Stable expression in HEK-293 cells of the rat alpha3/beta4 subtype of neuronal nicotinic acetylcholine receptor.

The alpha3/beta4 subtype of neuronal nicotinic acetylcholine receptor (nAChR) was stably expressed in human embryonic kidney (HEK) 293 cells that co-expressed a voltage-gated Ca2+ channel. alpha3/beta4-nAChR-expressing clones were identified using the fura-2 Ca2+ imaging technique, and were further characterised by single-cell and whole-cell patch-clamp studies. Acetylcholine (ACh) induced fast activating currents which showed desensitisation and inward rectification. The conductance of the ACh-activated channel was 29 pS. The order of potency of the nicotinic agonists tested was cytisine approximately = nicotine > acetylcholine. The EC50 value for ACh was 145 microM; the Hill coefficient was close to 2. The currents elicited by ACh were effectively blocked by nicotinic antagonists, but not by the muscarinic antagonist atropine. These properties are comparable to the pharmacological and physiological profile of ganglionic nicotinic receptors and type III currents of cultured hippocampal neurons.

The C-terminal bisphosphorylated proenkephalin-A-(209-237)-peptide from adrenal medullary chromaffin granules possesses antibacterial activity.

The chromaffin granules have been shown to be an excellent model to study the processing of proenkephalin-A and chromogranins. Recently, we reported a study dealing with the processing of chromogranin B/secretogranin I and the occurrence of the C-terminal chromogranin B-derived peptide 614-626 which was shown to have antibacterial activity [Strub, J.M., Garcia-Sablone, P., Looning, K., Taupenot, L., Hubert, P., Van Dorsselaer, A., Aunis, D. & Metz-Boutigue, M.H. (1995) Eur. J. Biochem. 229, 356-368]. We also observed that this new antibacterial activity present in chromaffin granules was associated with other endogenous protein-derived fragments yet to be characterized. The present study reports the isolation and characterization of a peptide which possesses antibacterial activity and which corresponds to the C-terminal 209-237 sequence of proenkephalin-A. A detailed study using microsequencing and matrix-assisted-laser-desorption time-of-flight mass spectrometry (MALD-TOF MS) allowed us to correlate the antibacterial activity of this peptide named enkelytin (FAEPLPSEEEGESYSKEVPEMEKRYGGFM) with post-translational modifications. Endogenous bisphosphorylated proenkephalin-A-(209-237) was active on Micrococcus luteus and Bacillus megaterium killing bacteria in the 0.2 - 0.4 microM range but was inactive in similar conditions towards Escherichia coli. Enkelytin shares sequence and structural similarities with the antibacterial C-terminal domain of diazepam-binding inhibitor. According to this similarity, a prediction of secondary structure is proposed for enkelytin and discussed in relationship to its biological activity.

Evidence for a phosphorylation-induced conformational change in phospholamban cytoplasmic domain by CD analysis.

Phospholamban (PLB), an integral membrane protein of cardiac sarcoplasmic reticulum (SR), is described as the regulator of the Ca(2+)-ATPase pump, via its phosphorylation-dephosphorylation of Ser-16. Recently it has been shown that a direct interaction between the N-terminal hydrophilic domain of PLB and Ca(2+)-ATPase may be one of the mechanisms of regulation. In order to show that this interaction could be modulated by a phosphorylation-induced conformational change in PLB, we ran CD studies on the synthetic peptide PLB(2-33) in its phosphorylated and non-phosphorylated forms, at various pHs, concentrations and in the absence or presence of trifluoroethanol. The results show a clear difference in structure of the phosphorylated and non-phosphorylated peptide.