Paul Blount | University of Texas Southwestern Medical Center at Dallas (original) (raw)

Papers by Paul Blount

Frontiers in chemistry, Feb 9, 2024

Editorial on the Research Topic In silico gating mechanism studies and modulator discovery for Ms... more Editorial on the Research Topic In silico gating mechanism studies and modulator discovery for MscL Mechanosensitive channel (MscL) proteins are viable pharmaceutical drug targets for development of precursors or antibiotics [

Membranes, 2021

Mechanosensitive channels respond to mechanical forces exerted on the cell membrane and play vita... more Mechanosensitive channels respond to mechanical forces exerted on the cell membrane and play vital roles in regulating the chemical equilibrium within cells and their environment. High-resolution structural information is required to understand the gating mechanisms of mechanosensitive channels. Protein-lipid interactions are essential for the structural and functional integrity of mechanosensitive channels, but detergents cannot maintain the crucial native lipid environment for purified mechanosensitive channels. Recently, detergent-free systems have emerged as alternatives for membrane protein structural biology. This report shows that while membrane-active polymer, SMA2000, could retain some native cell membrane lipids on the transmembrane domain of the mechanosensitive-like YnaI channel, the complete structure of the transmembrane domain of YnaI was not resolved. This reveals a significant limitation of SMA2000 or similar membrane-active copolymers. This limitation may come from...

Molecular Microbiology, 2019

SummaryThe bacterial MscL channel normally functions as an emergency release valve discharging cy... more SummaryThe bacterial MscL channel normally functions as an emergency release valve discharging cytoplasmic solutes upon osmotic stress. The channel opens and passes molecules up to 30 Å and its pore is the largest of any gated channel. Opening the MscL pore inappropriately is detrimental to the bacterial cell, suggesting MscL as a potential novel drug target. A small‐molecule compound, 011A, has been shown to increase sensitivity of the Escherichia coli MscL channel, slow growth, and even decrease viability of quiescent cultures. The mscL gene is highly conserved and found in the vast majority of bacterial species, including pathogens. Here, we test the hypothesis that 011A can influence the growth and viability of other bacterial species, specifically Staphylococcus aureus and Mycobacterium smegmatis, in a MscL‐dependent manner. Furthermore, we demonstrate that the 011A compound can increase potency of other antibiotics, presumably by permeabilizing the membrane and allowing easier...

Proceedings of the National Academy of Sciences, 2019

Significance Transient receptor potential (TRP) channels are key cation channels that respond to ... more Significance Transient receptor potential (TRP) channels are key cation channels that respond to thermal, chemical, electric, or mechanical stimuli. There are >60 mutations known in TrpV4 that cause defects in bone or neural development. Two mutations guided us to examine a cation–π bond, which we found to tie the four subunits together to help close the channel gate. This or a similar bond is seen in many structures of TRP channels. In experiments, breaking this bond favors opening and inactivation of TRPV4.

Biophysical Journal, 2019

Mechanosensitive channels from MscS and MscL families are major components of the tension-driven ... more Mechanosensitive channels from MscS and MscL families are major components of the tension-driven osmolyte efflux system regulating turgor pressure under day-today conditions and rescuing bacteria from rupture under abrupt osmotic down shock. MscL, which opens at higher tension, is considered to be an emergency valve acting at tensions approaching the lytic threshold. In patches taken from WT E. coli or Vibrio cholerae (Vc) spheroplasts, MscL is typically found in higher copy number than MscS, suggesting that the larger channel is the major pathway for expedient release of a larger spectrum of osmolytes in events of near-lethal shock. The smaller MscS was thought to regulate tension at moderate shocks and/or assist in termination of the osmotic permeability response due to its unique ability to inactivate. In this work we prepare the MscL KO construct containing the Kanamycin resistance cassette and 1 kb flanks and utilize the MuGENT technique to induce competence in Vibrio in order to introduce constructs into cells. Screening of 32 resistant candidates revealed a KO clone where the mscL ORF was undetectable. Surprisingly, osmotic shock viability of the KO clone was considerably higher than in WT and the rate of osmolyte release during osmotic shock measured with a millisecond resolution in light scattering stopped-flow experiments was also higher. Preliminary patch-clamp experiments indicated that MscL activities are not found in the KO, but the abundance of MscS increased more than twofold. The data indicate a strong compensatory reaction in Vc which responds to the absence of MscL with overexpression of the remaining MS channel. The work suggests the presence of physical mechanism allowing bacteria to detect the absence of MscL and trigger a compensatory reaction.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Jan 25, 2018

The bacterial mechanosensitive channel of large conductance (MscL) normally functions as an emerg... more The bacterial mechanosensitive channel of large conductance (MscL) normally functions as an emergency release valve discharging cytoplasmic solutes upon osmotic stress. Opening the large pore of MscL inappropriately is detrimental to the cell, and thus it has been speculated to be a potential antibiotic target. Although MscL is one of the best studied mechanosensitive channels, no chemical that influenced bacterial growth by modulating MscL is known. We therefore used a high-throughput screen to identify compounds that slowed growth in an MscL-dependent manner. We characterized 2 novel sulfonamide compounds identified in the screen. We demonstrated that, although both increase MscL gating, one of these compounds does not work through the folate pathway, as other antimicrobial sulfonamides; indeed, the sulfonamide portion of the compound is not needed for activity. The only mode of action appears to be MscL activation. The binding pocket is where an α-helix runs along the cytoplasmic...

Biophysical Journal, 2014

The Twik related potassium channel 1 (TREK-1) is one of the best studied mechanosensitive mammali... more The Twik related potassium channel 1 (TREK-1) is one of the best studied mechanosensitive mammalian channels. TREK-1 is known to play very important roles in depression, ischemia and vasoregulation. TREK-1 belongs to the family of background or "leak" potassium channels are constitutively open at rest and have a central role in the tuning of neuronal resting membrane potential, duration of action potential and regulation of neurotransmitter release. These K þ channels are members of the family of K2P channels subunits containing four transmembrane and two pore domains. The functional channel is formed by two subunits and is predicted to have a twofold symmetry. Here we show the feasibility of the use of microbial genetics to study the structure-function relationship of this mammalian channel. The advantage of this approach is that we can directly screen for channels with altered phenotypes and correlate this altered function with structural changes. We have successfully expressed a functional TREK-1 channel in bacterial cells, and show that it can partially rescue the slow growth phenotype of an E .coli strain deficient in three mayor potassium transporters. Furthermore, using random mutagenesis and bacterial screens we have isolated five mutants that better remediate the potassium deficiency of this bacterial strain. Because these mutants clustered in a stretch of 20 amino acids in the extracellular cap of the TREK-1, we think that we have found a functional "hot spot" by utilizing the power of bacterial genetics.

The Tachykinin Receptors, 1994

Page 1. CHAPTER 7 Molecular Biology of Receptors Structures, Expression, and Regulatory Mechanism... more Page 1. CHAPTER 7 Molecular Biology of Receptors Structures, Expression, and Regulatory Mechanisms James E. Krause, Paul Blount, and Bruce S. Sachais 1. Introduction ... 37. 37. 37. 37. 38' 396 bNK-2 382 hNK-3 432 rNK-3 419 SIL S L S L S L o W CKA Q TEP Hlil FGY l. L A ...

Molecular microbiology, 2004

Among players in neurobiology, ion channels are the demigods that underlie all our senses, behavi... more Among players in neurobiology, ion channels are the demigods that underlie all our senses, behaviour and intelligence. In animals, these 'gated pores' detect ligands, voltage, heat or stretch forces and emit electric or ionic signals. Patch clamp and genome sequencing now show that nearly all microbes also have these 'smart' molecules. Microbial channel proteins have yielded crystal structures so dear to neuroscientists. However, their natural roles in microbial physiology remain largely unknown. The intellectual and technical schisms between 'neuro' and 'micro' biology must be bridged before we know how we became so smart, and whether microbes are just as smart.

Gravitational and space biology bulletin : publication of the American Society for Gravitational and Space Biology, 1997

Receptor molecules that respond directly to gravity, touch, vibration, or osmotic pressure are in... more Receptor molecules that respond directly to gravity, touch, vibration, or osmotic pressure are inferred from their functions but not yet characterized as isolated proteins or products of cloned genes. These receptors are often in low abundance and in animal and plant tissues that are inaccessible, thus making biochemical analysis difficult. Yet, the application of the sensitive patch-clamp technique to measure transmembrane currents has demonstrated the ubiquity of ion channels whose opening is favored by membrane stretch forces. We have discovered in E. coli the activity of a mechanosensitive ion channel of large conductance (MscL), and have successfully isolated the corresponding protein and gene (Sukharev et al. 1994a). MscL channel appears to respond directly to stretch force in the lipid bilayer since it is active in artificial patches having only highly enriched MscL protein and lipids. Structurally, MscL is an integral membrane protein of only 136 amino-acid residues. Each ch...

Mechanosensitive Ion Channels, Part A, 2007

Publisher Summary Bacterial mechanosensitive (MS) channels are a means to study aspects of bacter... more Publisher Summary Bacterial mechanosensitive (MS) channels are a means to study aspects of bacterial physiology and are also the most advanced model system for studying MS channel function. They have thus emerged as a paradigm for studying the sense of a protein and respond to changes in its lipid environment. Among the bacterial MS channels, mechanosensitive channel of large conductance (MscL) has been the most tractable and is currently the best studied. Identification of the gene that encodes the MscL activity gave a first glimpse and chance for the genetic study of a channel that senses and responds to mechanical force. Models for the mechanisms of channel gating and the open structure have been generated and tested by several diverse approaches. Several studies have begun to determine the precise stimuli that are sensed by this channel. The data and projected models are providing a glimpse to the molecular mechanisms underlying an MS channel activity.

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Methods in Enzymology, 1999

Publisher Summary Although ion channel studies began with and continue to center on animals, a co... more Publisher Summary Although ion channel studies began with and continue to center on animals, a comprehensive study of ion channels must include microbes. Gated conductances, channel genes, proteins, and even crystal structures are amply documented in microbes. Traditionally, only a few large microbes such as Paramecium and Chara were amenable to direct electrophysiologic examination. However, the advent of patch-clamp techniques opened the field of ion channel studies in smaller cells including small animal, plant, or microbial cells. This chapter reviews electrophysiologic methods as applied to representative microbes. Several foreign channel genes have been expressed and analyzed in such microbial workhorses as Escherichia coli and the budding yeast Saccharomyces cerevisiae . These microbes allow mutant selection, based on agar-plate phenotypes after nondirected, random mutagenesis of channel genes that can be expressed in them. The strategies and methods of such studies are also reviewed here.

The Journal of cell biology, 1990

We have used fibroblast clones expressing muscle nicotinic acetylcholine receptor alpha and gamma... more We have used fibroblast clones expressing muscle nicotinic acetylcholine receptor alpha and gamma, and alpha and delta subunits to measure the kinetics of subunit assembly, and to study the properties of the partially assembled products that are formed. We demonstrate by coimmunoprecipitation that assembly intermediates in fibroblasts coexpressing alpha and delta subunits are formed in a time-dependent manner. The alpha and gamma- and the alpha and delta-producing transfected cells form complexes that, when labeled with 125I-alpha-bungarotoxin, migrate in sucrose gradients at 6.3S, a value consistent with a hetero-dimer structure. An additional peak at 8.5S is formed from the alpha and gamma subunits expressed in fibroblasts suggesting that gamma may have more than one binding site for alpha subunit. The stability and specificity of formation of these partially assembled complexes suggests that they are normal intermediates in the assembly of acetylcholine receptor. Comparison of th...

Science, 1982

The noncytopathic lymphocytic choriomeningitis virus displays a tropism for the anterior lobe of ... more The noncytopathic lymphocytic choriomeningitis virus displays a tropism for the anterior lobe of the murine pituitary gland. Virus replicates in cells that make growth hormone. This results in a diminished synthesis of growth hormone with a concomitant clinical picture of retarded growth and hypoglycemia. However, there is no morphologic evidence of either cell necrosis or inflammation in the anterior lobe of the pituitary. Hence, during infection in vivo, a noncytopathic virus may turn off the "differentiation" or "luxury" function of a cell while not killing that cell (loss of vital function). This is turn can disrupt homeostasis and cause disease. This model illustrates a novel way whereby viruses may cause disease.

Science, 1991

Neurotransmitter receptors are generally clustered in the postsynaptic membrane. The mechanism of... more Neurotransmitter receptors are generally clustered in the postsynaptic membrane. The mechanism of clustering was analyzed with fibroblast cell lines that were stably transfected with the four subunits for fetal (alpha, beta, gamma, delta) or adult (alpha, beta, epsilon, delta) type mouse muscle nicotinic acetylcholine receptors (AChRs). Immunofluorescent staining indicated that AChRs were dispersed on the surface of these cells. When transiently transfected with an expression construct encoding a 43-kilodalton protein that is normally concentrated under the postsynaptic membrane, AChRs expressed in these cells became aggregated in large cell-surface clusters, colocalized with the 43-kilodalton protein. This suggests that 43-kilodalton protein can induce AChR clustering and that cluster induction involves direct contact between AChR and 43-kilodalton protein.

Regulatory Peptides, 1993

Nature, 1986

The mechanism(s) by which infectious or malignant material is cleared by the host has long been a... more The mechanism(s) by which infectious or malignant material is cleared by the host has long been an area of intensive study. We have used the murine model of infection with lymphocytic choriomeningitis virus (LCMV) to look at immune clearance during persistent infection. LCMV was selected ...

Methods, 1994

Abstract A functional approach to molecular identification of the large conductance mechanosensit... more Abstract A functional approach to molecular identification of the large conductance mechanosensitive channel (MscL) from Escherichia coli cell envelope is described. Isolated membranes were solubilized in octylglucoside, and proteins were fractionated on several columns. Individual or pools of fractions were reconstituted into azolectin liposomes, which were then examined for the characteristic MscL activity (2.5-nS mechnoactivated currents) by the patch-clamp technique. Averaging the numbers of MscL conducting units in several patches allowed us to rule out inactive fractions and select MscL-containing fractions for subsequent steps of enrichment. We have thereby correlated MscL activity with the presence of a 17-kDa protein, whose gene, mscL , was then cloned. We confirmed a direct relationship between the mscL product and the channel activity by heterologous expression of this gene in two different systems. Here we describe the techniques used to record the characteristic MscL currents from liposomes reconstituted with material from MscL-expressing yeasts or with canine microsomes into which MscL protein was synthesized by a cell-free expression system.

Biophysical Journal, 2013

The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chl... more The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in the respiratory system and the gastrointestinal tract. Accordingly, human CLCA1 is a critical mediator of hypersecretory diseases that manifest mucus obstruction, such as asthma, COPD, and cystic fibrosis. It has been reported that hCLCA1 modulates calcium-activated chloride channels (CaCCs) in mammalian cell lines (Hamann et al., J Physiol 587: 2255-74; 2009), and that CLCAs are proteolytically processed during secretion (Patel et al., Annu Rev Physiol 71: 425-49; 2009); however, the precise molecular mechanisms of CLCAs remain unclear. To address this, we used a combination of sequence analysis, structure prediction, proteomics, and biochemical, biophysical and electrophysiological assays in HEK293 cells expressing several human and murine CLCA isoforms. We found that CLCAs are metalloproteases capable of both self-cleavage and cross-cleavage of other family members. We identified a novel zincin metalloprotease domain in the N-terminus of CLCA itself that is responsible for the self-proteolysis, and defined a consensus cleavage motif unique to the CLCA family. The activating effect of hCLCA1 on endogenous CaCCs was abolished in cells transfected with mutations that disrupt the metalloprotease activity or the cleavage site, and was recovered in cells transfected with the N-terminal fragment of the proteolysis, but not with the C-terminal fragment. Together, our data indicate that this unique CLCA self-cleavage event is required to unmask the N-terminal fragment of the protein, which is then responsible for the modulation of CaCCs. Our study provides a functional basis for CLCA1 self-cleavage, and a novel mechanism for regulation of chloride channel activity.