Concepcion Warner

Site-directed antibodies corresponding to conserved putative extracellular segments of sodium channels, coupled with binding studies of radiolabeled insect-selective scorpion neurotoxins, were employed to clarify the relationship between the toxins' receptor sites and the insect sodium channel. (1) The depressant insect toxin LqhIT2 was shown to possess two noninteracting binding sites in locust neuronal membranes: a high-affinity (KD1 = 0.9 +/- 0.6 nM) and low-capacity (Bmax1 = 0.1 +/- 0.07 pmol/mg) binding site as well as a low-affinity (KD2 = 185 +/- 13 nM) and high-capacity (Bmax2 = 10.0 +/- 0.6 pmol/mg) binding site. (2) The high-affinity site serves as a target for binding competition by the excitatory insect toxin AaIT. (3) The binding of LqhIT2 was significantly inhibited in a dose-dependent manner by each of four site-directed antibodies. The binding inhibition resulted from reduction in the number of binding sites. (4) The antibody-mediated inhibition of [125I]AaIT binding differs from that of LqhIT2: three out of the four antibodies which inhibited LqhIT2 binding only partially affected AaIT binding. Two antibodies, one corresponding to extracellular and one to intracellular segments of the channel, did not affect the binding of either toxin. These data suggest that the receptors to the depressant and excitatory insect toxins (a) comprise an integral part of the insect sodium channel, (b) are formed by segments of external loops in domains I, III, and IV of the sodium channel, and (c) are localized in close proximity but are not identical in spite of the competitive interaction between these toxins.

Translocation to the cytosol is an essential and rate-limiting step in the cytotoxicity of the potent plant toxin ricin. In an attempt to study the mechanism of ricin A-chain (RTA) translocation in a cell-free assay, we have partially purified Golgi and endoplasmic reticulum from Jurkat cells by discontinuous sucrose gradient fractionation. The membranes of the organelle fractions were solubilized by the addition of sodium cholate and reconstituted into proteoliposomes by dialyzing out the detergent. The resulting vesicles supported cell-free translocation of RTA (as assessed by an enzyme protection assay) at a rate which was linearly dependent on the concentration of the vesicle preparation. Ricin B-chain (RTB) neither translocated into the vesicles, nor increased the efficiency of RTA translocation. Liposomes prepared from purified phospholipids were not capable of supporting RTA translocation. Furthermore, protease treatment or concanavalin A adsorption of proteins from lysates prior to vesicle reconstitution resulted in abrogation of the translocation process, suggesting that the protein components of organelle membranes are required for RTA translocation. Reconstitution of translocation-competent proteoliposomes from detergent-solubilized membranes of endoplasmic reticulum- and Golgi-enriched fractions provides a convenient cell-free system to study the mechanism of RTA translocation. Translocation to the cytosol is an essential and rate-limiting step in the cytotoxicity of the potent plant toxin ricin. In an attempt to study the mechanism of ricin A-chain (RTA) translocation in a cell-free assay, we have partially purified Golgi and endoplasmic reticulum from Jurkat cells by discontinuous sucrose gradient fractionation. The membranes of the organelle fractions were solubilized by the addition of sodium cholate and reconstituted into proteoliposomes by dialyzing out the detergent. The resulting vesicles supported cell-free translocation of RTA (as assessed by an enzyme protection assay) at a rate which was linearly dependent on the concentration of the vesicle preparation. Ricin B-chain (RTB) neither translocated into the vesicles, nor increased the efficiency of RTA translocation. Liposomes prepared from purified phospholipids were not capable of supporting RTA translocation. Furthermore, protease treatment or concanavalin A adsorption of proteins from lysates prior to vesicle reconstitution resulted in abrogation of the translocation process, suggesting that the protein components of organelle membranes are required for RTA translocation. Reconstitution of translocation-competent proteoliposomes from detergent-solubilized membranes of endoplasmic reticulum- and Golgi-enriched fractions provides a convenient cell-free system to study the mechanism of RTA translocation.

Themolecular properties oftwosize forms of thealsubunit ofpurified skeletal muscle calcium channels wereanalyzed. Theminor, full-length, form, a1212, wasfound tohaveanapparent molecular massof214kDabyFerguson plot analysis, while themajor, truncated, form, nowdesignated al1o, hadanapparent molecular massof193kDa.Antibody mapping oftheC-terminal region ofallg0 with10anti-peptide antibodies placed theC terminus between residues 1685and 1699. Three consensus sites forcAMP-dependent protein phos- phorylation arepresent intheC-terminal region ofa1212 but notinal1g, andtheymaybeimportant fortheregulation of theionconductance activity ofthecalcium channel. Fourclasses ofvoltage-gated calcium channels havebeen defined onthebasis ofelectrophysiological andpharmaco- logical properties (1). L-type calcium channels mediate long- lasting calcium currents that aresensitive toinhibition by dihydropyridines (DHPs). Mostbiochemical studies have concentrated ontheL-type calcium channel fromskeletal muscle transverse tubules (T-tubules), whichcontain 50-to 100-fold moreDHP receptor sites thanother sources. The mostabundant form'4therabbit skeletal muscle calcium channel isacomplex offive subunits: al(175 kDa), a2(143 kDa), , (54kDa), y(30kDa), and8(24-27 kDa)(reviewed in refs. 2-4). Theal,A,andysubunits areproducts ofdistinct genes(5-7), while thea2and8subunits areencoded bythe samegene(8), whoseprotein product isproteolytically processed anddisulfide-linked togive thea28complex (9). Thealsubunit contains thereceptor sites forDHPsandother calcium channel modulating drugs. Thesequence ofits cDNApredicts aprotein of1873 amino acids (212 kDa)whose structure ishomologous tothesodium channel asubunit, with four internally homologous domains eachcontaining six predicted a-helical transmembrane segments (5). Thissub- unit alone issufficient toformfunctional calcium channels whenexpressed inmousefibroblast cells (10). TheDHP-sensitive calcium channel protein inskeletal muscle hasalso beenproposed toserve asthevoltage sensor forexcitation-contr action coupling, aprocess that doesnot