New Views of Multi-Ion Channels

Thus, most site-directed mutagenesis data render it untenable to consider that two or more roughly equivalent high affinity sites govern selectivity in multi-ion pores. The papers by Dang and McCleskey and Kiss et al. respond to this challenge by showing that a model with a single high affinity site, flanked by two binding sites of lower affinity close to the pore entrances, can generate much of the classical multi-ion behavior. The sites need not interact, and the two flanking sites could arise from one of several mechanisms: a featureless charged vestibule, a dehydration step, or a specific weak binding site. The multi-ion pore remains a cornerstone of permeation theory, but the new theory features only a single high affinity site and no mutual repulsion. The high flux rate occurs because ions pause at the flanking sites and reequilibrate thermally, gaining enough energy to move over the next barrier

Generation and evaluation of a large mutational library from the Escherichia coli mechanosensitive channel of large conductance, MscL - Implications for channel gating and evolutionary design

Random mutagenesis of the mechanosensitive channel of large conductance (MscL) from Escherichia coli coupled with a high-throughput functional screen has provided new insights into channel structure and function. Complementary interactions of conserved residues proposed in a computational model for gating have been evaluated, and important functional regions of the channel have been identified. Mutational analysis shows that the proposed S1 helix, despite having several highly conserved residues, can be heavily mutated without significantly altering channel function. The pattern of mutations that make MscL more difficult to gate suggests that MscL senses tension with residues located near the lipid headgroups of the bilayer. The range of phenotypical changes seen has implications for a proposed model for the evolutionary origin of mechanosensitive channels

Room-Temperature Alternative to the Arbuzov Reaction: The Reductive Deoxygenation of Acyl Phosphonates

The reductive deoxygenation of acyl phosphonates using a Wolff−Kishner-like sequence is described. This transformation allows direct access to alkyl phosphonates from acyl phosphonates at room temperature. The method can be combined with acyl phosphonate synthesis into a one pot, four-step procedure for the conversion of carboxylic acids into alkyl phosphonates. The methodology works well for a variety of aliphatic acids and shows a functional group tolerance similar to that of other hydrazone-forming reactions

Preparation of Translationally Competent tRNA by Direct Chemical Acylation

Nonsense codon suppression for unnatural amino acid incorporation requires the preparation of a suppressor aminoacyl-tRNA. Chemical acylation strategies are general but inefficient and arduous. A recent report (J. Am. Chem. Soc. 2007, 129, 15848) showed acylation of RNA mediated by lanthanum(III) using amino acid phosphate esters. The successful implementation of this methodology to full-length suppressor tRNA is described, and it is shown that the derived aminoacyl-tRNA is translationally competent in Xenopus oocytes

Establishing an Ion Pair Interaction in the Homomeric {rho}1 {gamma}-Aminobutyric Acid Type A Receptor That Contributes to the Gating Pathway

{gamma}-Aminobutyric acid type A (GABAA) receptors are members of the Cys-loop superfamily of ligand-gated ion channels. Upon agonist binding, the receptor undergoes a structural transition from the closed to the open state, but the mechanism of gating is not well understood. Here we utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study the gating interface of the human homopentameric {rho}1 GABAA receptor. We have identified an ion pair interaction between two conserved charged residues, Glu92 in loop 2 of the extracellular domain and Arg258 in the pre-M1 region. We hypothesize that the salt bridge exists in the closed state by kinetic measurements and free energy analysis. Several other charged residues at the gating interface are not critical to receptor function, supporting previous conclusions that it is the global charge pattern of the gating interface that controls receptor function in the Cys-loop superfamily

The Cation-π Interaction

The chemistry community now recognizes the cation−π interaction as a major force for molecular recognition, joining the hydrophobic effect, the hydrogen bond, and the ion pair in determining macromolecular structure and drug–receptor interactions. This Account provides the author’s perspective on the intellectual origins and fundamental nature of the cation−π interaction

Physical organic chemistry in the brain

A new tool for precise structural modification of ion channel proteins - the in vivo nonsense suppression technique for incorporating unnatural amino acids - has been developed. This new tool allows the kind of systematic structure-function correlation studies that are commonly employed in physical organic chemistry to be applied to the important proteins of molecular neurobiology. In particular, the method has been applied to the nicotinic acetylcholine receptor, The differing roles of the numerous aromatic amino acids present at the agonist binding site can be distinguished. In addition, high precision studies of the ion channel region have produced new insights into the channel gating mechanism

New high-spin π systems

Organic molecules and materials with large numbers of unpaired spins promise to display novel combinations of magnetic, optical, and electrical properties. Two examples from our lab demonstrate the potential for strongly coupling magnetic and optical properties and the possibility of designing magnetic materials that are electrically insulating

Subunit Stoichiometry of a Heteromultimeric G protein-coupled Inward-rectifier K^+ Channel

We investigated the stoichiometry of the heteromultimeric G protein-coupled inward-recitfier K^+ channel (GIRK) formed from GIRK1 and GIRK4 subunits. Multimeric GIRK constructs with several concatenated channel subunits were expressed in Xenopus oocytes. Coexpression of various trimeric constructs with different monomers clearly showed that the functional channel has stoichiometry (GIRK1)_2(GIRK4)_2. Efforts to establish a preferred arrangement of subunits around the channel pore suggest that more than one arrangement may be viable

A Stereochemical Test of a Proposed Structural Feature of the Nicotinic Acetylcholine Receptor

Understanding the gating mechanism of the nicotinic acetylcholine receptor (nAChR) and similar channels constitutes a significant challenge in chemical neurobiology. In the present work, we use a stereochemical probe to evaluate a proposed pin-into-hydrophobic socket mechanism for the αVal46 side chain of the nAChR. Utilizing nonsense suppression methodology we incorporated isoleucine (Ile), O-methyl threonine (Omt) and threonine (Thr) as well as their side chain epimers (the allo counterparts). Surprisingly, our results indicate that only the pro-S methyl group of the αVal46 side chain is sensitive to changes in hydrophobicity, consistent with the precise geometrical requirements of the pin-into-socket mechanism