Electrostatic Contributions of Aromatic Residues in the Local Anesthetic Receptor of Voltage-Gated Sodium Channels

Antiarrhythmics, anticonvulsants, and local anesthetics target voltage-gated sodium channels, decreasing excitability of nerve and muscle cells. Channel inhibition by members of this family of cationic, hydrophobic drugs relies on the presence of highly conserved aromatic residues in the pore-lining S6 segment of the fourth homologous domain of the channel. We tested whether channel inhibition was facilitated by an electrostatic attraction between lidocaine and {pi} electrons of the aromatic rings of these residues, namely a cation-{pi} interaction. To this end, we used the in vivo nonsense suppression method to incorporate a series of unnatural phenylalanine derivatives designed to systematically reduce the negative electrostatic potential on the face of the aromatic ring. In contrast to standard point mutations at the same sites, these subtly altered amino acids preserve the wild-type voltage dependence of channel activation and inactivation. Although these phenylalanine derivatives have no effect on low-affinity tonic inhibition by lidocaine or its permanently charged derivative QX-314 at any of the substituted sites, high-affinity use-dependent inhibition displays substantial cation-{pi} energetics for 1 residue only: Phe1579 in rNaV1.4. Replacement of the aromatic ring of Phe1579 by cyclohexane, for example, strongly reduces use-dependent inhibition and speeds recovery of lidocaine-engaged channels. Channel block by the neutral local anesthetic benzocaine is unaffected by the distribution of {pi} electrons at Phe1579, indicating that our aromatic manipulations expose electrostatic contributions to channel inhibition. These results fine tune our understanding of local anesthetic inhibition of voltage-gated sodium channels and will help the design of safer and more salutary therapeutic agents

A Cation–π Interaction between Extracellular TEA and an Aromatic Residue in Potassium Channels

Open-channel blockers such as tetraethylammonium (TEA) have a long history as probes of the permeation pathway of ion channels. High affinity blockade by extracellular TEA requires the presence of an aromatic amino acid at a position that sits at the external entrance of the permeation pathway (residue 449 in the eukaryotic voltage-gated potassium channel Shaker). We investigated whether a cation–{pi} interaction between TEA and such an aromatic residue contributes to TEA block using the in vivo nonsense suppression method to incorporate a series of increasingly fluorinated Phe side chains at position 449. Fluorination, which is known to decrease the cation–{pi} binding ability of an aromatic ring, progressively increased the inhibitory constant Ki for the TEA block of Shaker. A larger increase in Ki was observed when the benzene ring of Phe449 was substituted by nonaromatic cyclohexane. These results support a strong cation–{pi} component to the TEA block. The data provide an empirical basis for choosing between Shaker models that are based on two classes of reported crystal structures for the bacterial channel KcsA, showing residue Tyr82 in orientations either compatible or incompatible with a cation–{pi} mechanism. We propose that the aromatic residue at this position in Shaker is favorably oriented for a cation–{pi} interaction with the permeation pathway. This choice is supported by high level ab initio calculations of the predicted effects of Phe modifications on TEA binding energy

A Cation-π Interaction Discriminates among Sodium Channels That Are Either Sensitive or Resistant to Tetrodotoxin Block

Voltage-gated sodium channels control the upstroke of the action potential in excitable cells of nerve and muscle tissue, making them ideal targets for exogenous toxins that aim to squelch electrical excitability. One such toxin, tetrodotoxin (TTX), blocks sodium channels with nanomolar affinity only when an aromatic Phe or Tyr residue is present at a specific location in the external vestibule of the ion-conducting pore. To test whether TTX is attracted to Tyr401 of NaV1.4 through a cation-{pi} interaction, this aromatic residue was replaced with fluorinated derivatives of Phe using in vivo nonsense suppression. Consistent with a cation-{pi} interaction, increased fluorination of Phe401, which reduces the negative electrostatic potential on the aromatic face, caused a monotonic increase in the inhibitory constant for block. Trifluorination of the aromatic ring decreased TTX affinity by ~50-fold, a reduction similar to that caused by replacement with the comparably hydrophobic residue Leu. Furthermore, we show that an energetically equivalent cation-{pi} interaction underlies both use-dependent and tonic block by TTX. Our results are supported by high level ab initio quantum mechanical calculations applied to a model of TTX binding to benzene. Our analysis suggests that the aromatic side chain faces the permeation pathway where it orients TTX optimally and interacts with permeant ions. These results are the first of their kind to show the incorporation of unnatural amino acids into a voltage-gated sodium channel and demonstrate that a cation-{pi} interaction is responsible for the obligate nature of an aromatic at this position in TTX-sensitive sodium channels

Suprathermal electrons at Saturn's bow shock

The leading explanation for the origin of galactic cosmic rays is particle acceleration at the shocks surrounding young supernova remnants (SNRs), although crucial aspects of the acceleration process are unclear. The similar collisionless plasma shocks frequently encountered by spacecraft in the solar wind are generally far weaker (lower Mach number) than these SNR shocks. However, the Cassini spacecraft has shown that the shock standing in the solar wind sunward of Saturn (Saturn's bow shock) can occasionally reach this high-Mach number astrophysical regime. In this regime Cassini has provided the first in situ evidence for electron acceleration under quasi-parallel upstream magnetic conditions. Here we present the full picture of suprathermal electrons at Saturn's bow shock revealed by Cassini. The downstream thermal electron distribution is resolved in all data taken by the low-energy electron detector (CAPS-ELS, <28 keV) during shock crossings, but the higher energy channels were at (or close to) background. The high-energy electron detector (MIMI-LEMMS, >18 keV) measured a suprathermal electron signature at 31 of 508 crossings, where typically only the lowest energy channels (<100 keV) were above background. We show that these results are consistent with theory in which the "injection" of thermal electrons into an acceleration process involves interaction with whistler waves at the shock front, and becomes possible for all upstream magnetic field orientations at high Mach numbers like those of the strong shocks around young SNRs. A future dedicated study will analyze the rare crossings with evidence for relativistic electrons (up to ~1 MeV).Comment: 22 pages, 5 figures. Accepted for publication in Ap

An X-Ray Jet from a White Dwarf - Detection of the Collimated Outflow from CH Cygni with Chandra

Most symbiotic stars consist of a white dwarf accreting material from the wind of a red giant. An increasing number of these objects have been found to produce jets. Analysis of archival Chandra data of the symbiotic system CH Cygni reveals faint extended emission to the south, aligned with the optical and radio jets seen in earlier HST and VLA observations. CH Cygni thus contains only the second known white dwarf with an X-ray jet, after R Aquarii. The X-rays from symbiotic-star jets appear to be produced when jet material is shock-heated following collision with surrounding gas, as with the outflows from some protostellar objects and bipolar planetary nebulae.Comment: 4 & a bit pages, 4 figures, accepted by ApJL; uses emulateapj.cls and revtex4. Minor changes following referees report, & shortened to meet page limi

Site-Specific Incorporation of Unnatural Amino Acids into Receptors Expressed in Mammalian Cells

AbstractWe describe an approach to achieve unnatural amino acid incorporation into channels and receptors expressed in mammalian cells. We show that microelectroporation provides a general method to deliver DNA, mRNA, and tRNA simultaneously. In both CHO cells and cultured neurons, microelectroporation efficiently delivers an in vitro transcribed, serine amber suppressor tRNA, leading to nonsense suppression in a mutant EGFP gene. In CHO cells, both natural and unnatural amino acids chemically appended to a suppressor tRNA are site specifically incorporated into the nicotinic acetylcholine receptor (nAChR). Electrophysiology confirms the expected functional consequences of the unnatural residue. The microelectroporation strategy described here is more general, less tedious, and less damaging to mammalian neuronal and nonneuronal cells than previous approaches to nonsense suppression in small cells and provides the first example of unnatural amino acid incorporation in mammalian cells using chemically aminoacylated tRNA