant to levamisole identified the components of the L-AChR clustering machinery, in addition to proteins required for receptor biosynthesis or efficacy. This work further emphasizes the importance of pursuing ligand-gated channel characterization in their native environment.