James I. Dunlop

A cytoplasmic region determines single-channel conductance in 5-HT3 receptors.

5-Hydroxytryptamine type 3 (5-HT3) receptors are cation-selective transmitter-gated ion channels of the Cys-loop superfamily. The single-channel conductance of human recombinant 5-HT3 receptors assembled as homomers of 5-HT3A subunits, or heteromers of 5-HT3A and 5-HT3B subunits, are markedly different, being 0.4 pS (refs 6, 9) and 16 pS (ref. 7), respectively. Paradoxically, the channel-lining M2 domain of the 5-HT3A subunit would be predicted to promote cation conduction, whereas that of the 5-HT3B subunit would not. Here we describe a determinant of single-channel conductance that can explain these observations. By constructing chimaeric 5-HT3A and 5-HT3B subunits we identified a region (the ‘HA-stretch’) within the large cytoplasmic loop of the receptor that markedly influences channel conductance. Replacement of three arginine residues unique to the HA-stretch of the 5-HT3A subunit by their 5-HT3B subunit counterparts increased single-channel conductance 28-fold. Significantly, ultrastructural studies of the Torpedo nicotinic acetylcholine receptor indicate that the key residues might frame narrow openings that contribute to the permeation pathway. Our findings solve the conundrum of the anomalously low conductance of homomeric 5-HT3A receptors and indicate an important function for the HA-stretch in Cys-loop transmitter-gated ion channels.

Identification of Residues in the CH2/CH3 Domain Interface of IgA Essential for Interaction with the Human Fcα Receptor (FcαR) CD89

Cellular receptors for IgA (FcαR) mediate important protective functions. An extensive panel of site-directed mutant IgAs was used to identify IgA residues critical for FcαR (CD89) binding and triggering. Although a tailpiece-deleted IgA1 was able to bind and trigger CD89, antibodies featuring CH3 domain exchanges between human IgA1 and IgG1 could not, indicating that both domains but not the tailpiece are required for FcαR recognition. To further investigate the role of the interdomain region, numerous IgA1s, each with a point substitution in either of two interdomain loops (Leu-257—Gly-259 in Cα2; Pro-440—Phe-443 in Cα3), were generated. With only one exception (G259R), substitutions produced either ablation (L257R, P440A, A442R, F443R) or marked reduction (P440R) in CD89 binding and triggering. Further support for involvement of these interdomain loops was provided by interspecies comparisons of IgA. Thus a human IgA1 mutant, LA441–442MN, which mimicked the mouse IgA loop sequence through substitution of two adjacent residues in the Cα3 loop, was found, like mouse IgA, not to bind CD89. In contrast, bovine IgA1, identical to human IgA1 within these interdomain loops despite numerous differences elsewhere in the Fc region, did bind CD89. We have thus identified motifs in the interdomain region of IgA Fc critical for FcαR binding and triggering, significantly enhancing present understanding of the molecular basis of the IgA-FcαR interaction.

Common Determinants of Single Channel Conductance within the Large Cytoplasmic Loop of 5-Hydroxytryptamine Type 3 and α4β2 Nicotinic Acetylcholine Receptors

Homomeric 5-hydroxytryptamine type 3A receptors (5-HT3ARs) have a single channel conductance (γ) below the resolution of single channel recording (966 ± 75 fS, estimated by variance analysis). By contrast, heteromeric 5-HT3A/B and nicotinic acetylcholine receptors (nAChRs) have picosiemen range γ values. In this study, single channel recordings revealed that replacement of cytoplasmic membrane-associated (MA) helix arginine 432 (-4′), 436 (0′), and 440 (4′) residues by 5-HT3B (-4′Gln, 0′Asp, and 4′Ala) residues increases γ to 36.5 ± 1.0 pS. The 0′ residue makes the most substantial contribution to γ of the 5-HT3AR. Replacement of 0′Arg by aspartate, glutamate (α7 nAChR subunit MA 0′), or glutamine (β2 subunit MA 0′) increases γ to the resolvable range (>6 pS). By contrast, replacement of 0′Arg by phenylalanine (α4 subunit MA 0′) reduced γ to 416 ± 107 fS. In reciprocal experiments with α4β2 nAChRs (γ = 31.3 ± 0.8 pS), replacement of MA 0′ residues by arginine in α4β2(Q443R) and α4(F588R)β2 reduced γ slightly. By contrast, the γ of double mutant α4(F588R)β2(Q443R) was halved. The MA -4′ and 4′ residues also influenced γ of 5-HT3ARs. Replacement of nAChR α4 or β2 MA 4′ residues by arginine made current density negligible. By contrast, replacement of both -4′ residues by arginine produced functional nAChRs with substantially reduced γ (11.4 ± 0.5 pS). Homology models of the 5-HT3A and α4β2 nAChRs against Torpedo nAChR revealed MA -4′, 0′, and 4′ residues within five intracellular portals. This locus may be a common determinant of ion conduction throughout the Cys loop receptor family.

Common determinants of single channel conductance within the large cytoplasmic loop of 5-HT3 and α4β2 nicotinic acetylcholine receptors

Homomeric 5-hydroxytryptamine type 3A receptors (5-HT3ARs) have a single channel conductance (γ) below the resolution of single channel recording (966 ± 75 fS, estimated by variance analysis). By contrast, heteromeric 5-HT3A/B and nicotinic acetylcholine receptors (nAChRs) have picosiemen range γ values. In this study, single channel recordings revealed that replacement of cytoplasmic membrane-associated (MA) helix arginine 432 (-4′), 436 (0′), and 440 (4′) residues by 5-HT3B (-4′Gln, 0′Asp, and 4′Ala) residues increases γ to 36.5 ± 1.0 pS. The 0′ residue makes the most substantial contribution to γ of the 5-HT3AR. Replacement of 0′Arg by aspartate, glutamate (α7 nAChR subunit MA 0′), or glutamine (β2 subunit MA 0′) increases γ to the resolvable range (>6 pS). By contrast, replacement of 0′Arg by phenylalanine (α4 subunit MA 0′) reduced γ to 416 ± 107 fS. In reciprocal experiments with α4β2 nAChRs (γ = 31.3 ± 0.8 pS), replacement of MA 0′ residues by arginine in α4β2(Q443R) and α4(F588R)β2 reduced γ slightly. By contrast, the γ of double mutant α4(F588R)β2(Q443R) was halved. The MA -4′ and 4′ residues also influenced γ of 5-HT3ARs. Replacement of nAChR α4 or β2 MA 4′ residues by arginine made current density negligible. By contrast, replacement of both -4′ residues by arginine produced functional nAChRs with substantially reduced γ (11.4 ± 0.5 pS). Homology models of the 5-HT3A and α4β2 nAChRs against Torpedo nAChR revealed MA -4′, 0′, and 4′ residues within five intracellular portals. This locus may be a common determinant of ion conduction throughout the Cys loop receptor family.

National recommendations of the Technical UK DNA working group on mixture interpretation for the NDNAD and for court going purposes.

The Technical UK DNA working group comprises representatives from all of the major suppliers of the UK and Ireland who contribute to the UK national DNA database. The group has the following terms of reference:To act as a peer review body.To agree experimental designs, to provide advice to the custodian to facilitate the development of the NDNAD.To support the CJS by the development of a coordinated UK strategy.To be inclusive, rather than exclusive, with regard to the introduction and use of methods.To define best scientific practice.To define guidelines for analysis and interpretation of evidence.To produce guidance that can be used by the UK Accreditation Services (UKAS).The group falls under the European Network of Forensic Science Institutes (ENFSI) umbrella. We will feed back recommendations to the ENFSI group for further discussion in order to facilitate European Policy. The group recently met in order to consider in detail the ISFG DNA Commission recommendations on the interpretation of mixtures, to place them in the context of the UK jurisdictions.

Cleavage of a recombinant human immunoglobulin A2 (IgA2)-IgA1 hybrid antibody by certain bacterial IgA1 proteases.

ABSTRACT To understand more about the factors influencing the cleavage of immunoglobulin A1 (IgA1) by microbial IgA1 proteases, a recombinant human IgA2/IgA1 hybrid molecule was generated. In the hybrid, termed IgA2/A1 half hinge, a seven-amino-acid sequence corresponding to one half of the duplicated sequence making up the IgA1 hinge was incorporated into the equivalent site in IgA2. Insertion of the IgA1 half hinge into IgA2 did not affect antigen binding capacity or the functional activity of the hybrid molecule, as judged by its ability to bind to IgA Fcα receptors and trigger respiratory bursts in neutrophils. Although the IgA2/A1 hybrid contained only half of the IgA1 hinge, it was found to be cleaved by a variety of different bacterial IgA1 proteases, including representatives of those that cleave IgA1 in the different duplicated halves of the hinge, namely, those of Prevotella melaninogenica, Streptococcus pneumoniae, S. sanguis, Neisseria meningitidis types 1 and 2, N. gonorrhoeae types 1 and 2, and Haemophilus influenzae type 2. Thus, for these enzymes the recognition site for IgA1 cleavage is contained within half of the IgA1 hinge region; additional distal elements, if required, are provided by either an IgA1 or an IgA2 framework. In contrast, the IgA2/A1 hybrid appeared to be resistant to cleavage with S. oralis and some H. influenzae type 1 IgA1 proteases, suggesting these enzymes require additional determinants for efficient substrate recognition.