Background: A class of analgesic ␣-conotoxins potently inhibits N-type calcium channels. Results: The activity of ␣-conotoxins Vc1.1 and RgIA was reduced following knockdown of GABA B receptor expression in sensory neurons and could be reconstituted in HEK 293 cells expressing human GABA B receptors and Ca v 2.2. Conclusion: GABA B receptors are needed for inhibition of Ca v 2.2 by Vc1.1 and RgIA. Significance: These analgesic ␣-conotoxins activate human GABA B receptors.

Neuronal voltage-gated N-type (Ca v 2.2) calcium channels are expressed throughout the nervous system and regulate neurotransmitter release and hence synaptic transmission. They are predominantly modulated via G protein-coupled receptor activated pathways, and the well characterized Gbg subunits inhibit Ca v 2.2 currents. Analgesic a-conotoxin Vc1.1, a peptide from predatory marine cone snail venom, inhibits Ca v 2.2 channels by activating pertussis toxin-sensitive G i/o proteins via the GABA B receptor (GABA B R) and potently suppresses pain in rat models. Using a heterologous GABA B R expression system, electrophysiology, and mutagenesis, we showed a-conotoxin Vc1.1 modulates Ca v 2.2 via a different pathway from that of the GABA B R agonists GABA and baclofen. In contrast to GABA and baclofen, Vc1.1 changes Ca v 2.2 channel kinetics by increasing the rate of activation and shifting its halfmaximum inactivation to a more hyperpolarized potential. We then systematically truncated the GABA B1a C terminus and discovered that removing the proximal carboxyl terminus of the GABA B1a subunit significantly reduced Vc1.1 inhibition of Ca v 2.2 currents. We propose a novel mechanism by which Vc1.1 activates GABA B R and requires the GABA B1a proximal carboxyl terminus domain to inhibit Ca v 2.2 channels. These findings provide important insights into how GABA B Rs mediate Ca v 2.2 channel inhibition and alter nociceptive transmission.

Mechanosensitive (MS) ion channels are the primary molecular transducers of mechanical force into electrical and/or chemical intracellular signals in living cells. They have been implicated in innumerable mechanosensory physiological processes including touch and pain sensation, hearing, blood pressure control, micturition, cell volume regulation, tissue growth, or cellular turgor control. Much of what we know about the basic physical principles underlying the conversion of mechanical force acting upon membranes of living cells into conformational changes of MS channels comes from studies of MS channels reconstituted into artificial liposomes. Using bacterial MS channels as a model, we have shown by reconstituting these channels into liposomes that there is a close relationship between the physico-chemical properties of the lipid bilayer and structural dynamics bringing about the function of these channels.

Over recent years, the role of matrix vesicles in the initial stages of arterial calcification has been recognized. Matrix calcifying vesicles have been isolated from atherosclerotic arteries and the biochemical composition of calcified vesicles has been studied. No studies have yet been carried out to examine the fine structure of matrix vesicles in order to visualize the features of the consequent stages of their calcification in arteries. In the present work, a high resolution ultrastructural analysis has been employed and the study revealed that matrix vesicles in human atherosclerotic lesions are heterogeneous with two main types which we classified. Type I calcified vesicles were presented by vesicles surrounded by two electron-dense layers and these vesicles were found to be resistant to the calcification process in atherosclerotic lesions in situ. Type II matrix vesicles were presented by vesicles surrounded by several electron-dense layers and these vesicles were found to represent calcifying vesicles in atherosclerotic lesions. To test the hypothesis that calcification of matrix vesicles surrounded by multilayer sheets may occur simply as a physicochemical process, independently from the cell regulation, we produced multilamellar liposomes and induced their calcification in vitro in a manner similar to that occurring in matrix vesicles in atherosclerotic lesions in situ.