β Subunit M2–M3 Loop Conformational Changes Are Uncoupled from α1 β Glycine Receptor Channel Gating: Implications for Human Hereditary Hyperekplexia

Hereditary hyperekplexia, or startle disease, is a neuromotor disorder caused mainly by mutations that either prevent the surface expression of, or modify the function of, the human heteromeric α1 β glycine receptor (GlyR) chloride channel. There is as yet no explanation as to why hyperekplexia mutations that modify channel function are almost exclusively located in the α1 to the exclusion of β subunit. The majority of these mutations are identified in the M2–M3 loop of the α1 subunit. Here we demonstrate that α1 β GlyR channel function is less sensitive to hyperekplexia-mimicking mutations introduced into the M2–M3 loop of the β than into the α1 subunit. This suggests that the M2–M3 loop of the α subunit dominates the β subunit in gating the α1 β GlyR channel. A further attempt to determine the possible mechanism underlying this phenomenon by using the voltage-clamp fluorometry technique revealed that agonist-induced conformational changes in the β subunit M2–M3 loop were uncoupled from α1 β GlyR channel gating. This is in contrast to the α subunit, where the M2–M3 loop conformational changes were shown to be directly coupled to α1 β GlyR channel gating. Finally, based on analysis of α1 β chimeric receptors, we demonstrate that the structural components responsible for this are distributed throughout the β subunit, implying that the β subunit has evolved without the functional constraint of a normal gating pathway within it. Our study provides a possible explanation of why hereditary hyperekplexia-causing mutations that modify α1 β GlyR channel function are almost exclusively located in the α1 to the exclusion of the β subunit

Molecular dynamics simulation links conformation of a pore-flanking region to hyperekplexia-related dysfunction of the inhibitory glycine receptor.

AbstractInhibitory glycine receptors mediate rapid synaptic inhibition in mammalian spinal cord and brainstem. The previously identified hyperekplexia mutation GLRA1(P250T), located within the intracellular TM1-2 loop of the GlyR α1 subunit, results in altered receptor activation and desensitization. Here, elementary steps of ion channel function of α1(250) mutants were resolved and shown to correlate with hydropathy and molar volume of residue α1(250). Single-channel recordings and rapid activation kinetic studies using laser pulse photolysis showed reduced conductance but similar open probability of α1(P250T) mutant channels. Molecular dynamics simulation of a helix-turn-helix motif representing the intracellular TM1-2 domain revealed alterations in backbone conformation, indicating an increased flexibility in these mutants that paralleled changes in elementary steps of channel function. Thus, the architecture of the TM1-2 loop is a critical determinant of ion channel conductance and receptor desensitization

Symmetric dimeric adamantanes for exploring the structure of two viroporins: influenza virus M2 and hepatitis C virus p7

Yasmine M Mandour,1 Ulrike Breitinger,2 Chunlong Ma,3 Jun Wang,3 Frank M Boeckler,4 Hans-Georg Breitinger,2 Darius P Zlotos1 1Department of Pharmaceutical Chemistry, German University in Cairo, 2Department of Biochemistry, The German University in Cairo, Cairo, Egypt; 3Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ, USA; 4Department of Pharmaceutical and Medicinal Chemistry, Eberhard Karls University of Tübingen, Tübingen, Germany Background: Adamantane-based compounds have been identified to interfere with the ion-channel activity of viroporins and thereby inhibit viral infection. To better understand the difference in the inhibition mechanism of viroporins, we synthesized symmetric dimeric adamantane analogs of various alkyl-spacer lengths.Methods: Symmetric dimeric adamantane derivatives were synthesized where two amantadine or rimantadine molecules were linked by various alkyl-spacers. The inhibitory activity of the compounds was studied on two viroporins: the influenza virus M2 protein, expressed in Xenopus oocytes, using the two-electrode voltage-clamp technique, and the hepatitis C virus (HCV) p7 channels for five different genotypes (1a, 1b, 2a, 3a, and 4a) expressed in HEK293 cells using whole-cell patch-clamp recording techniques.Results: Upon testing on M2 protein, dimeric compounds showed significantly lower inhibitory activity relative to the monomeric amantadine. The lack of channel blockage of the dimeric amantadine and rimantadine analogs against M2 wild type and M2-S31N mutant was consistent with previously proposed drug-binding mechanisms and further confirmed that the pore-binding model is the pharmacologically relevant drug-binding model. On the other hand, these dimers showed similar potency to their respective monomeric analogs when tested on p7 protein in HCV genotypes 1a, 1b, and 4a while being 700-fold and 150-fold more potent than amantadine in genotypes 2a and 3a, respectively. An amino group appears to be important for inhibiting the ion-channel activity of p7 protein in genotype 2a, while its importance was minimal in all other genotypes.Conclusion: Symmetric dimeric adamantanes can be considered a prospective class of p7 inhibitors that are able to address the differences in adamantane sensitivity among the various genotypes of HCV. Keywords: adamantane, dimers, viroporins, p7, M2, HC

Using approximate matching to reduce the volume of digital data

Digital forensic investigators frequently have to search for relevant files in massive digital corpora – a task often compared to finding a needle in a haystack. To address this challenge, investigators typically apply cryptographic hash functions to identify known files. However, cryptographic hashing only allows the detection of files that exactly match the known file hash values or fingerprints. This paper demonstrates the benefits of using approximate matching to locate relevant files. The experiments described in this paper used three test images of Windows XP, Windows 7 and Ubuntu 12.04 systems to evaluate fingerprint-based comparisons. The results reveal that approximate matching can improve file identification – in one case, increasing the identification rate from 1.82% to 23.76%

Functional complementation of Glra1spd-ot, a glycine receptor subunit mutant, by independently expressed C-terminal domains

The oscillator mouse (Glra1spd-ot) carries a 9 bp microdeletion plus a 2 bp microinsertion in the glycine receptor α1 subunit gene, resulting in the absence of functional α1 polypeptides from the CNS and lethality 3 weeks after birth. Depending on differential use of two splice acceptor sites in exon 9 of the Glra1 gene, the mutant allele encodes either a truncated α1 subunit (spdot-trc) or a polypeptide with a C-terminal missense sequence (spdot-elg). During recombinant expression, both splice variants fail to form ion channels. In complementation studies, a tail construct, encoding the deleted C-terminal sequence, was coexpressed with both mutants. Coexpression with spdot-trc produced glycine-gated ion channels. Rescue efficiency was increased by inclusion of the wild-type motif RRKRRH. In cultured spinal cord neurons from oscillator homozygotes, viral infection with recombinant C-terminal tail constructs resulted in appearance of endogenous α1 antigen. The functional rescue of α1 mutants by the C-terminal tail polypeptides argues for a modular subunit architecture of members of the Cys-loop receptor family