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

Magnetotaxis and Magnetic Particles in Bacteria

Magnetotactic bacteria contain magnetic particles that constitute a permanent magnetic dipole and cause each cell to orient and migrate along geomagnetic field lines. Recent results relevant to the biomineralization process and to the function of magnetotaxis are discussed

A taxonomic guide to the mysids of the South Atlantic Bight.

Following the examination of extensive collections from the National Museum of Natural History (NMNH), the Southeastern Regional Taxonomic Center (SERTC), and other regional institutions, 18 species of the family Mysidae are recognized and described from the South Atlantic Bight (Cape Lookout, North Carolina to Cape Canaveral, Florida). This report includes synonymies of previous records, as well as new species distribution records. Previous regional accounts of Metamysidopsis munda and Metamysidopsis mexicana are attributed to Metamysidopsis swifti. New regional records are established for Amathimysis brattegardi, Heteromysis beetoni, and Siriella thompsonii. Two other species tentatively identified as Amathimysis sp. (nr. serrata) and Mysidopsis sp. (cf. mortenseni) may represent new taxa. Neobathymysis renoculata is included and discussed as a potential regional species. An illustrated key to the species currently known from the South Atlantic Bight is presented. Relevant taxonomic, distributional, and ecological information is also included for each species. (PDF file contains 45 pages.

COMPARISON OF HOP DOWNY MILDEW EPIDEMICS USING SPATIAL ANALYSIS

Methods of spatial analysis including distribution fitting, variance-to-mean ratios, Morisita\u27s index, doublet and runs analyses, Greig-Smith analysis and variography were used to investigate the spatial pattern of hop downy mildew. Use of these methods allowed examination of the spatial structure of hop downy mildew at three spatial scales: within hop hills, between nearby hop hills, and for hop hills more separated in space. The results obtained were in general agreement for methods of analysis which assessed spatial structure at the same spatial scale with the exception of Morisita\u27s index of clumping which did not identify clumps of diseased hills of the same size as Greig-Smith analysis and semi-variograms

Biomineralization of Magnetic Iron Minerals in Bacteria

Magnetotactic bacteria orient and migrate along magnetic field lines. This ability is based on a submicron assembly of single-magnetic domain iron mineral particles that elegantly solves the problem of how to construct a magnetic dipole that is large enough to be oriented in the geomagnetic field at ambient temperature, yet fit inside a micron-sized cell. The solution is based on the ability of the bacteria to accumulate high concentrations of iron, and control the deposition, size and orientation of a specific iron mineral at specific locations in the cell

AQP2 is Necessary for Vasopressin- and Forskolin-Mediated Filamentous Actin Depolymerization in Renal Epithelial Cells

Remodeling of the actin cytoskeleton is required for vasopressin (VP)‐induced aquaporin 2 (AQP2) trafficking. Here, we asked whether VP and forskolin (FK)‐mediated F‐actin depolymerization depends on AQP2 expression. Using various MDCK and LLC‐PK1 cell lines with different AQP2 expression levels, we performed F‐actin quantification and immunofluorescence staining after VP/FK treatment. In MDCK cells, in which AQP2 is delivered apically, VP/FK mediated F‐actin depolymerization was significantly correlated with AQP2 expression levels. A decrease of apical membrane associated F‐actin was observed upon VP/FK treatment in AQP2 transfected, but not in untransfected cells. There was no change in basolateral actin staining under these conditions. In LLC‐PK1 cells, which deliver AQP2 basolaterally, a significant VP/FK mediated decrease in F‐actin was also detected only in AQP2 transfected cells. This depolymerization response to VP/FK was significantly reduced by siRNA knockdown of AQP2. By immunofluorescence, an inverse relationship between plasma membrane AQP2 and membrane‐associated F‐actin was observed after VP/FK treatment again only in AQP2 transfected cells. This is the first report showing that VP/FK mediated F‐actin depolymerization is dependent on AQP2 protein expression in renal epithelial cells, and that this is not dependent on the polarity of AQP2 membrane insertion