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Binding of -Conotoxin-PVIIA to Open and Closed Shaker K-Channels Are

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Binding of -Conotoxin-PVIIA to Open and Closed Shaker K-Channels Are marine drugs Article Binding of κ-Conotoxin-PVIIA to Open and Closed Shaker K-Channels Are Differentially Affected by the Ionic Strength David Naranjo 1,* and Ignacio Díaz-Franulic 2,3 1 Instituto de Neurociencia, Facultad de Ciencias, Universidad de Valparaíso, Valparaíso 2360103, Chile 2 Center for Bioinformatics and Integrative Biology, Facultad de Ciencias de la Vida, Universidad Andrés Bello, Santiago 8370146, Chile; [email protected] 3 Centro Interdisciplinario de Neurociencia de Valparaíso, Facultad de Ciencias, Universidad de Valparaíso, Valparaíso 2360103, Chile * Correspondence: [email protected]; Tel.: +56-32-2508024 Received: 24 August 2020; Accepted: 13 September 2020; Published: 26 October 2020 Abstract: κ-Conotoxin-PVIIA (κ-PVIIA) is a potassium-channel blocking peptide from the venom of the fish-hunting snail, Conus purpurascens, which is essential for quick prey’s excitotoxic immobilization. Binding of one κ-PVIIA to Shaker K-channels occludes the K+-conduction pore without additional conformational effects. Because this 27-residue toxin is +4-charged at neutral pH, we asked if electrostatic interactions play a role in binding. With Voltage-Clamp electrophysiology, we tested how ionic strength (IS) affects κ-PVIIA blockade to Shaker. When IS varied from ~0.06 to ~0.16 M, the dissociation constant for open and closed channels increased by ~5- and ~16-fold, respectively. While the association rates decreased equally, by ~4-fold, in open and closed channels, the dissociation rates increased 4–5-fold in closed channels but was IS-insensitive in open channels. To explain this differential IS-dependency, we propose that the bound κ-PVIIA wobbles, so that in open channels the intracellular environment, via ion-conduction pore, buffers the imposed IS-changes in the toxin-channel interface. A Brønsted-Bjerrum analysis on the rates predicts that if, instead of fish, the snail preyed on organisms with seawater-like lymph ionic composition, a severely harmless toxin, with >100-fold diminished affinity, would result. Thus, considerations of the native ionic environment are essential for conotoxins evaluation as pharmacological leads. Keywords: Kv-channel; peptide toxin; conotoxin; association rate; dissociation rate; Brønsted-Bjerrum equation; predator-prey 1. Introduction Conotoxins form a family of peptides produced by marine predatory snails of genus Conus that bind to a wide variety of transporters, ion channels, G protein-coupled receptors and enzymes, providing a rich source of molecular templates and therapeutic leads for drug developing [1]. In fish hunting snails, a group of conotoxins acting in concert triggers an early excitotoxic shock; causing prey immobilization by axonal depolarization and repetitive firing near the venom injection lesion, followed by a long-term motor inhibition. The rapid elevated excitability arises because the venom blocks potassium channels and delays sodium channels inactivation [2]. κ-conotoxin-PVIIA (κ-PVIIA) is a 27-residue peptide forming part of the venom of Conus purpurascens, a piscivorous marine snail, that possibly is partially responsible for the excitotoxic shock in fish [2]. The toxin inhibition mechanism on voltage gated K-channels is well-known. As proposed for both scorpion α-KTx toxins and anemone toxins, κ-PVIIA inhibits voltage gated potassium channels by plugging the pore, thus, interrupting ion conduction [3–5]. Additionally, as proposed for the α-KTx chaybdotoxin (CTX), no induced Mar. Drugs 2020, 18, 533; doi:10.3390/md18110533 www.mdpi.com/journal/marinedrugs Mar. Drugs 2020, 18, 533 2 of 10 conformational effects in the channel have been detected upon toxin binding [5–7]. Thus, the inhibition mechanism is the simplest possible; the toxin binding just occludes the pore. In addition, kinetic analyses of the toxin binding to potassium channels have revealed that κ-PVIIA blocks the pore with a 1 1 very high association rate of 10–100 s− µM− , a value common among α-KTx toxins and in the range of diffusion limited protein associations [8,9]. Such mechanism of inhibition should be crucial for the early poisonous excitotoxic immobilizing shock of the prey, which is usually faster than the predatory snail [2]. As other animal toxins of the same family, κ-PVIIA is endowed with an excess of positive charges at its surface [10,11]. The spatial localization of positive charged residues in κ-PVIIA exhibits striking mimicry to that of CTX [10]. Thus, some of these charges may participate in the K-channel recognition surface. On the other hand, the potassium channel external pore entrance is dominated by negatively charged residues. Thus, both the molecular steering and recognition should be electrostatically facilitated [12]. To our knowledge, this hypothesis has not been tested. If so, this type of toxin would be largely ineffective for orthologous K-channels in marine animals having high ionic strength plasma, as some mollusks, echinoderms or arthropods. In this work we study the effects of ionic strength in the kinetics of κ-PVIIA blockade on Shaker K-channels. We found that the association rate is very sensitive to the ionic strength, which is a result consistent with our hypothesis that the protein-protein recognition is mediated by through-space electrostatic interactions. Interestingly, the dissociation rate is also sensitive to the ionic strength in closed channels, but in open channels is ionic strength insensitive. We suggest that this lack of sensitivity in open channels is due to the fact that the toxin wobbles in the bound state [5]. 2. Results In normal amphibian plasma ionic strength, heterologous expression of voltage activated Shaker K-channels in Xenopus oocytes was detected as positive (outward) currents in response to positive-going voltage pulses between 80 and +50 mV from a holding potential of 90 mV (Figure1). These voltage − − pulses drag the membrane potential to values more positive than the potassium equilibrium potential, thus they induce outward movement of K+-ions and, therefore positive currents. As the voltage pulse is made more positive, both the number of channels recruited and the speed with which they activate increase. Figure1A–C shows current traces obtained from oocytes expressing Shaker K-channels in the absence and in the presence of 100 nM of κ-PVIIA perfused with three different recording solutions differing in ionic strength (50-Na+, 100-Na+ and 150-Na+; A, B and C, respectively; See methods). In the presence of κ-PVIIA (right traces), the activation kinetics appears delayed and the currents at the end of the voltage pulse slightly diminished in relation to their controls. As the ionic strength increases, the toxin induced inhibition gets weaker. Unfortunately, we had to limit the ionic strength excursion up to 0.16 M because higher ion concentrations induce osmotic stress that damages the oocytes and the quality of the recordings. We have shown before that the apparent effect on the kinetics is due to the voltage dependence of the toxin/channel binding equilibrium [3,5,13]. Thus, 10 ms after the beginning of the voltage pulse, the observed time dependent relaxations are dominated by the transition from a high-affinity/binding equilibrium in closed channel, to a low-affinity, and voltage dependent, equilibrium in open channels. Figure2 (left panel) shows these relaxations, obtained by computing point-by-point quotients between the traces with/without toxin from Figure1, for di fferent voltages (See methods). All quotient traces start from approximately the same level of inhibition at the beginning of the voltage pulse (top of Figure2A, left panel), which represents closed /resting channel inhibition, that later reach a different voltage dependent equilibrium inhibition after an exponential relaxation. Each trace was well described by a single exponential function that provided three parameters for each voltage pulse (blue traces): (a) the fraction of inhibited resting channels, which was estimated by extrapolating the fitting curve to the beginning of the voltage pulse (arrow; open symbols in Figure2, middle panel), Mar. Drugs 2020, 18, 533 3 of 10 (b) the asymptotic fraction of inhibited open channels at each voltage (iTx/iCon; filled circles in Figure2, Mar.middle Drugs panel), 2020, 18 and, x FOR (c) thePEER time REVIEW constants for the voltage dependent relaxations (τ, Figure2, right panel).3 of 10 Figure 1.1.Ionic Ionic strength strength effects effects on the onShaker theblockade Shaker byblockadeκ-Conotoxin-PVIIA by κ-Conotoxin-PVIIA (κ-PVIIA). Two-electrode (κ-PVIIA). voltageTwo-electrode clamp tracesvoltage records clamp obtained traces re withcords pulses obtained between with pulses80 and between+50 mV −80 with and 10 +50 mV mV increments with 10 − mVfrom increments a holding voltage from a ofholding90 mV voltage (top). of External −90 mV solutions (top). External in (A) 50-Na solutions+;(B )in 100-Na (A) 50-Na+; and+; ( (BC)) 100-Na 150-Na++; − wereand ( madeC) 150-Na changing+ were the made NaCl concentrationchanging the but NaCl preserving concentr allation other but ionic preserving concentration. all other For 50-Na ionic+, 100concentration. mM mannitol For was 50-Na added+, 100 to mM the solutionmannitol for was osmolarity added to compensation. the solution for Discontinuous osmolarity compensation. lines indicate Discontinuouszero-current level. lines indicate zero-current level. AllAs thequotient ionic traces strength start increases, from approximately the inhibition the of sa restingme level and of openinhibition channels at the decreases. beginning But of the voltageionic strength pulse (top effect of on Figure the inhibition 2A, left panel), at resting which seems represents to be much closed/resting more intense channel thanthat inhibition, of the open that + + laterstate. reach Thus, a fordifferent example, voltage theresting dependent fractional equilibrium current inhibition rises from after ~0.15 an at exponential 50-Na to ~0.7relaxation. at 150-Na Each, whichtrace was represents well described a ~14-fold by rise a single in the exponential dissociation fu constant,nction that KD; provided from ~17 nMthree to parameters 230 nM.
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