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

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

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

Developing an Effective Instrument for Assessing the Performance of Public University Presidents

Conducting a worthwhile assessment of the performance of university presidents poses unique challenges for higher education institutions. One of the most difficult issues is determining the content and format of the assessment instrument. Due to the breadth and complexity of the job, the list of potential criteria that could serve as content for the assessment instrument is almost limitless. There are also many format options with respect to the arrangement of the assessment instrument and plan for administering it. In order to fill an apparent gap in research on university president assessment, this study proposes a model for determining the content and format of an assessment instrument — the Assessment Instrument Development Approach (AIDA). The AIDA model incorporates a mixed-methods research design aimed at identifying the content and format of an assessment instrument that will produce reliable and valid results. Addressing the question, What approach can UNM and other public universities use to develop an effective performance assessment instrument for their president?\u27 this study critiqued the initial AIDA model and suggests a revised model based on study findings. Addressing the question, \u27What is the preferred content and format for a president performance assessment instrument?\u27 this study proposed an initial framework for the relationships among variables derived from pertinent theory and concepts pertaining to university president assessment. Considering the study results, a revised framework was developed along with example hypotheses that should be tested in future research to gain additional insights into performance assessment for university presidents. The results of this study reveal there are many candidate criteria and formats for assessing performance. What appears to be lacking in literature and in practice is a means to identify the best criteria and formats that will produce reliable, valid, and useful results for assessment of university presidents. The methods and findings described in this study provide additional insight into the \u27means\u27 for developing an assessment instrument and the \u27ends\u27 which are fair, equitable, and productive assessments of university president performance

Dissecting the Functions of Conserved Prolines within Transmembrane Helices of the D2 Dopamine Receptor

G protein-coupled receptors (GPCRs) contain a number of conserved proline residues in their transmembrane helices, and it is generally assumed these play important functional and/or structural roles. Here we use unnatural amino acid mutagenesis, employing α-hydroxy acids and proline analogues, to examine the functional roles of five proline residues in the transmembrane helices of the D2 dopamine receptor. The well-known tendency of proline to disrupt helical structure is important at all sites, while we find no evidence for a functional role for backbone amide cis–trans isomerization, another feature associated with proline. At most proline sites, the loss of the backbone NH is sufficient to explain the role of the proline. However, at one site, P210^(5.50), a substituent on the backbone N appears to be essential for proper function. Interestingly, the pattern in functional consequences that we see is mirrored in the pattern of structural distortions seen in recent GPCR crystal structures

Comparing and contrasting Escherichia coli and Mycobacterium tuberculosis mechanosensitive channels (MscL) - New gain of function mutations in the loop region

Sequence analysis of 35 putative MscL homologues was used to develop an optimal alignment for Escherichia coli and Mycobacterium tuberculosis MscL and to place these homologues into sequence subfamilies. By using this alignment, previously identified E. coli MscL mutants that displayed severe and very severe gain of function phenotypes were mapped onto the M. tuberculosis MscL sequence. Not all of the resulting M. tuberculosis mutants displayed a gain of function phenotype; for instance, normal phenotypes were noted for mutations at Ala20, the analogue of the highly sensitive Gly22 site in E. coli. A previously unnoticed intersubunit hydrogen bond in the extracellular loop region of the M. tuberculosis MscL crystal structure has been analyzed. Cross-linkable residues were substituted for the residues involved in the hydrogen bond, and cross-linking studies indicated that these sites are spatially close under physiological conditions. In general, mutation at these positions results in a gain of function phenotype, which provides strong evidence for the importance of the loop region in MscL channel function. No analogue to this interesting interaction could be found in E. coli MscL by sequence alignment. Taken together, these results indicate that caution should be exercised in using the M. tuberculosis MscL crystal structure to analyze previous functional studies of E. coli MscL

Expression and Circular Dichroism Studies of the Extracellular Domain of the alpha Subunit of the Nicotinic Acetylcholine Receptor

To provide material suitable for structural studies of the nicotinic acetylcholine receptor, we have expressed and purified the NH2-terminal extracellular domain of the mouse muscle alpha subunit. Several constructs were initially investigated using Xenopus oocytes as a convenient small scale expression system. A fusion protein (alpha210GPI) consisting of the 210 NH2-terminal amino acids of the alpha subunit and a glycosylphosphatidylinositol anchorage sequence conferred surface alpha-bungarotoxin binding in oocytes. Coexpression of alpha210GPI with an analogous construct made from the delta subunit showed no evidence of heterodimer formation. The alpha210GPI protein was chosen for large scale expression in transfected Chinese hamster ovary cells. The alpha210GPI protein was cleaved from these cells and purified on an immunoaffinity column. Gel and column chromatography show that the purified protein is processed as expected and exists as a monomer. The purified protein also retains the two distinct, conformation-specific binding sites expected for the correctly folded alpha subunit. Circular dichroism studies of alpha210GPI suggest that this region of the receptor includes considerable beta-sheet secondary structure, with a small proportion of alpha-helix

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

From ab initio quantum mechanics to molecular neurobiology: A cation-pi binding site in the nicotinic receptor

The nicotinic acetylcholine receptor is the prototype ligand-gated ion channel. A number of aromatic amino acids have been identified as contributing to the agonist binding site, suggesting that cation-pi interactions may be involved in binding the quaternary ammonium group of the agonist, acetylcholine. Here we show a compelling correlation between: (i) ab initio quantum mechanical predictions of cation-pi binding abilities and (ii) EC50 values for acetylcholine at the receptor for a series of tryptophan derivatives that were incorporated into the receptor by using the in vivo nonsense-suppression method for unnatural amino acid incorporation. Such a correlation is seen at one, and only one, of the aromatic residues-tryptophan-149 of the alpha subunit. This finding indicates that, on binding, the cationic, quaternary ammonium group of acetylcholine makes van der Waals contact with the indole side chain of alpha tryptophan-149, providing the most precise structural information to date on this receptor. Consistent with this model, a tethered quaternary ammonium group emanating from position alpha 149 produces a constitutively active receptor