Stephan A Pless

IT University of Copenhagen, København, Capital Region, Denmark

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Publications (38)185.93 Total impact

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    ABSTRACT: ATP-sensitive potassium (KATP) channels are heteromultimeric complexes of an inwardly-rectifying Kir channel (Kir6.x) and sulfonylurea receptors (SUR). Their regulation by intracellular ATP and ADP generates electrical signals in response to changes in cellular metabolism. We investigated channel elements that control the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels re-open after rapid washout of ATP with a time constant of approximately 60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp68, Lys170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp68 or Lys170 markedly slow the kinetics of channel opening (500 ms and 700 ms for Trp68Leu and Lys170Asn, respectively), while increasing channel open probability. Examining the functional effects of these residues using phi-value analysis revealed a steep negative slope. This finding implies that these residues play a role in lowering the transition state energy barrier between open and closed channel states. Using unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68 for normal channel gating, potentially necessary to localize the ε-amine of Lys170 in the PIP2 binding site. Overall, our findings identify a discrete pair of highly conserved residues with an essential role for controlling gating kinetics of Kir channels. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; DOI:10.1074/jbc.M114.631960
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    ABSTRACT: Unnatural amino acid incorporation into ion channels has proven to be a valuable approach to interrogate detailed hypotheses arising from atomic resolution structures. In this short review, we provide a brief overview of some of the basic principles and methods for incorporation of unnatural amino acids into proteins. We also review insights into the function and pharmacology of voltage-gated ion channels that have emerged from unnatural amino acid mutagenesis approaches.This article is protected by copyright. All rights reserved
    The Journal of Physiology 01/2015; DOI:10.1113/jphysiol.2014.287714
  • Biophysical Journal 01/2015; 108(2):20a-21a. DOI:10.1016/j.bpj.2014.11.136
  • Stephan A. Pless
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    ABSTRACT: In this issue of Structure, Ulens and colleagues demonstrate how an elegant combination of complementary functional and structural approaches can uncover both binding sites and conformational consequences associated with the Alzheimer’s drug memantine binding to an ion channel.
    Structure 10/2014; 22(10):1373–1374. DOI:10.1016/j.str.2014.09.003
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    ABSTRACT: Voltage-gated sodium (NaV) channels mediate electrical excitability in animals. Despite strong sequence conservation among the voltage-sensor domains (VSDs) of closely related voltage-gated potassium (KV) and NaV channels, the functional contributions of individual side chains in Nav VSDs remain largely enigmatic. To this end, natural and unnatural side chain substitutions were made in the S2 hydrophobic core (HC), the extracellular negative charge cluster (ENC), and the intracellular negative charge cluster (INC) of the four VSDs of the skeletal muscle sodium channel isoform (NaV1.4). The results show that the highly conserved aromatic side chain constituting the S2 HC makes distinct functional contributions in each of the four NaV domains. No obvious cation-pi interaction exists with nearby S4 charges in any domain, and natural and unnatural mutations at these aromatic sites produce functional phenotypes that are different from those observed previously in Kv VSDs. In contrast, and similar to results obtained with Kv channels, individually neutralizing acidic side chains with synthetic derivatives and with natural amino acid substitutions in the INC had little or no effect on the voltage dependence of activation in any of the four domains. Interestingly, countercharge was found to play an important functional role in the ENC of DI and DII, but not DIII and DIV. These results suggest that electrostatic interactions with S4 gating charges are unlikely in the INC and only relevant in the ENC of DI and DII. Collectively, our data highlight domain-specific functional contributions of highly conserved side chains in NaV VSDs.
    The Journal of General Physiology 05/2014; 143(5):645-56. DOI:10.1085/jgp.201311036
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    Timothy Lynagh, Stephan A Pless
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    ABSTRACT: Cys-loop receptors are ligand-gated ion channels that are activated by a structurally diverse array of neurotransmitters, including acetylcholine, serotonin, glycine, and GABA. After the term "chemoreceptor" emerged over 100 years ago, there was some wait until affinity labeling, molecular cloning, functional studies, and X-ray crystallography experiments identified the extracellular interface of adjacent subunits as the principal site of agonist binding. The question of how subtle differences at and around agonist-binding sites of different Cys-loop receptors can accommodate transmitters as chemically diverse as glycine and serotonin has been subject to intense research over the last three decades. This review outlines the functional diversity and current structural understanding of agonist-binding sites, including those of invertebrate Cys-loop receptors. Together, this provides a framework to understand the atomic determinants involved in how these valuable therapeutic targets recognize and bind their ligands.
    Frontiers in Physiology 04/2014; 5:160. DOI:10.3389/fphys.2014.00160
  • Biophysical Journal 01/2014; 106(2):143a. DOI:10.1016/j.bpj.2013.11.828
  • Biophysical Journal 01/2014; 106(2):541a-542a. DOI:10.1016/j.bpj.2013.11.3018
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    ABSTRACT: Voltage-gated potassium (Kv) channels enable potassium efflux and membrane repolarization in excitable tissues. Many Kv channels undergo a progressive loss of ion conductance in the presence of a prolonged voltage stimulus, termed slow inactivation, but the atomic determinants that regulate the kinetics of this process remain obscure. Using a combination of synthetic amino acid analogs and concatenated channel subunits we establish two H-bonds near the extracellular surface of the channel that endow Kv channels with a mechanism to time the entry into slow inactivation: an intra-subunit H-bond between Asp447 and Trp434 and an inter-subunit H-bond connecting Tyr445 to Thr439. Breaking of either interaction triggers slow inactivation by means of a local disruption in the selectivity filter, while severing the Tyr445-Thr439 H-bond is likely to communicate this conformational change to the adjacent subunit(s). DOI:
    eLife Sciences 12/2013; 2:e01289. DOI:10.7554/eLife.01289
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    ABSTRACT: Voltage-gated potassium channels elicit membrane hyperpolarization through voltage-sensor domains that regulate the conductive status of the pore domain. To better understand the inherent basis for the open-closed equilibrium in these channels, we undertook an atomistic scan using synthetic fluorinated derivatives of aromatic residues previously implicated in the gating of Shaker potassium channels. Here we show that stepwise dispersion of the negative electrostatic surface potential of only one site, Phe481, stabilizes the channel open state. Furthermore, these data suggest that this apparent stabilization is the consequence of the amelioration of an inherently repulsive open-state interaction between the partial negative charge on the face of Phe481 and a highly co-evolved acidic side chain, Glu395, and this interaction is potentially modulated through the Tyr485 hydroxyl. We propose that the intrinsic open-state destabilization via aromatic repulsion represents a new mechanism by which ion channels, and likely other proteins, fine-tune conformational equilibria.
    Nature Communications 04/2013; 4:1784. DOI:10.1038/ncomms2761
  • Stephan A Pless, Christopher A Ahern
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    ABSTRACT: G protein-coupled receptors and ion channels couple a wide range of external stimuli to cellular growth and division, metabolism, motility, and a myriad of intra- and intercellular signaling pathways. G protein-coupled receptors initiate complex, interrelated downstream signaling cascades, whereas rapid ionic flux through channels directly supports membrane excitability and mediates cellular functions through second messengers. Because of these characteristics, these ubiquitous transmembrane proteins are valuable therapeutic targets and have provided fertile ground for the development of leading-edge synthetic and chemical biological approaches. Here we summarize recent advances in the use of site-directed incorporation of unnatural amino acids and chemical probes to study ligand-receptor interactions, determine the location of binding sites, and examine the downstream conformational consequences of ligand binding in G protein-coupled receptors and ion channels.
    Annual Review of Pharmacology 01/2013; 53:211-29. DOI:10.1146/annurev-pharmtox-011112-140343
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    ABSTRACT: Transient receptor potential vanilloid subfamily member 1 channels are polymodal sensors of noxious stimuli and integral players in thermosensation, inflammation and pain signaling. It has been shown previously that under prolonged stimulation, these channels show dynamic pore dilation, providing a pathway for large and otherwise relatively impermeant molecules. Further, we have shown recently that these nonselective cation channels, when activated by capsaicin, are potently and reversibly blocked by external application of quaternary ammonium compounds and local anesthetics. Here we describe a novel phenomenon in transient receptor potential channel pharmacology whereby their expression levels in Xenopus laevis oocytes, as assessed by the magnitude of macroscopic currents, are negatively correlated with extracellular blocker affinity: small current densities give rise to nanomolar blockade by quaternary ammoniums and this affinity decreases linearly as current density increases. Possible mechanisms to explain these data are discussed in light of similar observations in other channels and receptors.
    Channels (Austin, Tex.) 01/2013; 7(1):47-50. DOI:10.4161/chan.23105
  • Stephan A. Pless, Christopher A. Ahern
    Biophysical Journal 01/2013; 104(2):542-. DOI:10.1016/j.bpj.2012.11.3001
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    Biophysical Journal 01/2013; 104(2):122-. DOI:10.1016/j.bpj.2012.11.707
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    ABSTRACT: Voltage sensor domains (VSDs) regulate ion channels and enzymes by undergoing conformational changes depending on membrane electrical signals. The molecular mechanisms underlying the VSD transitions are not fully understood. Here, we show that some mutations of I241 in the S1 segment of the Shaker Kv channel positively shift the voltage dependence of the VSD movement and alter the functional coupling between VSD and pore domains. Among the I241 mutants, I241W immobilized the VSD movement during activation and deactivation, approximately halfway between the resting and active states, and drastically shifted the voltage activation of the ionic conductance. This phenotype, which is consistent with a stabilization of an intermediate VSD conformation by the I241W mutation, was diminished by the charge-conserving R2K mutation but not by the charge-neutralizing R2Q mutation. Interestingly, most of these effects were reproduced by the F244W mutation located one helical turn above I241. Electrophysiology recordings using nonnatural indole derivatives ruled out the involvement of cation-Π interactions for the effects of the Trp inserted at positions I241 and F244 on the channel's conductance, but showed that the indole nitrogen was important for the I241W phenotype. Insight into the molecular mechanisms responsible for the stabilization of the intermediate state were investigated by creating in silico the mutations I241W, I241W/R2K, and F244W in intermediate conformations obtained from a computational VSD transition pathway determined using the string method. The experimental results and computational analysis suggest that the phenotype of I241W may originate in the formation of a hydrogen bond between the indole nitrogen atom and the backbone carbonyl of R2. This work provides new information on intermediate states in voltage-gated ion channels with an approach that produces minimum chemical perturbation.
    The Journal of General Physiology 12/2012; 140(6):635-52. DOI:10.1085/jgp.201210827
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    ABSTRACT: Transient receptor potential vanilloid subtype 1 (TRPV1) channels are essential nociceptive integrators in primary afferent neurons. These nonselective cation channels are inhibited by local anesthetic compounds through an undefined mechanism. Here, we show that lidocaine inhibits TRPV1 channels expressed in Xenopus laevis oocytes, while the neutral local anesthetic, benzocaine, does not, suggesting a titratable amine is required for high affinity inhibition. Consistent with this possibility, extracellular tetraethylammonium (TEA) and tetramethylammonium (TMA) application produces potent, voltage-dependent pore block. Alanine substitutions at F649 and E648, residues in the putative TRPV1 pore region, significantly abrogated the concentration-dependent TEA inhibition. The results suggest that large cations, shown previously to enter cells through activated TRP channels, can also act as channel blockers.
    Molecular pharmacology 09/2012; 82(6). DOI:10.1124/mol.112.079277
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    Biophysical Journal 01/2012; 102(3):326-. DOI:10.1016/j.bpj.2011.11.1788
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    Biophysical Journal 01/2012; 102(3):13a. DOI:10.1016/j.bpj.2011.11.095

Publication Stats

322 Citations
185.93 Total Impact Points


  • 2014–2015
    • IT University of Copenhagen
      København, Capital Region, Denmark
  • 2011–2013
    • University of British Columbia - Vancouver
      • • Department of Anesthesiology, Pharmacology and Therapeutics
      • • Department of Cellular and Physiological Sciences
      Vancouver, British Columbia, Canada
  • 2008–2011
    • University of Queensland
      • School of Biomedical Sciences
      Brisbane, Queensland, Australia