Annie Schmid

Properties of structure and interaction of the receptor for ω-conotoxin, a polypeptide active on Ca2+ channels

Binding properties of omega-conotoxin (GVIA) to avian and mammalian neuronal Ca2+ channels were investigated using a radioiodinated toxin derivative. An exceptionally high affinity of 0.6 to 2 pM was found both from equilibrium and kinetics measurements. Only one class of non-interacting binding sites was detected. In chick brain, dissucinimidyl suberate specifically cross-linked the toxin to 170 kDa component that comprises a 140 kDa peptide disulfide linked to a 30 kDa peptide, very similar to the heavily glycosylated component of the L-type DHP-sensitive Ca2+ channel. A large peptide of 210-220 kDa was labelled using the azidonitrobenzoyloxy derivative of omega-conotoxin as cross-linking reagent but not the 170/140+30 kDa component. The results suggest that the neuronal Ca2+ channel could be composed by the association of two distinct high molecular weight peptides of 220 kDa and 170/140+30 kDa.

Photoaffinity labelling and phosphorylation of a 165 kilodalton peptide associated with dihydropyridine and phenylalkylamine-sensitive calcium channels

Partially purified fractions of dihydropyridine and phenylalkylamine receptors associated with voltage-dependent calcium channels in rabbit skeletal muscle were found to contain two glycopeptides of similar molecular weight. A peptide of approximately 165 kDa was photoaffinity labelled with an arylazido-phenylalkylamine Ca channel inhibitor and also was phosphorylated with cAMP-dependent protein kinase. Another peptide of 170 kDa could be distinguished from the 165 kDa peptide by peptide mapping and differences in electrophoretic mobility. The results suggest that the 165 kDa peptide contains the sites responsible for regulation of calcium channel activity by calcium channel inhibitors as well as by neurotransmitters that regulate its activity in a cAMP-dependent manner.

Ontogenic appearance of Ca2+ channels characterized as binding sites for nitredipine during development of nervous, skeletal and cardiac muscle systems in the rat

The appearance of specific receptors for the Ca2+ channel antagonist nitrendipine has been followed during the fetal and post‐natal development of rat brain without cerebellum, cerebellum, skeletal muscle and cardiac muscle. The number of nitrendipine receptors is low at the fetal stage and increases drastically during post‐natal development of brain, cerebellum, skeletal muscle and cardiac muscle. The time course of this increase is different for each type of tissue studied. No significant change in receptor ligand dissociation constant (K d) can be detected over the development period studied. The results are discussed in relation with the known properties of the differentiation process in the four types of excitable tissues studied.

Activation of the voltage-dependent Ca2+ channel in rat heart cells by dihydropyridine derivatives.

The two dihydropyridines Bay K8644 and CGP 28392 increase 45Ca2+ influx in cultured rat cardiac cells with half-maximal effects at 2 nM and 30 nM respectively at a membrane potential of -75 mV. This stimulation of Ca2+ uptake is inhibited by nitrendipine, verapamil and bepridil. Ca2+ channel activation produced by Bay K8644 and CGP 28392 has been compared with Ca2+ channel activation produced by depolarization. There is no addition between the effects of drugs activating the Ca2+ channel and the effects of depolarization suggesting that Bay K8644 and CGP 28392 work preferentially on polarized membranes. 45Ca2+ flux experiments yielded results which are in excellent agreement with electrophysiological and contraction data obtained with the same cells in culture. Dose-response curves for the physiological effects of the drugs are observed over the same range of concentrations as their inhibition of [3H]nitrendipine binding to its receptor.

Comparative changes of levels of nitrendipine Ca2+ channels, of tetrodotoxin-sensitive Na+ channels and of ouabain-sensitive (Na+ + K+)-ATPase following denervation of rat and chick skeletal muscle

Three major ion transport systems, the nitrendipine‐sensitive Ca2+ channels, the tetrodotoxin‐sensitive Na+ channel and the ouabain‐sensitive (Na+ + K+‐ATPase, have been studied in skeletal muscle from rat and chick after chronic denervation. It is shown that the situation found for the Ca2+ channel differs dramatically from that found for the Na2+ channel and the (Na2+ + K+)‐ATPase and that regulation of the nitrendipine‐sensitive Ca2+ channel in denervated muscle also differs widely from that of the tetrodotoxinsensitive Na+ channel and the ouabain‐sensitive (Na+ + K+‐ATPase which show a quite similar evolution.

Antibodies reveal the cytolocalization and subunit structure of the 1,4-dihydropyridine component of the neuronal Ca2+ channel

Rabbit brain synaptosomes bind the 1,4-dihydropyridine derivative (+)[3H]-PN 200-110 with an equilibrium dissociation constant of 0.04 nM and a maximal binding capacity of 400 fmol/mg of protein. Using polyclonal antibodies raised against the different components of the skeletal muscle 1,4-dihydropyridine receptor, we have demonstrated that the brain and muscle receptors share the same subunit composition comprising a large polypeptide chain of Mr 140,000 associated by disulfide bridges with a smaller peptide of Mr 32,000. These antibodies have been used in immunofluorescence staining of brain sections. They reveal a distribution of the Ca2+ channel protein similar to that of 1,4-dihydropyridine binding sites with (+)[3H]PN 200-110 by the autoradiographic technique.

A polypeptide toxin from the coral Goniopora Purification and action on Ca2+ channels

A polypeptide toxin has been isolated from Goniopora coral with an Mr of 19000. Goniopora toxin has the following properties: (i) it induces contraction of guinea pig ileum and this contraction is prevented by Ca2+-channel blockers; (ii) it stimulates 45Ca2+ influx in cardiac cells in culture and this stimulation is abolished by Ca2+-channel blockers; (iii) it prevents binding of (+)-[3H]PN 200-110 to the Ca2+-channel protein of skeletal muscle T-tubule membranes. All these results taken together suggest that Goniopora toxin is a Ca2+-channel activator.A polypeptide toxin has been isolated from Goniopora coral with an M r of 19000. Goniopora toxin has the following properties: (i) it induces contraction of guinea pig ileum and this contraction is prevented by Ca2+‐channel blockers; (ii) it stimulates 45Ca2+ influx in cardiac cells in culture and this stimulation is abolished by Ca2+‐channel blockers; (iii) it prevents binding of (+)‐[3H]PN 200‐110 to the Ca2+‐channel protein of skeletal muscle T‐tubule membranes. All these results taken together suggest that Goniopora toxin is a Ca2+‐channel activator.

Appearance and Function of Voltage‐Dependent Ca2+ Channels during Pre‐ and Postnatal Development of Cardiac and Skeletal Muscles

The most prominent feature of many excitable cells is their ability to respond to voltage applied across their plasma membranes. Usually, the response consists of a change in membrane permeability to specific cations due to the opening or closing of ionic channels. It has been shown that the ionic properties of most of the cell types studied are often different at the early stage of development than in the adult. Furthermore, during the course of development, the ionic response changes in reaction to a change in the cationic channels involved? These observations have raised several questions: (1) Are the changes observed common to all excitable cell types? (2) Are these changes developmentally regulated, and if so what mechanism controls them? (3) What is the role of these ionic channels in the differentiation process and in maturation of the cells? This review tries to provide partial answers to these questions using the example of the voltage-sensitive, slow Ca2+ channel, which is sensitive to dihydropyridines,8 and those throughout the development of cardiac and skeletal muscles. These two cell types were chosen for the study of the differentiation process, because many pharmacological and biochemical properties were already known for adult cells and t i s~ues .~ . ~ This has made it easier to investigate the time course and appearance of structures that are responsible for cell excitability and to detail changes in the biochemical and functional properties of the slow, voltage-dependent Ca2+ channels from the earliest to the latest stage of development.

Molecular Properties of Structure and Regulation of the Calcium Channel

Voltage-dependent calcium channels are known to play important roles in excitationcontraction coupling in cardiac and smooth muscles and also in a number of neurosecretory processes. They have recently become accessible to biochemical study as a result of the availability of a number of tritiated calcium channel inhibitors belonging to the dihydropyridine and the phenylalkylamine series.’ Dihydropyridine derivatives such as nitrendipine and other calcium inhibitors such as verapamil, bepridil, and diltiazem are very important therapeutic agents in the treatment of cardiovascular disorders.’ One of the difficulties associated with the biochemical characterization of macromolecules that confer electrical excitability to biological membranes, such as the voltage-sensitive calcium channel, is that they are almost always present in very low amounts in membranes. For most membrane preparations in which the voltagesensitive calcium channel has been characterized, the dihydropyridine receptor is only present at a density in the range of 0.1 to 1 pmol/mg protein.’ Therefore, the transverse tubule (T-tubule) membrane preparation isolated from rabbit skeletal muscle appears to be exceptional in that it contains more than 50 pmol [3H]dihydropyridine-binding sites/mg p r ~ t e i n . ~ Voltage-clamp analyses of skeletal muscle have shown that essentially all specific Ca’+ conductances are localized in the transverse tubular system and that the channel responsible for these conductances is inhibited by low concentrations

Biochemistry, molecular pharmacology, and functional control of Ca2+ channels.

Most of what we now know of the structure of the Ca2+ channel comes from work that has been carried out with skeletal muscle membranes. This is because it was shown a number of years ago that the richest source of receptors for Cd+ channel blockers and particularly for 1,Cdihydropyridines (DHP) is the skeletal muscle Ttubule membrane system. Two main types of Ca2+ channels are found in skeletal muscle. One of them is the low threshold, or T-type, Ca*+ channel, the other one is the high threshold or L-type Ca2+ ~hannel.~, Patch-clamp analysis has actually identified two different subtypes of L-type Ca2+ channels in mammalian skeletal muscle2; both are blocked by 1,4-dihydropyridines, but they have different voltage sensitivities of the activation process and different inactivation kinetics. In muscular dysgenesis, a mouse muscle disease corresponding to an absence of contraction, receptor sites for Ca2+ channel blockers are nearly absent in muscle membranes and the functional expression of the Ca2+ channel is lacking.In vitro