Biology subjects

Kern, D. M.

Publications and source records attributed to Kern, D. M..

7 recordsLinked to original sources

bioRxiv2024

Assembly and lipid-gating of LRRC8A:D volume-regulated anion channels

Volume-regulated anion channels (VRACs) are ubiquitously expressed vertebrate ion channels that open in response to hypotonic swelling. VRACs assemble as heteromers of LRRC8A and LRRC8B-E subunits, with different subunit combinations resulting in channels with different properties. Recent studies have described the structures of LRRC8A:C VRACs, but how other VRACs assemble, and which structural features are conserved or variant across channel assemblies remains unknown. Herein, we used cryo-EM to determine structures of a LRRC8A:D VRAC with a 4:2 subunit stoichiometry, which we captured in two conformations. The presence of LRRC8D subunits increases hydrophobicity and widens the selectivity filter, which may explain the unique substrate selectivity of LRRC8D-containing VRACs. The structures reveal lipids bound inside the channel pore, similar to those observed in LRRC8A:C VRACs. Using electrophysiological experiments, we confirmed that pore lipids block conduction in the closed state, demonstrating that lipid-gating is a general property of VRACs. Finally, we observe that LRRC8D subunit incorporation disrupts packing of the cytoplasmic LRR domains, increasing channel dynamics and opening lateral fenestrations, which we speculate are necessary for pore lipid evacuation and channel activation.

biophysics↗

bioRxiv2022

Structural basis for assembly and lipid-mediated gating of LRRC8A:C volume-regulated anion channels

Leucine-rich repeat-containing protein 8 (LRRC8) family members form volume regulated anion channels activated by hypoosmotic cell swelling. LRRC8 channels are ubiquitously expressed in vertebrate cells as heteromeric assemblies of LRRC8A (Swell1) and LRRC8B-E subunits. Channels of different subunit composition have distinct properties that explain the functional diversity of LRRC8 currents implicated in a broad range of physiology. However, the basis for heteromeric LRRC8 channel assembly and function is unknown. Here, we leverage a fiducial-tagging strategy to determine single-particle cryo-electron microscopy structures of heterohexameric LRRC8A:C channels in detergent micelles and lipid nanodiscs in three conformations. LRRC8A:C channels show pronounced changes in channel architecture compared to homomeric channels due to heterotypic cytoplasmic LRR interactions that displace LRRs and the LRRC8C subunit away from the conduction axis and poise the channel for activation. The structures and associated functional studies further reveal that lipids embedded in the channel pore block ion conduction in the closed state. Together, our results provide insight into determinants for heteromeric LRRC8 channel assembly, activity, and gating by lipids.

biophysics↗

bioRxiv2022

Structure of SARS-CoV-2 M protein in lipid nanodiscs

SARS-CoV-2 encodes four structural proteins incorporated into virions, spike (S), envelope (E), nucleocapsid (N), and membrane (M). M plays an essential role in viral assembly by organizing other structural proteins through physical interactions and directing them to sites of viral budding. As the most abundant protein in the viral envelope and a target of patient antibodies, M is a compelling target for vaccines and therapeutics. Still, the structure of M and molecular basis for its role in virion formation are unknown. Here, we present the cryo-EM structure of SARS-CoV-2 M in lipid nanodiscs to 3.5 [A] resolution. M forms a 50 kDa homodimer that is structurally related to the SARS-CoV-2 ORF3a viroporin, suggesting a shared ancestral origin. Structural comparisons reveal how intersubunit gaps create a small, enclosed pocket in M and large open cavity in ORF3a, consistent with a structural role and ion channel activity, respectively. M displays a strikingly electropositive cytosolic surface that may be important for interactions with N, S, and viral RNA. Molecular dynamics simulations show a high degree of structural rigidity and support a role for M homodimers in scaffolding viral assembly. Together, these results provide insight into roles for M in coronavirus assembly and structure.

biophysics↗

bioRxiv2021

Small molecule SWELL1-LRRC8 complex induction improves glycemic control and nonalcoholic fatty liver disease in murine Type 2 diabetes

Type 2 diabetes (T2D) is associated with insulin resistance, impaired insulin secretion from the pancreatic {beta}-cell, and nonalcoholic fatty liver disease (NAFLD). SWELL1 (LRRC8a) ablation impairs adipose and skeletal muscle insulin-pAKT2 signaling, {beta}-cell insulin secretion and glycemic control - suggesting that SWELL1-LRRC8 complex dysfunction contributes to T2D pathogenesis. Here, we show that ICl,SWELL and SWELL1 protein are reduced in adipose and {beta}-cells in murine and human T2D. Combining cryo-electron microscopy, molecular docking, medicinal chemistry, and functional studies, we define a structure activity relationship to rationally-designed active derivatives (SN-40X) of a SWELL1 channel inhibitor (DCPIB/SN-401), that bind the SWELL1-LRRC8 hexameric complex, restore SWELL1-LRRC8 protein, plasma membrane trafficking, signaling and islet insulin secretion via SWELL1-dependent mechanisms. In vivo, SN-401 and active SN-40X compounds restore glycemic control and prevents NAFLD by improving insulin-sensitivity and insulin secretion in murine T2D. These findings demonstrate that small molecule SWELL1 modulators restore SWELL1-dependent insulin-sensitivity and insulin secretion in T2D and may represent a first-in-class therapeutic approach for T2D and NAFLD.

physiology↗

bioRxiv2020

Cryo-EM structure of the SARS-CoV-2 3a ion channel in lipid nanodiscs

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes the coronavirus disease 2019 (COVID-19). SARS-CoV-2 encodes three putative ion channels: E, 8a, and 3a1,2. 3a is expressed in SARS patient tissue and anti-3a antibodies are observed in patient plasma3-6. 3a has been implicated in viral release7, inhibition of autophagy8, inflammasome activation9, and cell death10,11 and its deletion reduces viral titer and morbidity in mice1, raising the possibility that 3a could be an effective vaccine or therapeutic target3,12. Here, we present the first cryo-EM structures of SARS-CoV-2 3a to 2.1 [A] resolution and demonstrate 3a forms an ion channel in reconstituted liposomes. The structures in lipid nanodiscs reveal 3a dimers and tetramers adopt a novel fold with a large polar cavity that spans halfway across the membrane and is accessible to the cytosol and the surrounding bilayer through separate water- and lipid-filled openings. Electrophysiology and fluorescent ion imaging experiments show 3a forms Ca2+-permeable non-selective cation channels. We identify point mutations that alter ion permeability and discover polycationic inhibitors of 3a channel activity. We find 3a-like proteins in multiple Alphacoronavirus and Betacoronavirus lineages that infect bats and humans. These data show 3a forms a functional ion channel that may promote COVID-19 pathogenesis and suggest targeting 3a could broadly treat coronavirus diseases.

biophysics↗

bioRxiv2019

Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs

Cation-chloride-cotransporters (CCCs) catalyze transport of Cl- with K+ and/or Na+ across cellular membranes. CCCs play roles in volume regulation, neural development and function, audition, blood pressure regulation, and renal function. CCCs are targets of drugs including loop diuretics and their disruption is implicated in pathophysiologies including epilepsy, hearing loss, and the genetic disorders Andermann, Gitelman, and Bartter syndromes. Here we present the cryo-EM structure of a CCC, the Mus musculus K+-Cl- cotransporter KCC4, in lipid nanodiscs. The structure, captured in an inside-open conformation, reveals the architecture of KCCs including an extracellular domain poised to regulate transport activity through an outer gate. We further identify substrate K+ and Cl- binding sites and provide a structural explanation for differences in substrate specificity and transport ratio between CCCs. These results provide mechanistic insight into the function and regulation of a physiologically important transporter family.

biophysics↗

bioRxiv2018

Cryo-EM structures of the DCPIB-inhibited volume-regulated anion channel LRRC8A in lipid nanodiscs

Hypoosmotic conditions activate volume-regulated anion channels in vertebrate cells. These channels are formed by leucine-rich repeat-containing protein 8 (LRRC8) family members and contain LRRC8A in homo- or hetero-hexameric assemblies. Here we present single-particle cryo-electron microscopy structures of LRRC8A in complex with the inhibitor DCPIB reconstituted in lipid nanodiscs. DCPIB plugs the channel like a cork in a bottle - binding in the extracellular selectivity filter and sterically occluding ion conduction. Constricted and expanded structures reveal coupled dilation of cytoplasmic LRRs and the channel pore, suggesting a mechanism for channel gating by internal stimuli. Conformational and symmetry differences between LRRC8A structures determined in detergent micelles and lipid bilayers related to reorganization of intersubunit lipid binding sites demonstrate a critical role for the membrane in determining channel structure. These results provide insight into LRRC8 gating and inhibition and the role of lipids in the structure of an ionic-strength sensing ion channel.

neuroscience↗