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Patch-seq reveals new neuronal subtypes by combining electrophysiological and RNA-seq data on single neurons in situ. Traditionally, neuroscientists have defined the identity of neurons by the cells' location, morphology, connectivity and excitability. However, the direct relationship between these parameters and the molecular phenotypes has remained largely unexplored. Here, we present a method for obtaining full transcriptome data from single neocortical pyramidal cells and interneurons after whole-cell patch-clamp recordings in mouse brain slices. In our approach, termed Patch-seq, a patch-clamp stimulus protocol is followed by the aspiration of the entire somatic compartment into the recording pipette, reverse transcription of RNA including addition of unique molecular identifiers, cDNA amplification, Illumina library preparation and sequencing. We show that Patch-seq reveals a close link between electrophysiological characteristics, responses to acute chemical challenges and RNA expression of neurotransmitter receptors and channels. Moreover, it distinguishes neuronal subpopulations that correspond to both well-established and, to our knowledge, hitherto undescribed neuronal subtypes. Our findings demonstrate the ability of Patch-seq to precisely map neuronal subtypes and predict their network contributions in the brain.

A central question in mechanobiology is how mechanical forces acting in or on cells are transmitted to mechanically-gated PIEZO channels that convert these forces into biochemical signals. Here we examined the role of the intracellular domains of PIEZO2, which account for 25% of the channel, and demonstrate that these domains fine-tune properties such as poking and stretch-sensitivity, velocity coding and single channel conductance. Moreover, we show that the intrinsically disordered linker between the transmembrane helices twelve and thirteen (IDR5) is required for the activation of PIEZO2 by cytoskeleton-transmitted forces. The deletion of IDR5 abolishes PIEZO2-mediated inhibition of neurite outgrowth, while it only partially affected its sensitivity to cell indentation and does not alter its stretch sensitivity. Thus, we propose that PIEZO2 is a polymodal mechanosensor that detects different types of mechanical stimuli via different force transmission pathways, which highlights the importance of utilizing multiple complementary assays when investigating PIEZO function. A key question in mechanobiology is how mechanical forces are transmitted to PIEZO ion channels. Here, Verkest et al. identify an intracellular channel domain that is required for the activation of PIEZO2 by cytoskeleton-transmitted forces.

Lysosomes are critical for cellular metabolism and are heterogeneously involved in various cellular processes. The ability to measure lysosomal metabolic heterogeneity is essential for understanding their physiological roles. We therefore built a single-lysosome mass spectrometry (SLMS) platform integrating lysosomal patch-clamp recording and induced nano-electrospray ionization (nanoESI)/mass spectrometry (MS) that enables concurrent metabolic and electrophysiological profiling of individual enlarged lysosomes. The accuracy and reliability of this technique were validated by supporting previous findings, such as the transportability of lysosomal cationic amino acids transporters such as PQLC2 and the lysosomal trapping of lysosomotropic, hydrophobic weak base drugs such as lidocaine. We derived metabolites from single lysosomes in various cell types and classified lysosomes into five major subpopulations based on their chemical and biological divergence. Senescence and carcinoma altered metabolic profiles of lysosomes in a type-specific manner. Thus, SLMS can open more avenues for investigating heterogeneous lysosomal metabolic changes during physiological and pathological processes.Single-lysosome mass spectrometry (SLMS) integrates lysosomal patch-clamp recording and induced nanoESI/MS for concurrent metabolic and electrophysiological profiling of individual enlarged lysosomes.

A cryo-electron microscopy structure shows that the mucolipin domain of the lysosomal calcium channel TRPML3 binds phosphatidylinositol-3,5-bisphosphate and gates the channel. Closing in on ion channels Numerous ion channels sit in the membranes of intracellular organelles and are responsible for maintaining concentration gradients and ionic signalling. The transient receptor potential mucolipin (TRPML) channels are Ca( II )-releasing channels that are crucial to endolysosomal function. While TRPML channels regulate physiological processes including membrane trafficking and exocytosis, mutations of TRPML1 cause the lysosomal storage disorder mucolipidosis type IV. Three papers in this issue of Nature report the structure of TRPML channels by cryo-electron microscopy. Seok-Yong Lee and colleagues report the structure of TRPML3, while studies from teams led by Xiaochun Li and Youxing Jiang present the structure of TRPML1. Together, these studies reveal the open and closed states of the TRPML family, indicating the regulatory mechanisms of these channels. As with most TRP channels, TRPML can be gated by specific lipids, and these studies provide insights into substrate binding and channel activation. The modulation of ion channel activity by lipids is increasingly recognized as a fundamental component of cellular signalling. The transient receptor potential mucolipin (TRPML) channel family belongs to the TRP superfamily 1 , 2 and is composed of three members: TRPML1–TRPML3. TRPMLs are the major Ca 2+ -permeable channels on late endosomes and lysosomes (LEL). They regulate the release of Ca 2+ from organelles, which is important for various physiological processes, including organelle trafficking and fusion 3 . Loss-of-function mutations in the MCOLN1 gene, which encodes TRPML1, cause the neurodegenerative lysosomal storage disorder mucolipidosis type IV, and a gain-of-function mutation (Ala419Pro) in TRPML3 gives rise to the varitint–waddler ( Va ) mouse phenotype 4 , 5 , 6 . Notably, TRPML channels are activated by the low-abundance and LEL-enriched signalling lipid phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P 2 ), whereas other phosphoinositides such as PtdIns(4,5)P 2 , which is enriched in plasma membranes, inhibit TRPMLs 7 , 8 . Conserved basic residues at the N terminus of the channel are important for activation by PtdIns(3,5)P 2 and inhibition by PtdIns(4,5)P 2 8 . However, owing to a lack of structural information, the mechanism by which TRPML channels recognize PtdIns(3,5)P 2 and increase their Ca 2+ conductance remains unclear. Here we present the cryo-electron microscopy (cryo-EM) structure of a full-length TRPML3 channel from the common marmoset ( Callithrix jacchus ) at an overall resolution of 2.9 Å. Our structure reveals not only the molecular basis of ion conduction but also the unique architecture of TRPMLs, wherein the voltage sensor-like domain is linked to the pore via a cytosolic domain that we term the mucolipin domain. Combined with functional studies, these data suggest that the mucolipin domain is responsible for PtdIns(3,5)P 2 binding and subsequent channel activation, and that it acts as a ‘gating pulley’ for lipid-dependent TRPML gating.

This protocol describes how to integrate whole-cell patch-clamp in single neurons from mouse brain tissue slices with single-cell RNA sequencing and morphological recovery. Neurons exhibit a rich diversity of morphological phenotypes, electrophysiological properties, and gene-expression patterns. Understanding how these different characteristics are interrelated at the single-cell level has been difficult because of the lack of techniques for multimodal profiling of individual cells. We recently developed Patch-seq, a technique that combines whole-cell patch-clamp recording, immunohistochemistry, and single-cell RNA-sequencing (scRNA-seq) to comprehensively profile single neurons from mouse brain slices. Here, we present a detailed step-by-step protocol, including modifications to the patching mechanics and recording procedure, reagents and recipes, procedures for immunohistochemistry, and other tips to assist researchers in obtaining high-quality morphological, electrophysiological, and transcriptomic data from single neurons. Successful implementation of Patch-seq allows researchers to explore the multidimensional phenotypic variability among neurons and to correlate gene expression with phenotype at the level of single cells. The entire procedure can be completed in ∼2 weeks through the combined efforts of a skilled electrophysiologist, molecular biologist, and biostatistician.

Modulation of voltage-gated potassium (Kv) channels by auxiliary subunits is central to the physiological function of channels in the brain and heart 1 , 2 . Native Kv4 tetrameric channels form macromolecular ternary complexes with two auxiliary β-subunits—intracellular Kv channel-interacting proteins (KChIPs) and transmembrane dipeptidyl peptidase-related proteins (DPPs)—to evoke rapidly activating and inactivating A-type currents, which prevent the backpropagation of action potentials 1 – 5 . However, the modulatory mechanisms of Kv4 channel complexes remain largely unknown. Here we report cryo-electron microscopy structures of the Kv4.2–DPP6S–KChIP1 dodecamer complex, the Kv4.2–KChIP1 and Kv4.2–DPP6S octamer complexes, and Kv4.2 alone. The structure of the Kv4.2–KChIP1 complex reveals that the intracellular N terminus of Kv4.2 interacts with its C terminus that extends from the S6 gating helix of the neighbouring Kv4.2 subunit. KChIP1 captures both the N and the C terminus of Kv4.2. In consequence, KChIP1 would prevent N-type inactivation and stabilize the S6 conformation to modulate gating of the S6 helices within the tetramer. By contrast, unlike the reported auxiliary subunits of voltage-gated channel complexes, DPP6S interacts with the S1 and S2 helices of the Kv4.2 voltage-sensing domain, which suggests that DPP6S stabilizes the conformation of the S1–S2 helices. DPP6S may therefore accelerate the voltage-dependent movement of the S4 helices. KChIP1 and DPP6S do not directly interact with each other in the Kv4.2–KChIP1–DPP6S ternary complex. Thus, our data suggest that two distinct modes of modulation contribute in an additive manner to evoke A-type currents from the native Kv4 macromolecular complex. Cryo-electron microscopy structures of the voltage-gated potassium channel Kv4.2 alone and in complex with auxiliary subunits (DPP6S and/or KChIP1) reveal the distinct mechanisms of these two different subunits in modulating channel activity.

Binding of cAMP to Hyperpolarization activated cyclic nucleotide gated (HCN) channels facilitates pore opening. It is unclear why the isolated cyclic nucleotide binding domain (CNBD) displays in vitro lower affinity for cAMP than the full-length channel in patch experiments. Here we show that HCN are endowed with an affinity switch for cAMP. Alpha helices D and E, downstream of the cyclic nucleotide binding domain (CNBD), bind to and stabilize the holo CNBD in a high affinity state. These helices increase by 30-fold cAMP efficacy and affinity measured in patch clamp and ITC, respectively. We further show that helices D and E regulate affinity by interacting with helix C of the CNBD, similarly to the regulatory protein TRIP8b. Our results uncover an intramolecular mechanism whereby changes in binding affinity, rather than changes in cAMP concentration, can modulate HCN channels, adding another layer to the complex regulation of their activity. Cyclic AMP modulation of HCN channels underlies beta adrenergic stimulation of heart rate. Here, authors describe an intramolecular mechanism that controls cAMP affinity of the cyclic nucleotide binding domain of these channels.

Two-pore channels are endo-lysosomal cation channels with malleable selectivity filters that drive endocytic ion flux and membrane traffic. Here we show that TPC2 can differentially regulate its cation permeability when co-activated by its endogenous ligands, NAADP and PI(3,5)P 2 . Whereas NAADP rendered the channel Ca 2+ -permeable and PI(3,5)P 2 rendered the channel Na + -selective, a combination of the two increased Ca 2+ but not Na + flux. Mechanistically, this was due to an increase in Ca 2+ permeability independent of changes in ion selectivity. Functionally, we show that cell permeable NAADP and PI(3,5)P 2 mimetics synergistically activate native TPC2 channels in live cells, globalizing cytosolic Ca 2+ signals and regulating lysosomal pH and motility. Our data reveal that flux of different ions through the same pore can be independently controlled and identify TPC2 as a likely coincidence detector that optimizes lysosomal Ca 2+ signaling. TPC2 is a lysosomal ion channel permeable to both calcium and sodium ions. Here, the authors show that TPC2 can selectively increase its calcium permeability when simultaneously challenged by both its natural activators- NAADP and PI(3,5)P 2 .

Impaired protein stability or trafficking underlies diverse ion channelopathies and represents an unexploited unifying principle for developing common treatments for otherwise dissimilar diseases. Ubiquitination limits ion channel surface density, but targeting this pathway for the purposes of basic study or therapy is challenging because of its prevalent role in proteostasis. We developed engineered deubiquitinases (enDUBs) that enable selective ubiquitin chain removal from target proteins to rescue the functional expression of disparate mutant ion channels that underlie long QT syndrome (LQT) and cystic fibrosis (CF). In an LQT type 1 (LQT1) cardiomyocyte model, enDUB treatment restored delayed rectifier potassium currents and normalized action potential duration. CF-targeted enDUBs synergistically rescued common (ΔF508) and pharmacotherapy-resistant (N1303K) CF mutations when combined with the US Food and Drug Administation (FDA)-approved drugs Orkambi (lumacaftor/ivacaftor) and Trikafta (elexacaftor/tezacaftor/ivacaftor and ivacaftor). Altogether, targeted deubiquitination via enDUBs provides a powerful protein stabilization method that not only corrects diverse diseases caused by impaired ion channel trafficking, but also introduces a new tool for deconstructing the ubiquitin code in situ. Engineered deubiquitinases (enDUBs) use nanobodies to bring deubiquitinases to target proteins for selective ubiquitin chain removal. enDUBs can rescue the functional expression of mutant ion channels like those responsible for cystic fibrosis.