Amino acid sequence of the alpha subunit and computer modelling of the alpha and beta subunits of echicetin from the venom of Echis carinatus (saw-scaled viper)

Echicetin, a heterodimeric protein from the venom of Echis carinatus, binds to platelet glycoprotein Ib (GPIb) and so inhibits platelet aggregation or agglutination induced by various platelet agonists acting via GPIb. The amino acid sequence of the beta subunit of echicetin has been reported and found to belong to the recently identified snake venom subclass of the C-type lectin protein family. Echicetin alpha and beta subunits were purified. N-terminal sequence analysis provided direct evidence that the protein purified was echicetin. The paper presents the complete amino acid sequence of the alpha subunit and computer models of the alpha and beta subunits. The sequence of alpha echicetin is highly similar to the alpha and beta chains of various heterodimeric and homodimeric C-type lectins. Neither of the fully reduced and alkylated alpha or beta subunits of echicetin inhibited the platelet agglutination induced by von Willebrand factor-ristocetin or alpha-thrombin. Earlier reports about the inhibitory activity of reduced and alkylated echicetin beta subunit might have been due to partial reduction of the protein.

The alpha and beta subunits of the metalloprotease meprin are expressed in separate layers of human epidermis, revealing different functions in keratinocyte proliferation and differentiation

The zinc endopeptidase meprin (EC 3.4.24.18) is expressed in brush border membranes of intestine and kidney tubules, intestinal leukocytes, and certain cancer cells, suggesting a role in epithelial differentiation and cell migration. Here we show by RT-PCR and immunoblotting that meprin is also expressed in human skin. As visualized by immunohistochemistry, the two meprin subunits are localized in separate cell layers of the human epidermis. Meprin alpha is expressed in the stratum basale, whereas meprin beta is found in cells of the stratum granulosum just beneath the stratum corneum. In hyperproliferative epidermis such as in psoriasis vulgaris, meprin alpha showed a marked shift of expression from the basal to the uppermost layers of the epidermis. The expression patterns suggest distinct functions for the two subunits in skin. This assumption is supported by diverse effects of recombinant meprin alpha and beta on human adult low-calcium high-temperature keratinocytes. Here, beta induced a dramatic change in cell morphology and reduced the cell number, indicating a function in terminal differentiation, whereas meprin alpha did not affect cell viability, and may play a role in basal keratinocyte proliferation.

Sudden infant death syndrome-associated mutations in the sodium channel beta subunits.

BACKGROUND Approximately 10% of sudden infant death syndrome (SIDS) cases may stem from potentially lethal cardiac channelopathies, with approximately half of channelopathic SIDS involving the Na(V)1.5 cardiac sodium channel. Recently, Na(V) beta subunits have been implicated in various cardiac arrhythmias. Thus, the 4 genes encoding Na(V) beta subunits represent plausible candidate genes for SIDS. OBJECTIVE This study sought to determine the spectrum, prevalence, and functional consequences of sodium channel beta-subunit mutations in a SIDS cohort. METHODS In this institutional review board-approved study, mutational analysis of the 4 beta-subunit genes, SCN1B to 4B, was performed using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing of DNA derived from 292 SIDS cases. Engineered mutations were coexpressed with SCN5A in HEK 293 cells and were whole-cell patch clamped. One of the putative SIDS-associated mutations was similarly studied in adenovirally transduced adult rat ventricular myocytes. RESULTS Three rare (absent in 200 to 800 reference alleles) missense mutations (beta3-V36M, beta3-V54G, and beta4-S206L) were identified in 3 of 292 SIDS cases. Compared with SCN5A+beta3-WT, beta3-V36M significantly decreased peak I(Na) and increased late I(Na), whereas beta3-V54G resulted in a marked loss of function. beta4-S206L accentuated late I(Na) and positively shifted the midpoint of inactivation compared with SCN5A+beta4-WT. In native cardiomyocytes, beta4-S206L accentuated late I(Na) and increased the ventricular action potential duration compared with beta4-WT. CONCLUSION This study provides the first molecular and functional evidence to implicate the Na(V) beta subunits in SIDS pathogenesis. Altered Na(V)1.5 sodium channel function due to beta-subunit mutations may account for the molecular pathogenic mechanism underlying approximately 1% of SIDS cases.

Loss-of-function mutation of the SCN3B-encoded sodium channel {beta}3 subunit associated with a case of idiopathic ventricular fibrillation.

AIMS Loss-of-function mutations in the SCN5A-encoded sodium channel SCN5A or Nav1.5 have been identified in idiopathic ventricular fibrillation (IVF) in the absence of Brugada syndrome phenotype. Nav1.5 is regulated by four sodium channel auxiliary beta subunits. Here, we report a case with IVF and a novel mutation in the SCN3B-encoded sodium channel beta subunit Navbeta3 that causes a loss of function of Nav1.5 channels in vitro. METHODS AND RESULTS Comprehensive open reading frame mutational analysis of KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, GPD1L, four sodium channel beta subunit genes (SCN1-4B), and targeted scan of RYR2 was performed. A novel missense mutation, Navbeta3-V54G, was identified in a 20-year-old male following witnessed collapse and defibrillation from VF. The ECG exhibited epsilon waves, and imaging studies demonstrated a structurally normal heart. The mutated residue was highly conserved across species, localized to the Navbeta3 extracellular domain, and absent in 800 reference alleles. We found that HEK-293 cells had endogenous Navbeta3, but COS cells did not. Co-expression of Nav1.5 with Navbeta3-V54G (with or without co-expression of the Navbeta1 subunit) in both HEK-293 cells and COS cells revealed a significant decrease in peak sodium current and a positive shift of inactivation compared with WT. Co-immunoprecipitation experiments showed association of Navbeta3 with Nav1.5, and immunocytochemistry demonstrated a dramatic decrease in trafficking to the plasma membrane when co-expressed with mutant Navbeta3-V54G. CONCLUSION This study provides molecular and cellular evidence implicating mutations in Navbeta3 as a cause of IVF.

Impact of subunit positioning on GABAA receptor function

The major isoforms of the GABAA (gamma-aminobutyric acid type A) receptor are composed of two alpha, two beta and one gamma subunit. Thus alpha and beta subunits occur twice in the receptor pentamer. As it is well documented that different isoforms of alpha and beta subunits can co-exist in the same pentamer, the question is raised whether the relative position of a subunit isoform affects the functional properties of the receptor. We have used subunit concatenation to engineer receptors of well-defined subunit arrangement to study this question. Although all five subunits may be concatenated, we have focused on the combination of triple and dual subunit constructs. We review here what is known so far on receptors containing simultaneously alpha1 and alpha6 subunits and receptors containing beta1 and beta2 subunits. Subunit concatenation may not only be used to study receptors containing two different subunit isoforms, but also to introduce a point mutation into a defined position in receptors containing either two alpha or beta subunits, or to study the receptor architecture of receptors containing unconventional GABAA receptor subunits. Similar approaches may be used to characterize other members of the pentameric ligand-gated ion channel family, including nicotinic acetylcholine receptors, glycine receptors and 5-HT3 (5-hydroxytryptamine) receptors.

Snake C-type lectin-like proteins and platelet receptors

Snake venoms are complex mixtures of biologically active proteins and peptides. Many affect haemostasis by activating or inhibiting coagulant factors or platelets, or by disrupting endothelium. Snake venom components are classified into various families, such as serine proteases, metalloproteinases, C-type lectin-like proteins, disintegrins and phospholipases. Snake venom C-type lectin-like proteins have a typical fold resembling that in classic C-type lectins such as the selectins and mannose-binding proteins. Many snake venom C-type lectin-like proteins have now been characterized, as heterodimeric structures with alpha and beta subunits that often form large molecules by multimerization. They activate platelets by binding to VWF or specific receptors such as GPIb, alpha2beta1 and GPVI. Simple heterodimeric GPIb-binding molecules mainly inhibit platelet functions, whereas multimeric ones activate platelets. A series of tetrameric snake venom C-type lectin-like proteins activates platelets by binding to GPVI while another series affects platelet function via integrin alpha2beta1. Some act by inducing VWF to bind to GPIb. Many structures of these proteins, often complexed with their ligands, have been determined. Structure-activity studies show that these proteins are quite complex despite similar backbone folding. Snake C-type lectin-like proteins often interact with more than one platelet receptor and have complex mechanisms of action.

The promiscuous role of the epsilon subunit in GABA(A) receptor biogenesis

The formation of alpha1beta2gamma2epsilon receptors suggests that the epsilon subunit does not displace the single gamma2 subunit in alpha1beta2gamma2 receptors. Thus, epsilon must replace alpha and/or beta subunit(s) if the pentameric receptor structure is to be preserved. To assess the potential for which subunit is replaced in alphabetaepsilon and alphabetagammaepsilon receptors we analyzed the assembly and functional expression of the epsilon subunit with respect to alpha1, beta2 and gamma2 subunits. Using concatenated subunits, we have determined that epsilon is capable of substituting for either (but not both) of the alpha subunits, one of the beta subunits, and possibly the gamma2 subunit. However, the most likely sites at which the epsilon subunit may contribute to receptor function appears to be at position 1 (replaces alpha1) in alphabetagammaepsilon (varepsilon-beta2-alpha1-beta2-gamma2) receptors, or at position 4 (replaces beta2) in alphabetaepsilon (alpha1-beta2-alpha1-varepsilon-beta2) receptors. In both cases, it appears that only a single GABA binding site is present.

Increased expression of the auxiliary beta2-subunit of ventricular L-type Ca2+ channels leads to single-channel activity characteristic of heart failure

BACKGROUND: Increased activity of single ventricular L-type Ca(2+)-channels (L-VDCC) is a hallmark in human heart failure. Recent findings suggest differential modulation by several auxiliary beta-subunits as a possible explanation. METHODS AND RESULTS: By molecular and functional analyses of human and murine ventricles, we find that enhanced L-VDCC activity is accompanied by altered expression pattern of auxiliary L-VDCC beta-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac beta-subunits: Unlike beta(1) or beta(3) isoforms, beta(2a) and beta(2b) induce a high-activity channel behavior typical of failing myocytes. In accordance, beta(2)-subunit mRNA and protein are up-regulated in failing human myocardium. In a model of heart failure we find that mice overexpressing the human cardiac Ca(V)1.2 also reveal increased single-channel activity and sarcolemmal beta(2) expression when entering into the maladaptive stage of heart failure. Interestingly, these animals, when still young and non-failing ("Adaptive Phase"), reveal the opposite phenotype, viz: reduced single-channel activity accompanied by lowered beta(2) expression. Additional evidence for the cause-effect relationship between beta(2)-subunit expression and single L-VDCC activity is provided by newly engineered, double-transgenic mice bearing both constitutive Ca(V)1.2 and inducible beta(2) cardiac overexpression. Here in non-failing hearts induction of beta(2)-subunit overexpression mimicked the increase of single L-VDCC activity observed in murine and human chronic heart failure. CONCLUSIONS: Our study presents evidence of the pathobiochemical relevance of beta(2)-subunits for the electrophysiological phenotype of cardiac L-VDCC and thus provides an explanation for the single L-VDCC gating observed in human and murine heart failure.

SCN4B-encoded sodium channel beta4 subunit in congenital long-QT syndrome.

BACKGROUND Congenital long-QT syndrome (LQTS) is potentially lethal secondary to malignant ventricular arrhythmias and is caused predominantly by mutations in genes that encode cardiac ion channels. Nearly 25% of patients remain without a genetic diagnosis, and genes that encode cardiac channel regulatory proteins represent attractive candidates. Voltage-gated sodium channels have a pore-forming alpha-subunit associated with 1 or more auxiliary beta-subunits. Four different beta-subunits have been described. All are detectable in cardiac tissue, but none have yet been linked to any heritable arrhythmia syndrome. METHODS AND RESULTS We present a case of a 21-month-old Mexican-mestizo female with intermittent 2:1 atrioventricular block and a corrected QT interval of 712 ms. Comprehensive open reading frame/splice mutational analysis of the 9 established LQTS-susceptibility genes proved negative, and complete mutational analysis of the 4 Na(vbeta)-subunits revealed a L179F (C535T) missense mutation in SCN4B that cosegregated properly throughout a 3-generation pedigree and was absent in 800 reference alleles. After this discovery, SCN4B was analyzed in 262 genotype-negative LQTS patients (96% white), but no further mutations were found. L179F was engineered by site-directed mutagenesis and heterologously expressed in HEK293 cells that contained the stably expressed SCN5A-encoded sodium channel alpha-subunit (hNa(V)1.5). Compared with the wild-type, L179F-beta4 caused an 8-fold (compared with SCN5A alone) and 3-fold (compared with SCN5A + WT-beta4) increase in late sodium current consistent with the molecular/electrophysiological phenotype previously shown for LQTS-associated mutations. CONCLUSIONS We provide the seminal report of SCN4B-encoded Na(vbeta)4 as a novel LQT3-susceptibility gene.

Two alpha subunits and one beta subunit of meprin zinc-endopeptidases are differentially expressed in the zebrafish Danio rerio

Meprins are members of the astacin family of metalloproteases expressed in epithelial tissues, intestinal leukocytes and certain cancer cells. In mammals, there are two homologous subunits, which form complex glycosylated disulfide-bonded homo- and heterooligomers. Both human meprin alpha and meprin beta cleave several basement membrane components, suggesting a role in epithelial differentiation and cell migration. There is also evidence that meprin beta is involved in immune defence owing to its capability of activating interleukin-1beta and the diminished mobility of intestinal leukocytes in meprin beta-knockout mice. Here we show for the first time by reverse transcription PCR, immunoblotting and immunofluorescence analyses that meprins are expressed not only in mammals, but also in the zebrafish Danio rerio. In contrast to the human, mouse and rat enzymes, zebrafish meprins are encoded by three genes, corresponding to two homologous alpha subunits and one beta subunit. Observations at both the mRNA and protein level indicate a broad distribution of meprins in zebrafish. However, there are strikingly different expression patterns of the three subunits, which is consistent with meprin expression in mammals. Hence, D. rerio appears to be a suitable model to gain insight into the basic physiological functions of meprin metalloproteases.

A GABA(A) receptor of defined subunit composition and positioning: concatenation of five subunits

We show that the five subunits of a gamma-aminobutyric acid type A receptor (GABA(A) receptor) can be concatenated to yield a functional receptor. This concatenated receptor alpha(1)-beta(2)-alpha(1)-gamma(2)-beta(2) has the advantage of a known subunit arrangement. Most of its functional properties are not significantly different from a receptor formed by individual subunits. Extent of expression amounted to about 40% of that of non-concatenated receptors in Xenopus oocytes, after injection of oocytes with comparable amounts of cRNA coding for concatenated and non-concatenated receptors. The ability to express receptors consisting of five subunits enables detailed studies of GABA(A) receptor subtype selective compounds.

Replacement of histidine in position 105 in the alpha(5) subunit by cysteine stimulates zolpidem sensitivity of alpha(5)beta(2)gamma(2) GABA(A) receptors

Zolpidem is a positive allosteric modulator of GABA(A) receptors with sensitivity to subunit composition. While it acts with high affinity and efficacy at GABA(A) receptors containing the alpha(1) subunit, it has a lower affinity to GABA(A) receptors containing alpha(2), alpha(3), or alpha(5) subunits and has a very weak efficacy at receptors containing the alpha(5) subunit. Here, we show that replacing histidine in position 105 in the alpha(5) subunit by cysteine strongly stimulates the effect of zolpidem in receptors containing the alpha(5) subunit. The side chain volume of the amino acid residue in this position does not correlate with the modulation by zolpidem. Interestingly, serine is not able to promote the potentiation by zolpidem. The homologous residues to alpha(5)H105 in alpha(1), alpha(2), and alpha(3) are well-known determinants of the action of classical benzodiazepines. Other studies have shown that replacement of these histidines alpha(1)H101, alpha(2)H101, and alpha(3)H126 by arginine, as naturally present in alpha(4) and alpha(6), leads to benzodiazepine insensitivity of these receptors. Thus, the nature of the amino acid residue in this position is not only crucial for the action of classical benzodiazepines but in alpha(5) containing receptors also for the action of zolpidem.

Mechanism of Ca(v)1.2 channel modulation by the amino terminus of cardiac beta2-subunits

L-type calcium channels are composed of a pore, alpha1c (Ca(V)1.2), and accessory beta- and alpha2delta-subunits. The beta-subunit core structure was recently resolved at high resolution, providing important information on many functional aspects of channel modulation. In this study we reveal differential novel effects of five beta2-subunits isoforms expressed in human heart (beta(2a-e)) on the single L-type calcium channel current. These splice variants differ only by amino-terminal length and amino acid composition. Single-channel modulation by beta2-subunit isoforms was investigated in HEK293 cells expressing the recombinant L-type ion conducting pore. All beta2-subunits increased open probability, availability, and peak current with a highly consistent rank order (beta2a approximately = beta2b > beta2e approximately = beta2c > beta2d). We show graded modulation of some transition rates within and between deep-closed and inactivated states. The extent of modulation correlates strongly with the length of amino-terminal domains. Two mutant beta2-subunits that imitate the natural span related to length confirm this conclusion. The data show that the length of amino termini is a relevant physiological mechanism for channel closure and inactivation, and that natural alternative splicing exploits this principle for modulation of the gating properties of calcium channels.