Laszlo Nadasdi

Role of Q-type Ca2+ Channels in Vasopressin Secretion From Neurohypophysial Terminals of the Rat

1 The nerve endings of rat neurohypophyses were acutely dissociated and a combination of pharmacological, biophysical and biochemical techniques was used to determine which classes of Ca2+ channels on these central nervous system (CNS) terminals contribute functionally to arginine vasopressin (AVP) and oxytocin (OT) secretion. 2 Purified neurohypophysial plasma membranes not only had a single high‐affinity binding site for the N‐channel‐specific ω‐conopeptide MVIIA, but also a distinct high‐affinity site for another ω‐conopeptide (MVIIC), which affects both N‐ and P/Q‐channels. 3 Neurohypophysial terminals exhibited, besides L‐ and N‐type currents, another component of the Ca2+ current that was only blocked by low concentrations of MVIIC or by high concentrations of ω‐AgaIVA, a P/Q‐channel‐selective spider toxin. 4 This Ca2+ current component had pharmacological and biophysical properties similar to those described for the fast‐inactivating form of the P/Q‐channel class, suggesting that in the neurohypophysial terminals this current is mediated by a ‘Q’‐type channel. 5 Pharmacological additivity studies showed that this Q‐component contributed to rises in intraterminal Ca2+ concentration ([Ca2+]i) in only half of the terminals tested. 6 Furthermore, the non‐L‐ and non‐N‐component of Ca2+‐dependent AVP release, but not OT release, was effectively abolished by the same blockers of Q‐type current. 7 Thus Q‐channels are present on a subset of the neurohypophysial terminals where, in combination with N‐ and L‐channels, they control AVP but not OT peptide neurosecretion.

Solution structure of ω-conotoxin MVIIA using 2D NMR spectroscopy

The solution structure of ω‐conotoxin MVIIA (SNX‐111), a peptide toxin from the fish hunting cone snail Conus magus and a high‐affinity blocker of N‐type calcium channels, was determined by 2D NMR spectroscopy. The backbones of the best 44 structures match with an average pairwise RMSD of 0.59 angstroms. The structures contain a short segment of triple‐stranded β‐sheet involving residues 6–8, 20–21, and 24–25. The structure of this toxin is very similar to that of ω‐conotoxin GVIA with which is has only 40% sequence homology, but very similar calcium channel binding affinity and selectivity.

Bioavailability of Ziconotide in brain: influx from blood, stability, and diffusion.

Ziconotide is a selective peptide antagonist of the N-type calcium channel currently in clinical trials for analgesia. Ziconotide reached a maximal brain concentration of between 0.003 and 0.006% of the injected material per gram of tissue at 3-20 min after i.v. injection, and this decayed to below 0.001%/g after 2 h. The structurally distinct conopeptide SNX-185 (synthetic TVIA) was considerably more persistent in brain after i.v. administration, with 0.0035% of the injected material present at 2-4 h after i.v. injection, and 0.0015% present at 24 h. Similar results (i.e. greater persistence of SNX-185) were obtained when the peptides were perfused through in vivo dialysis probes implanted into the hippocampus. Image analysis and serial sectioning showed that diffusion of Ziconotide in the extracellular fluid around the dialysis probe was minimal, with the peptide located within 1 mm of the probe after 2 h. In vitro diffusion through cultured bovine brain microvessel endothelial cells (BBMEC) verified that a close structural analog of Ziconotide (SNX-194) passed through this blood-brain barrier (BBB) model as expected for peptides of similar physical properties (permeability coefficient of 6.5 x 10(-4) cm/g). Passage from blood to brain was also verified by in situ perfusion through the carotid artery. A statistically greater amount of radioactivity was found to cross the BBB after perfusion of radioiodinated Ziconotide compared to [14C]inulin. Capillary depletion experiments and HPLC analysis defined the brain location and stability.

STRUCTURE AND FUNCTION OF G PROTEIN COUPLED RECEPTORS

The G protein coupled receptors (GPC-Rs) comprise a large superfamily of genes encoding numerous receptors which all show common structural features, e.g., seven putative membrane spanning domains. Their biological functions are extremely diverse, ranging from vision and olfaction to neuronal and endocrine signaling. The GPC-Rs couple via multiple G proteins to a growing number of recognized second messenger pathway, e.g., cAMP and phosphatidyl inositol turnover. This review summarizes our current knowledge of the molecular mechanisms by which the GPC-Rs activate second messenger systems, and it addresses their regulation and structure.