Mechanical unfolding and refolding may regulate the molecular elasticity of modular proteins with mechanical functions. The development of the atomic force microscopy (AFM) has recently enabled the dynamic measurement of these processes at the single-molecule level. Protein engineering techniques allow the construction of homomeric polyproteins for the precise analysis of the mechanical unfolding of single domains. alpha-Helical domains are mechanically compliant, whereas beta-sandwich domains, particularly those that resist unfolding with backbone hydrogen bonds between strands perpendicular to the applied force, are more stable and appear frequently in proteins subject to mechanical forces. The mechanical stability of a domain seems to be determined by its hydrogen bonding pattern and is correlated with its kinetic stability rather than its thermodynamic stability. Force spectroscopy using AFM promises to elucidate the dynamic mechanical properties of a wide variety of proteins at the single molecule level and provide an important complement to other structural and dynamic techniques (e.g., X-ray crystallography, NMR spectroscopy, patch-clamp).

Glial cells actively participate in synaptic transmission. They clear molecules from the synaptic cleft, receive signals from neurons and, in turn, release molecules that can modulate signaling between neuronal elements. Whether glial-derived transmitters can contribute to enduring changes in postsynaptic efficacy, however, remains to be established. In rat hypothalamic paraventricular nucleus, we demonstrate an increase in the amplitude of miniature excitatory postsynaptic currents in response to norepinephrine that requires the release of ATP from glial cells. The increase in quantal efficacy, which likely results from an insertion of AMPA receptors, is secondary to the activation of P2X(7) receptors, an increase in postsynaptic calcium and the activation of phosphatidylinositol 3-kinase. The gliotransmitter ATP, therefore, contributes directly to the regulation of postsynaptic efficacy at glutamatergic synapses in the CNS.

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A three-dimensional culture system supports the development of primate preantral follicles to the antral stage with appreciable steroid production. This study assessed i) whether in vitro developmental competence of follicles is age dependent, ii) the role of gonadotropins and insulin in supporting folliculogenesis, and iii) anti-Müllerian hormone (AMH) and vascular endothelial growth factor (VEGF) production by growing follicles. Ovaries were obtained from prepubertal, young, and older adult rhesus macaques. Secondary follicles were encapsulated into alginate beads and cultured individually for 40 days in media containing 0.05 or 5 μg/ml insulin, with or without recombinant human (rh) FSH (500 mIU/ml). No follicles survived in the culture without rhFSH. In the presence of rhFSH, survival was lower for follicles from older animals, whereas growth, i.e. follicle diameter, was less by day 40 for follicles from prepubertal animals. The surviving follicles were categorized as no-grow (NG;≤250 μm), slow-grow (SG; 250–500 μm), and fast-grow (FG;≥500 μm) according to their diameters. SG follicles cultured with 5 μg/ml insulin produced more ovarian steroids than those cultured with 0.05 μg/ml insulin by week 5. SG and FG follicles produced more AMH and VEGF than the NG, and levels peaked at weeks 2 and 5 respectively. After 100 ng/ml rh chorionic gonadotropin treatment for 34 h, more healthy oocytes were retrieved from young adults whose follicles were cultured with 5 μg/ml insulin. This culture system offers an opportunity to characterize the endocrine and paracrine function of primate follicles that influence follicle growth and oocyte maturation.

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The afterdischarge of Aplysia bag cell neurons has served as a model system for the study of phosphorylation-mediated changes in neuronal excitability. The nature of the depolarization generating the afterdischarge, however, has remained unclear. We now have found that venom from Conus textile triggers a similar prolonged discharge, and we have identified a slow inward current and corresponding channel, the activation of which seems to contribute to the onset of the discharge. The slow inward current is voltage-dependent and Ca(2+)-sensitive, reverses at potentials slightly positive to O mV, exhibits a selectivity of K approximately equal to Na >> Tris > N-methyl-D-glucamine (NMDG), and is blocked by high concentrations of tetrodotoxin. Comparison of these features with those observed in channel recordings provides evidence that a Ca(2+)-sensitive, nonspecific cation channel is responsible for a slow inward current that regulates spontaneous repetitive firing and suggests that modulation of the cation channel underlies prolonged changes in neuronal response properties.

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By mediating the Ca(2+) influx that triggers exocytotic fusion, Ca(2+) channels play a central role in a wide range of secretory processes. Ca(2+) channels consist of a complex of protein subunits, including an alpha(1) subunit that constitutes the voltage-dependent Ca(2+)-selective membrane pore, and a group of auxiliary subunits, including beta, gamma, and alpha(2)-delta subunits, which modulate channel properties such as inactivation and channel targeting. Subtypes of Ca(2+) channels are constituted by different combinations of alpha(1) subunits (of which 10 have been identified) and auxiliary subunits, particularly beta (of which 4 have been identified). Activity-secretion coupling is determined not only by the biophysical properties of the channels involved, but also by the relationship between channels and the exocytotic apparatus, which may differ between fast and slow types of secretion. Colocalization of Ca(2+) channels at sites of fast release may depend on biochemical interactions between channels and exocytotic proteins. The aim of this article is to review recent work on Ca(2+) channel structure and function in exocytotic secretion. We discuss Ca(2+) channel involvement in selected types of secretion, including central neurotransmission, endocrine and neuroendocrine secretion, and transmission at graded potential synapses. Several different Ca(2+) channel subtypes are involved in these types of secretion, and their function is likely to involve a variety of relationships with the exocytotic apparatus. Elucidating the relationship between Ca(2+) channel structure and function is central to our understanding of the fundamental process of exocytotic secretion.