5-hydroxytryptamine type 3 (5-HT3) receptors are cation-selective transmitter-gated ion channels of the Cys-loop superfamily. The single-channel conductance of human recombinant 5-HT3 receptors assembled as homomers of 5-HT3A subunits, or heteromers of 5-HT3A and 5-HT3B subunits, are markedly different, being 0.4 pS (refs 6, 9) and 16 pS (ref. 7), respectively. Paradoxically, the channel-lining M2 domain of the 5-HT3A subunit would be predicted to promote cation conduction, whereas that of the 5-HT3B subunit would not. Here we describe a determinant of single-channel conductance that can explain these observations. By constructing chimaeric 5-HT3A and 5-HT3B subunits we identified a region (the 'HA-stretch') within the large cytoplasmic loop of the receptor that markedly influences channel conductance. Replacement of three arginine residues unique to the HA-stretch of the 5-HT3A subunit by their 5-HT3B subunit counterparts increased single-channel conductance 28-fold. Significantly, ultrastructural studies of the Torpedo nicotinic acetylcholine receptor indicate that the key residues might frame narrow openings that contribute to the permeation pathway. Our findings solve the conundrum of the anomalously low conductance of homomeric 5-HT3A receptors and indicate an important function for the HA-stretch in Cys-loop transmitter-gated ion channels.

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.

There are five families of vertebrate Cys loop receptors as follows: the nicotinic acetylcholine receptor (nAChR), 3 the 5-hydroxytryptamine type 3 receptor (5-HT 3 R), the zinc-activated ion channel, the ␥-aminobutyric acid type A receptor, and the strychnine-sensitive glycine receptor (1, 2). Structural analysis by cryo-EM of tubular crystals prepared from the Torpedo marmorata electric organ revealed that five subunits combine in nAChRs, forming a rosette around the central ion channel (3). The second transmembrane (M2) domain of each subunit participates in lining the channel pore, and collectively they present a hydrophobic constriction adjacent to what is traditionally believed to be the rate-limiting portion of the ion conduction pathway that controls single channel conductance (␥) and ionic selectivity (2, 4).The homomeric 5-HT 3A receptor is unique among Cys loop receptors, having a ␥ below the resolution of single channel recording, estimated by variance analysis to be in the femtosiemen range. The incorporation of the 5-HT 3B subunit into human heteromeric 5-HT 3A/B receptors increases ␥ to 16 pS, enabling direct observation of events by single channel recording from outside-out patches (5). The use of chimeric 5-HT 3A -5-HT 3B constructs and site-directed mutagenesis revealed a critical role of three arginine residues within the MA helix of the M3-M4 cytoplasmic loop in determining ␥ (6, 7). This has prompted speculation that the MA helix may participate in the control of ␥ in other Cys loop receptors (4,7,8). In support of this interpretation, cryo-EM analysis revealed portals within the Torpedo nAChR formed, in part, by the MA helices of adjacent subunits that may participate in the ion conduction pathway (9).In this study we investigated the influence of the 5-HT 3A subunit's MA helix Arg-432 (Ϫ4Ј), Arg-436 (0Ј), and Arg-440 (4Ј) residues in the control of ␥. We investigated the effect of introducing arginine into the equivalent locations within the nAChR ␣ 4 and ␤ 2 subunits. Our data confirm the critical role of MA Ϫ4Ј, 0Ј, and 4Ј residues in controlling ␥ of 5-HT 3A receptors and support the hypothesis that the MA helix also forms part of the ion conduction pathway of nAChRs. Our functional data provide support for the existence of cytoplasmic portals depicted in the 4 Å structural model of the nAChR (10). EXPERIMENTAL PROCEDURES DNA Constructs and Transient Transfection of Subunit cDNAs-cDNAs encoding rat wild-type (WT) nAChR ␣ 4 and ␤ 2 subunits (Dr. J. M. Boulter, Department of Psychiatry and Biobehavioral Sciences, UCLA), human WT 5-HT 3A subunits, and mutant nAChR and 5-HT 3A subunits were cloned into pGW1 (11). Point mutations were introduced using standard molecular biological techniques (7). All cDNAs were sequenced to confirm fidelity. Transfection of tsA-201, or HEK-293 cells, with subunit cDNAs, at equimolar ratios when appropriate, was performed by either the calcium phosphate precipitation method or electroporation (400 V, infinite resistance, 125 microfarads) using a Bio-Rad gene ...