Examinando por Autor "Naranjo, D."

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    Binding of κ-Conotoxin-PVIIA to Open and Closed Shaker K-Channels Are Differentially Affected by the Ionic Strength
    (NLM (Medline), 2020-10) Naranjo, D.; Diaz-Franulic, I.
    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.
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    Effective pore size and radius of capture for K+ ions in K-channels
    (NATURE PUBLISHING GROUP, 2016-02) Moldenhauer, H.; Diaz-Franulic, I.; Gonzalez-Nilo, F.; Naranjo, D.
    Reconciling protein functional data with crystal structure is arduous because rare conformations or crystallization artifacts occur. Here we present a tool to validate the dimensions of open pore structures of potassium-selective ion channels. We used freely available algorithms to calculate the molecular contour of the pore to determine the effective internal pore radius (r(E)) in several K-channel crystal structurss. r(E) was operationally defined as the radius of the biggest sphere able to enter the pore from the cytosolic side. We obtained consistent r(E) estimates for MthK and Kv1.2/2.1 structures, with r(E) = 5.3-5.9 angstrom and r(E) = 4.5-5.2 angstrom, respectively. We compared these structural estimates with functional assessments of the internal mouth radii of capture (r(C)) for two electrophysiological counterparts, the large conductance calcium activated K-channel (r(C) = 2.2 angstrom) and the Shaker K-v-channel (r(C) = 0.8 angstrom), for MthK and Kv1.2/2.1 structures, respectively. Calculating the difference between r(E) and r(C), produced consistent size radii of 3.1-3.7 angstrom and 3.6-4.4 angstrom for hydrated K+ ions. These hydrated K+ estimates harmonize with others obtained with diverse experimental and theoretical methods. Thus, these findings validate MthK and the Kv1.2/2.1 structures as templates for open BK and Kv-channels, respectively.
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    Gating-induced large aqueous volumetric remodeling and aspartate tolerance in the voltage sensor domain of Shaker K+ channels
    (National Academy of Sciences, 2018-08) Díaz-Franulic, I.; González-Pérez, V.; Moldenhauer, H.; Navarro-Quezada, N.; Naranjo, D.
    Neurons encode electrical signals with critically tuned voltage-gated ion channels and enzymes. Dedicated voltage sensor domains (VSDs) in these membrane proteins activate coordinately with an unresolved structural change. Such change conveys the transmembrane translocation of four positively charged arginine side chains, the voltage-sensing residues (VSRs; R1–R4). Countercharges and lipid phosphohead groups likely stabilize these VSRs within the low-dielectric core of the protein. However, the role of hydration, a sign-independent charge stabilizer, remains unclear. We replaced all VSRs and their neighboring residues with negatively charged aspartates in a voltage-gated potassium channel. The ensuing mild functional effects indicate that hydration is also important in VSR stabilization. The voltage dependency of the VSR aspartate variants approached the expected arithmetic summation of charges at VSR positions, as if negative and positive side chains faced similar pathways. In contrast, aspartates introduced between R2 and R3 did not affect voltage dependence as if the side chains moved outside the electric field or together with it, undergoing a large displacement and volumetric remodeling. Accordingly, VSR performed osmotic work at both internal and external aqueous interfaces. Individual VSR contributions to volumetric works approached arithmetical additivity but were largely dissimilar. While R1 and R4 displaced small volumes, R2 and R3 volumetric works were massive and vectorially opposed, favoring large aqueous remodeling during VSD activation. These diverse volumetric works are, at least for R2 and R3, not compatible with VSR translocation across a unique stationary charge transfer center. Instead, VSRs may follow separated pathways across a fluctuating low-dielectric septum. © National Academy of Sciences. All rights reserved.
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    Pore dimensions and the role of occupancy in unitary conductance of Shaker K channels
    (ROCKEFELLER UNIV PRESS, 2015-08) Díaz-Franulic, I.; Sepúlveda, R.V.; Navarro-Quezada, N.; González-Nilo, F.; Naranjo, D.
    K channels mediate the selective passage of K+ across the plasma membrane by means of intimate interactions with ions at the pore selectivity filter located near the external face. Despite high conservation of the selectivity filter, the K+ transport properties of different K channels vary widely, with the unitary conductance spanning a range of over two orders of magnitude. Mutation of Pro475, a residue located at the cytoplasmic entrance of the pore of the small-intermediate conductance K channel Shaker (Pro475Asp (P475D) or Pro475Gln (P475Q)), increases Shaker's reported. 20-pS conductance by approximately six-and approximately threefold, respectively, without any detectable effect on its selectivity. These findings suggest that the structural determinants underlying the diversity of K channel conductance are distinct from the selectivity filter, making P475D and P475Q excellent probes to identify key determinants of the K channel unitary conductance. By measuring diffusion-limited unitary outward currents after unilateral addition of 2 M sucrose to the internal solution to increase its viscosity, we estimated a pore internal radius of capture of 0.82 for all the three Shaker variants (wild type, P475D, and P475Q). This estimate is consistent with the internal entrance of the Kv1.2/2.1 structure if the effective radius of hydrated K+ is set to 4 A. Unilateral exposure to sucrose allowed us to estimate the internal and external access resistances together with that of the inner pore. We determined that Shaker resistance resides mainly in the inner cavity, whereas only similar to 8% resides in the selectivity filter. To reduce the inner resistance, we introduced additional aspartate residues into the internal vestibule to favor ion occupancy. No aspartate addition raised the maximum unitary conductance, measured at saturating [K+], beyond that of P475D, suggesting an similar to 200-pS conductance ceiling for Shaker. This value is approximately one third of the maximum conductance of the large conductance K (BK) channel (the K channel of highest conductance), reducing the energy gap between their K+ transport rates to similar to 1 kT. Thus, although Shaker's pore sustains ion translocation as the BK channel's does, higher energetic costs of ion stabilization or higher friction with the ion's rigid hydration cage in its narrower aqueous cavity may entail higher resistance.