Vol. 61, Issue 1, 43-54, January 2002

A Novel Class of Peptides with Facilitating Action on Neuronal Nicotinic Receptors of Rat Chromaffin Cells in Vitro: Functional and Molecular Dynamics Studies

Silvia Di Angelantonio, Valeria Costa, Paolo Carloni, Luigi Messori, and Andrea Nistri

Biophysics (S.D.A., V.C., A.N.) and Condensed Matter Sectors (P.C.) and Istituto Nazionale per la Fisica della Materia Unit (S.D.A., V.C., P.C., A.N.), International School for Advanced Studies, Trieste, Italy; and Department of Chemistry, University of Florence, Firenze, Italy (L.M.)

Peptides related to the N-terminal region of calcitonin gene-related peptide (CGRP) were tested for their ability to modulate neuronal nicotinic acetylcholine receptors (nAChRs) of rat cultured chromaffin cells under whole cell patch-clamp conditions. Although CGRP_1-7 and CGRP_2-7 depressed responses mediated by nAChRs, CGRP_1-6, CGRP_1-5, or CGRP_1-4 rapidly and reversibly potentiated submaximal nicotine currents while sparing maximal currents. CGRP_1-3 was inactive. The threshold concentration for the enhancing effect of CGRP_1-6 was 0.1 µM. CGRP_1-5 or CGRP_1-4 were less effective than CGRP_1-6. Coapplication of CGRP_1-6 and of the allosteric potentiator physostigmine (0.5 µM) gave additive effects on nicotine currents. CGRP_1-6 did not enhance responses generated by muscle-type nicotinic receptors of cultured myoblasts or by -aminobutyric acid_A receptors expressed by human embryonic kidney cells. Molecular dynamics (MD) simulations suggested that CGRP_1-7 exhibited a relatively rigid ring structure imparted by the disulfide bridge between Cys₂ and Cys₇. The circular dichroism (CD) spectrum recorded from the same peptide was in agreement with this result. Shorter peptides, missing such a bridge, exhibited propensity for -helix configuration. Replacing Cys₇ with Ala yielded CGRP_1-7A, a fragment with partial -helix structure and ability to enhance nicotine currents. CD measurements on CGRP_1-6 were compatible with these MD structural findings. Short terminal fragments of CGRP represent a novel class of substances with selective, rapid, and reversible potentiation of nAChRs.