Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels and cAMP-Dependent Modulation of Exocytosis in Cultured Rat Lactotrophs
- Ana I. Calejo1,2,*,
- Jernej Jorgačevski2,4,*,
- Boštjan Rituper2,
- Alenka Guček2,
- Patrícia M. Pereira1,
- Manuel A.S. Santos1,
- Maja Potokar2,4,
- Nina Vardjan2,4,
- Marko Kreft2,3,4,
- Paula P. Gonçalves1, and
- Robert Zorec2,4
- 1Centre for Environmental and Marine Studies & Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro, Portugal,
- 2Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, and
- 3Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia, and
- 4Celica Biomedical Center, 1000 Ljubljana, Slovenia
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Author contributions: A.I.C., J.J., B.R., A.G., P.M.P., M.A.S.S., P.P.G., and R.Z. designed research; A.I.C., J.J., B.R., A.G., and P.M.P. performed research; M.K. contributed unpublished reagents/analytic tools; A.I.C., J.J., B.R., P.M.P., and M.P. analyzed data; A.I.C., J.J., B.R., A.G., P.M.P., M.A.S.S., M.P., N.V., P.P.G., and R.Z. wrote the paper.
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↵*A.I.C. and J.J. contributed equally to this work.
Abstract
Hormone and neurotransmitter release from vesicles is mediated by regulated exocytosis, where an aqueous channel-like structure, termed a fusion pore, is formed. It was recently shown that second messenger cAMP modulates the fusion pore, but the detailed mechanisms remain elusive. In this study, we asked whether the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are activated by cAMP, are involved in the regulation of unitary exocytic events. By using the Western blot technique, a real-time PCR, immunocytochemistry in combination with confocal microscopy, and voltage-clamp measurements of hyperpolarizing currents, we show that HCN channels are present in the plasma membrane and in the membrane of secretory vesicles of isolated rat lactotrophs. Single vesicle membrane capacitance measurements of lactotrophs, where HCN channels were either augmented by transfection or blocked with an HCN channel blocker (ZD7288), show modulated fusion pore properties. We suggest that the changes in local cation concentration, mediated through HCN channels, which are located on or near secretory vesicles, have an important role in modulating exocytosis.
- Received December 18, 2013.
- Revision received September 30, 2014.
- Accepted October 6, 2014.
- Copyright © 2014 the authors 0270-6474/14/3315638-10$15.00/0
