Alternative splicing of Nav1.5: an electrophysiological comparison of 'neonatal' and 'adult' isoforms and critical involvement of a lysine residue

In developmentally regulated D1:S3 splicing of Nav1.5, there are 31 nucleotide differences between the 5'-exon ('neonatal') and the 3'-exon ('adult') forms, resulting in 7 amino acid differences in D1:S3-S3/S4 linker. In particular, splicing replaces a conserved negative aspartate residue in the 'adult' with a positive lysine. Here, 'neonatal' and 'adult' Nav1.5 alpha-subunit splice variants were stably transfected into EBNA-293 cells and their electrophysiological properties investigated by whole-cell patch-clamp recording. Compared with the 'adult' isoform, the 'neonatal' channel exhibited (1) a depolarized threshold of activation and voltage at which the current peaked; (2) much slower kinetics of activation and inactivation; (3) 50% greater transient charge (Na(+)) influx; (4) a stronger voltage dependence of time to peak; and (5) a slower recovery from inactivation. Tetrodotoxin sensitivity and VGSCbeta1-4 mRNA expression levels did not change. The significance of the charge-reversing aspartate to lysine substitution was investigated by mutating the lysine in the 'neonatal' channel back to aspartate. In this 'neonatal K211D' mutant, the electrophysiological parameters studied strongly shifted back towards the 'adult', that is the lysine residue was primarily responsible for the electrophysiological effects of Nav1.5 D1:S3 splicing. Taken together, these data suggest that the charge reversal in 'neonatal' Nav1.5 would (1) modify the channel kinetics and (2) prolong the resultant current, allowing greater intracellular Na(+) influx. Developmental and pathophysiological consequences of such differences are discussed.     

Forced unfolding of coiled-coils in fibrinogen by single-molecule AFM

Fibrinogen is a blood plasma protein that, after activation by thrombin, assembles into fibrin fibers that form the elastic network of blood clots. We used atomic force microscopy to study the forced unfolding of engineered linear oligomers of fibrinogen, and we show that forced extension of the oligomers produces sawtooth patterns with a peak-to-peak length consistent with the independent unfolding of the coiled-coils in a cooperative two-state manner. In contrast with force plateaus seen for myosin coiled-coils that suggested rapid refolding of myosin, Monte Carlo simulations of fibrinogen unfolding confirm that fibrinogen refolding is negligible on experimental timescales. The distinct behavior of fibrinogen seems to be due to its topologically complex coiled-coils and an interaction between fibrinogen's αC-domains and its central region.     

Quantitative metabolomic profiling of serum and urine in DSS-induced ulcerative colitis of mice by (1)H NMR spectroscopy

Quantitative profiling of a large number of metabolic compounds is a promising method to detect biomarkers in inflammatory bowel diseases (IBD), such as ulcerative colitis (UC). We induced an experimental form of UC in mice by treatment with dextran sulfate sodium (DSS) and characterized 53 serum and 69 urine metabolites by use of (1)H NMR spectroscopy and quantitative ("targeted") analysis to distinguish between diseased and healthy animals. Hierarchical multivariate orthogonal partial least-squares (OPLS) models were developed to detect and predict separation of control and DSS-treated mice. DSS treatment resulted in weight loss, colonic inflammation, and increase in myeloperoxidase activity. Metabolomic patterns generated from the OPLS data clearly separated DSS-treated from control mice with a slightly higher predictive power (Q(2)) for serum (0.73) than urine (0.71). During DSS colitis, creatine, carnitine, and methylamines increased in urine while in serum, maximal increases were observed for ketone bodies, hypoxanthine, and tryptophan. Antioxidant metabolites decreased in urine whereas in serum, glucose and Krebs cycle intermediates decreased strongly. Quantitative metabolic profiling of serum and urine thus discriminates between healthy and DSS-treated mice. Analysis of serum or urine seems to be equally powerful for detecting experimental colitis, and a combined analysis offers only a minor improvement.     

Analysis of Chemotaxis in White Blood Cells

We wish to report the development of an assay system for the study of white blood cells in vitro. With this system we have demonstrated that a yet unidentified substance found in red blood cell membranes and cyclic adenosine monophosphate (cAMP) cause the chemotactic response in white blood cells. We have not yet determined whether the substance released from the membrane is cAMP.     

Computationally designed peptide inhibitors of protein-protein interactions in membranes

We recently reported a computational method (CHAMP) for designing sequence-specific peptides that bind to the membrane-embedded portions of transmembrane proteins. We successfully applied this method to design membrane-spanning peptides targeting the transmembrane domains of the alpha IIb subunit of integrin alpha IIbbeta 3. Previously, we demonstrated that these CHAMP peptides bind specifically with reasonable affinity to isolated transmembrane helices of the targeted transmembrane region. These peptides also induced integrin alpha IIbbeta 3 activation due to disruption of the helix-helix interactions between the transmembrane domains of the alpha IIb and beta 3 subunits. In this paper, we show the direct interaction of the designed anti-alpha IIb CHAMP peptide with isolated full-length integrin alpha IIbbeta 3 in detergent micelles. Further, the behavior of the designed peptides in phospholipid bilayers is essentially identical to their behavior in detergent micelles. In particular, the peptides assume a membrane-spanning alpha-helical conformation that does not disrupt bilayer integrity. The activity and selectivity of the CHAMP peptides were further explored in platelets, comfirming that anti-alpha IIb activates wild-type alpha IIbbeta 3 in whole cells as a result of its disruption of the protein-protein interactions between the alpha and beta subunits in the transmembrane regions. These results demonstrate that CHAMP is a successful chemical biology approach that can provide specific tools for probing the transmembrane domains of proteins.     

Complementary combining site contact residue mutations of the anti-digoxin Fab 26-10 permit high affinity wild-type binding

Antibody 26-10, obtained in a secondary immune response, binds digoxin with high affinity (K(a) = 1.3 x 10(10) M(-1)) because of extensive shape complementarity. We demonstrated previously that mutations of the hapten contact residue HTrp-100 to Arg (where H refers to the heavy chain) resulted in increased specificity for digoxin analogs substituted at the cardenolide 16 position. However, mutagenesis of H:CDR1 did not result in such a specificity change despite the proximity of the H:CDR1 hapten contact residue Asn-35 to the cardenolide 16 position. Here we constructed a bacteriophage-displayed library containing randomized mutations at H chain residues 30-35 in a 26-10 mutant containing Arg-100 (26-10-RRALD). Phage were selected by panning against digoxin, gitoxin (16-OH), and 16-acetylgitoxin coupled to bovine serum albumin. Clones that retained wild-type Asn at position 35 showed preferred binding to gitoxin, like the 26-10-RRALD parent. In contrast, clones containing Val-35 selected mainly on digoxin-bovine serum albumin demonstrated a shift back to wild-type specificity. Several clones containing Val-35 bound digoxin with increased affinity, approaching that of the wild type in a few instances, in contrast to the mutation Val-35 in the wild-type 26-10 background, which reduces affinity for digoxin 90-fold. It has therefore proven possible to reorder the 26-10 binding site by mutations including two major contact residues on opposite sides of the site and yet to retain high affinity for binding for digoxin. Thus, even among antibodies that have undergone affinity maturation in vivo, different structural solutions to high affinity binding may be revealed.     

Clockwise or anticlockwise? Turning the centriole triplets in the right direction!

Centrosomes are small cytoplasmic macromolecular assemblies composed from two major components, centrioles and pericentriolar material, each with its own complex architecture. This organelle is of interest because it plays a role in a number of fundamental cellular processes and defects in these processes have recently been correlated with variety of human disease. Increasingly, what is known about the structure of this organelle has been overshadowed by the increasing wealth of information on its biochemistry. In this short review, we highlight some of the common centriole structural errors found in the literature and define a set of rules that define centriole structure.     

Resolving Two-dimensional Kinetics of the Integrin αIIbβ3-Fibrinogen Interactions Using Binding-Unbinding Correlation Spectroscopy

Using a combined experimental and theoretical approach named binding-unbinding correlation spectroscopy (BUCS), we describe the two-dimensional kinetics of interactions between fibrinogen and the integrin αIIbβ3, the ligand-receptor pair essential for platelet function during hemostasis and thrombosis. The methodology uses the optical trap to probe force-free association of individual surface-attached fibrinogen and αIIbβ3 molecules and forced dissociation of an αIIbβ3-fibrinogen complex. This novel approach combines force clamp measurements of bond lifetimes with the binding mode to quantify the dependence of the binding probability on the interaction time. We found that fibrinogen-reactive αIIbβ3 pre-exists in at least two states that differ in their zero force on-rates (k_on1 = 1.4 × 10⁻⁴ and k_on2 = 2.3 × 10⁻⁴ μm²/s), off-rates (k_off1 = 2.42 and k_off2 = 0.60 s⁻¹), and dissociation constants (K_d1 = 1.7 × 10⁴ and K_d2 = 2.6 × 10³ μm⁻²). The integrin activator Mn²⁺ changed the on-rates and affinities (K_d1 = 5 × 10⁴ and K_d2 = 0.3 × 10³ μm⁻²) but did not affect the off-rates. The strength of αIIbβ3-fibrinogen interactions was time-dependent due to a progressive increase in the fraction of the high affinity state of the αIIbβ3-fibrinogen complex characterized by a faster on-rate. Upon Mn²⁺-induced integrin activation, the force-dependent off-rates decrease while the complex undergoes a conformational transition from a lower to higher affinity state. The results obtained provide quantitative estimates of the two-dimensional kinetic rates for the low and high affinity αIIbβ3 and fibrinogen interactions at the single molecule level and offer direct evidence for the time- and force-dependent changes in αIIbβ3 conformation and ligand binding activity, underlying the dynamics of fibrinogen-mediated platelet adhesion and aggregation.