Biology subjects

Pless, S. A.

Publications and source records attributed to Pless, S. A..

12 recordsLinked to original sources

bioRxiv2025

Coupling between acid-sensing ion channel 1a and the monocarboxylate transporter family shapes cellular pH response

Proton-mediated activation of acid-sensing ion channel 1a (ASIC1a) leads to influx of sodium and, to a lesser extent, calcium, followed by rapid and complete desensitization. However, various ASIC1a-expressing cell types display atypical proton-induced currents characterized by altered proton sensitivity and slow and incomplete desensitization. The origin of this functional diversity and its physiological relevance remain unclear. Here, we show that the monocarboxylate transporter 1 (MCT1) interacts with ASIC1a, leading to subtype-specific modulation of proton sensitivity and desensitization. We delineate the molecular mechanism of the ASIC1a-MCT1 coupling and find that the presence of MCT1 is required for the distinct ASIC1a-mediated currents observed in glioblastoma cells, the most lethal brain cancer in adults. Strikingly, presence of ASIC1a together with MCT1 significantly prolongs the lifespan of mice intracranially injected with glioblastoma cells. We thus uncover the basis for altered ASIC1a function in glioblastoma and highlight the importance of deciphering the cell-type specific ASIC1a interactome.

biophysics↗

bioRxiv2024

Conformational plasticity of human acid-sensing ion channel 1a

Acid-sensing ion channels (ASICs) are typically activated by acidic environments and contribute to nociception and synaptic plasticity. ASIC1a is the most abundant subunit in the central nervous system and forms homomeric channels permeable to Na+ and Ca2+, making it a compelling therapeutic target for acidotic pathologies including stroke and traumatic brain injury. However, a complete conformational library of human ASIC1a in its various functional states has yet to be described. Using cryo-EM, we obtained hASIC1a structures across a pH range between 8.5 and 5.7, as well as in the presence of a toxin agonist and a gating-modulating mutation. We identify six major conformations that establish linear transmembrane helices to be associated with an open state, delineate mechanistic differences between proton and toxin activation, and demonstrate that desensitization leads to unexpected conformational diversity in the transmembrane domain. Together, they provide a three-dimensional rationalization of decades of structure-function studies on ASIC.

biochemistry↗

bioRxiv2024

The sodium leak channel NALCN is regulated by neuronal SNARE complex proteins

The sodium leak channel NALCN is vital for the regulation of electrical activity in neurons and other excitable cells, and mutations in the channel or its auxiliary proteins lead to severe neurodevelopmental disorders. Here we show that the neuronal SNARE complex proteins syntaxin and SNAP25, which enable synaptic transmission in the nervous system, inhibit the activity of the NALCN channel complex in both heterologous systems and primary neurons. The existence of this interaction suggests that the neurotransmitter release machinery can regulate electrical signalling directly, and therefore modulate the threshold for its own activity. We further find that reduction of NALCN currents is sufficient to promote cell survival in syntaxin-depleted cells. This suggests that disinhibited NALCN may cause the puzzling phenomenon of rapid neuronal cell death in the absence of syntaxin. This interaction may offer opportunities for future drug development against genetic diseases linked to both NALCN- and SNARE protein-containing complexes.

neuroscience↗

bioRxiv2023

Dynamic conformational changes of acid-sensing ion channels in different desensitizing conditions

Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to fast synaptic transmission and have roles in fear conditioning and nociception. Apart from activation at low pH, ASIC1a also undergoes several types of desensitization, including acute desensitization that terminates activation, steady-stated desensitization that occurs at sub-activating proton concentrations and limits subsequent activation, and tachyphylaxis that results in a progressive decrease in response during a series of activations. Structural insights from a desensitized state of ASIC1 have provided great spatial detail, but dynamic insights into conformational changes in different desensitizing conditions are largely missing. Here, we use electrophysiology and voltage-clamp fluorometry to follow the functional changes of the pore along with conformational changes at several positions in the extracellular and upper transmembrane domain via cysteine-labeled fluorophores. Acute desensitization terminates activation in wild-type but introducing an N414K mutation in the {beta}11-12 linker of mouse ASIC1a interfered with this process. The mutation also affected steady-state desensitization and led to pronounced tachyphylaxis. Common to all types of desensitization was that the extracellular domain remained sensitive to pH and underwent pH-dependent conformational changes. These conformational changes did, however, not necessarily lead to desensitization. N414K-containing channels remained sensitive to known peptide modulators that increased steady-state desensitization, indicating that the mutation only reduced, but not precluded, desensitization. Together, this study contributes to understanding the fundamental properties of ASIC1a desensitization, emphasizing the complex interplay between the conformational changes of the ECD and the pore during channel activation and desensitization. Statement of significanceAcid-sensing ion channels (ASICs) are proton-gated ion channels that contribute to synaptic activity and play roles in acidosis-related diseases. Prolonged acidosis can lead to desensitization in ASIC1a, and modulators that affect this desensitization have shown beneficial effects in pain and stroke. In this study, we investigated the functional and conformational changes during acute desensitization, steady-state desensitization, and tachyphylaxis through a mutation in the {beta}11-12 linker of ASIC1a. We found that the mutation retained pH-dependent conformational changes of the extracellular domain (ECD) but largely disconnected these movements from the channel pore. Collectively, our work emphasizes the critical role of the {beta}11-12 linker for the pH-dependent conformational interplay between the ECD and the channel pore.

molecular biology↗

bioRxiv2023

Protein semisynthesis underscores role of a conserved lysine in activation and desensitization of acid-sensing ion channels

Acid-sensing ion channels (ASICs) are trimeric ion channels that open a cation-conducting pore in response to proton binding. Excessive ASIC activation during prolonged acidosis in conditions such as inflammation and ischemia is linked to pain and stroke. A conserved lysine in the extracellular domain (Lys211 in mASIC1a) is suggested to play a key role in ASIC function. However, the precise contributions are difficult to dissect with conventional mutagenesis, as replacement of Lys211 with naturally occurring amino acids invariably changes multiple physico-chemical parameters. Here, we study the contribution of Lys211 to mASIC1a function using tandem protein-trans splicing (tPTS) to incorporate non-canonical lysine analogs. We conduct optimization efforts to improve splicing and functionally interrogate semisynthetic mASIC1a. In combination with molecular modeling, we show that Lys211 charge and side chain length are crucial to activation and desensitization, thus emphasizing that tPTS can enable atomic-scale interrogations of membrane proteins in live cells.

biophysics↗

bioRxiv2021

Divergent functional and conformational outcomes in an ion channel-peptide interaction

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels involved in fast synaptic transmission. Pharmacological inhibition of ASIC1a reduces neurotoxicity and stroke infarct volumes, with the cysteine knot toxin Psalmotoxin-1 (PcTx1) being one of the most potent and selective inhibitors. PcTx1 binds at the subunit interface in the extracellular domain (ECD), but the mechanism and conformational consequences of the interaction, as well as the number of toxin molecules required for inhibition remain unknown. Here we use voltage-clamp fluorometry and subunit concatenation to decipher the mechanism and stoichiometry of PcTx1 inhibition of ASIC1a. Besides the known inhibitory binding mode, we propose PcTx1 to have at least two additional binding modes that are decoupled from the pore. One of these modes induces a long-lived ECD conformation that reduces the activity of an endogenous neuropeptide. This long-lived conformational state is proton-dependent and can be destabilized by a mutation that decreases PcTx1 sensitivity. Lastly, the use of concatemeric channel constructs reveal that disruption of a single PcTx1 binding site is sufficient to destabilize the toxin-induced conformation, while functional inhibition is not impaired until two or more binding sites are mutated. Together, our work provides insight into the mechanism of PcTx1 inhibition of ASICs and uncovers a prolonged conformational change with possible pharmacological implications.

biophysics↗

bioRxiv2021

Disrupting inter-subunit interactions favors channel opening in P2X2 receptors

Background and PurposeP2X receptors (P2XRs) are trimeric ligand-gated ion channels (LGICs) that open a cation-selective pore in response to ATP binding to their large extracellular domain (ECD). The seven known P2XR subtypes can assemble as homo- or heterotrimeric complexes and contribute to numerous physiological functions, including nociception, inflammation and hearing. The overall structure of P2XRs is well established, but little is known about the spectrum and prevalence of human genetic variations and the functional implications in specific domains. Experimental ApproachHere we examine the impact of P2X2 receptor (P2X2R) inter-subunit interface missense variants identified in the human population or through structural predictions. We test both single and double mutants through electrophysiological and biochemical approaches. Key resultsWe demonstrate that predicted ECD inter-subunit interfaces display a higher-than-expected density of missense variations and that the majority of mutations that disrupt putative inter-subunit interactions result in channels with higher apparent ATP affinity. Lastly, we show that double mutants at the subunit interface show significant energetic coupling, especially if located in close proximity. Conclusions and ImplicationsWe provide the first structural mapping of the mutational burden across the human population in a LGIC and show that the density of missense mutations is constrained between protein domains, indicating evolutionary selection at the domain level. Our data may indicate that, unlike other LGICs, P2X2Rs have evolved an intrinsically high threshold for activation, possibly to allow for additional modulation or as a cellular protection mechanism against overstimulation. Bullet point summary What is already known: O_LIP2X2 receptors are ATP-activated ion channels implicated in hearing and nociceptice pathways C_LI What this study adds: O_LIA structural mapping of missense variants observed in the human population C_LIO_LIWe identify the intersubunit-interface as a variant hotspot and decipher functional impact of mutations C_LI Clinical significance: O_LIThe development of both inhibitors and activators of P2X2 receptor function may be required C_LI

biophysics↗

bioRxiv2021

Differential contributions of M1 and pre-M1 to ion selectivity in ASICs and ENaCs

The ability to discriminate between different ionic species, termed ion selectivity, is a key feature of ion channels and forms the basis for their physiological function. Members of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily of trimeric ion channels are typically sodium selective, but to a surprisingly variable degree. While acid-sensing ion channels (ASICs) are weakly sodium selective (sodium:potassium around 10:1), ENaCs show a remarkably high preference for sodium over potassium (>500:1). The most obvious explanation for this discrepancy may be expected to originate from differences in the pore-lining second transmembrane segment (M2). However, these show a relatively high degree of sequence conservation between ASICs and ENaCs and previous functional and structural studies could not unequivocally establish that differences in M2 alone can account for the disparate degrees of ion selectivity. By contrast, surprisingly little is known about the contributions of the first transmembrane segment (M1) and the preceding pre-M1 region. In this study, we use conventional and non-canonical amino acid-based mutagenesis in combination with a variety of electrophysiological approaches to show that the pre-M1 and M1 regions of mASIC1a channels are major determinants of ion selectivity. Mutational investigations of the corresponding regions in hENaC show that they contribute less to ion selectivity, despite affecting ion conductance. In conclusion, our work supports the notion that the remarkably different degrees of sodium selectivity in ASICs and ENaCs are achieved through different mechanisms. The results further highlight how M1 and pre-M1 are likely to differentially affect pore structure in these related channels.

biophysics↗

bioRxiv2020

Functional crosstalk between phosphorylation and disease-causing mutations in the cardiac sodium channel Nav1.5

The voltage-gated sodium channel Nav1.5 initiates the cardiac action potential. Alterations of its activation and inactivation properties due to mutations can cause severe, life-threatening arrhythmias. Yet despite intensive research efforts, many functional aspects of this cardiac channel remain poorly understood. For instance, Nav1.5 undergoes extensive post-translational modification in vivo, but the functional significance of these modifications is largely unexplored, especially under pathological conditions. This is because most conventional approaches are unable to insert metabolically stable post-translational modification mimics, thus preventing a precise elucidation of the contribution by these modifications to channel function. Here, we overcome this limitation by using protein semi-synthesis of Nav1.5 in live cells and carry out complementary molecular dynamics simulations. We introduce metabolically stable phosphorylation mimics on both WT and two pathogenic long-QT mutant channel backgrounds and decipher functional and pharmacological effects with unique precision. We elucidate the mechanism by which phosphorylation of Y1495 impairs steady-state inactivation in WT Nav1.5. Surprisingly, we find that while the Q1476R patient mutation does not affect inactivation on its own, it enhances the impairment of steady-state inactivation caused by phosphorylation of Y1495 through enhanced unbinding of the inactivation particle. We also show that both phosphorylation and patient mutations can impact Nav1.5 sensitivity towards the clinically used anti-arrhythmic drugs quinidine and ranolazine, but not flecainide. The data highlight that functional effects of Nav1.5 phosphorylation can be dramatically amplified by patient mutations. Our work is thus likely to have implications for the interpretation of mutational phenotypes and the design of future drug regimens. Significance statementThe cardiac sodium channel (Nav1.5) is crucial for generating a regular heartbeat. It is thus not surprising that mutations in its sequence have been linked to life-threatening arrhythmias. Interestingly, Nav1.5 activity can also be altered by posttranslational modifications, such as tyrosine phosphorylation. Our combination of protein engineering and molecular modeling studies has revealed that the detrimental effect of a long QT3 patient mutation is only exposed when a proximal tyrosine is phosphorylated. This suggests a dynamic crosstalk between the genetic mutation and a neighboring phosphorylation, a phenomenon that could be important in other classes of proteins. Additionally, we show that phosphorylation can affect the channels sensitivity towards clinically-relevant drugs, a finding that may prove important when devising patient-specific treatment plans.

biophysics↗

bioRxiv2020

High-throughput characterization of 309 photocrosslinker-bearing ASIC1a variants maps residues critical for channel function and pharmacology

Incorporation of non-canonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing hASIC1a (human acid-sensing ion channel 1a) variants in transiently transfected mammalian cells. We introduce three different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch-clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay and live-cell crosslinking provides insight into the hASIC1a-psalmotoxin-1 interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells.

biophysics↗

bioRxiv2020

Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

Manipulation of proteins by chemical modification is a powerful way to decipher their function or harness that function for therapeutic purposes. Despite recent progress in ribosome-dependent and semi-synthetic chemical modifications, these techniques sometimes have limitations in the number and type of modifications that can be simultaneously introduced or their application in live eukaryotic cells. Here we present a new approach to incorporate single or multiple post-translational modifications or non-canonical amino acids into soluble and membrane proteins expressed in eukaryotic cells. We insert synthetic peptides into proteins of interest via tandem protein trans-splicing using two orthogonal split intein pairs and validate our approach by investigating different aspects of GFP, NaV1.5 and P2X2 receptor function. Because the approach can introduce virtually any chemical modification into both intracellular and extracellular regions of target proteins, we anticipate that it will overcome some of the drawbacks of other semi-synthetic or ribosome-dependent methods to engineer proteins.

synthetic biology↗

bioRxiv2019

The sodium leak channel complex is modulated by voltage and extracellular calcium

The sodium leak channel (NALCN) is essential for survival in mammals: NALCN mutations are life-threatening in humans and knockout is lethal in mice. However, the basic functional and pharmacological properties of NALCN have remained elusive. Here, we found that the robust function of NALCN in heterologous systems requires co-expression of UNC79, UNC80 and FAM155A. The resulting NALCN channel complex is constitutively active, conducts monovalent cations but is blocked by physiological concentrations of extracellular divalent cations. Our data support the notion that NALCN is directly responsible for the increased excitability observed in a variety of neurons in reduced extracellular Ca2+. Despite the smaller number of voltage-sensing residues in the putative voltage sensors of NALCN, the channel complex shows voltage-dependent modulation of the constitutive current, suggesting that voltage-sensing domains can give rise to a broader range of gating phenotypes than previously anticipated. Our work points towards formerly unknown contributions of NALCN to neuronal excitability and opens avenues for pharmacological targeting. HighlightsO_LIFunction of NALCN requires UNC79, UNC80 and FAM155A C_LIO_LIThe complex is permeable to monovalent cations, but is blocked by divalent cations C_LIO_LIThe complex displays a constitutively active, voltage-modulated current phenotype C_LIO_LIPositively charged side chains in S4 of NALCN VSD I and II confer voltage sensitivity C_LI

neuroscience↗