TY - JOUR
T1 - Optical control of calcium-regulated exocytosis
AU - Izquierdo-Serra, Mercè
AU - Trauner, Dirk
AU - Llobet, Artur
AU - Gorostiza, Pau
N1 - Funding Information:
We are grateful to Ehud Y. Isacoff for the gift of the GluK2-L439C-eGFP clone, to M. A. Valverde for the Ca v 2.1 clone, to C. Manzo and I. Rimmaudo for the help with data processing, and to M. Segovia and G. Álvarez de Toledo for the help with amperometric recordings. We are indebted to M. Ruíz-Mejías for performing initial experiments, and to C. Solsona, G. Álvarez de Toledo and E. Y. Isacoff for helpful discussions and comments on the manuscript. We acknowledge financial support from the Human Frontier Science Program through a Career Development Award, from the European Research Council through a Starting Grant, from the European Commission through a ICT-FET grant, from the Ministry of Education through a grant (to P.G.) and through a FPU fellowship (to M.I.), from CIBER-BBN through a young investigator fellowship (to M.I.) and from the RecerCaixa foundation .
PY - 2013/3
Y1 - 2013/3
N2 - Background Neurons signal to each other and to non-neuronal cells as those in muscle or glands, by means of the secretion of neurotransmitters at chemical synapses. In order to dissect the molecular mechanisms of neurotransmission, new methods for directly and reversibly triggering neurosecretion at the presynaptic terminal are necessary. Here we exploit the calcium permeability of the light-gated channel LiGluR in order to reversibly manipulate cytosolic calcium concentration, thus controlling calcium-regulated exocytosis. Methods Bovine chromaffin cells expressing LiGluR were stimulated with light. Exocytic events were detected by amperometry or by whole-cell patch-clamp to quantify membrane capacitance and calcium influx. Results Amperometry reveals that optical stimulation consistently triggers exocytosis in chromaffin cells. Secretion of catecholamines can be adjusted between zero and several Hz by changing the wavelength of illumination. Differences in secretion efficacy are found between the activation of LiGluR and native voltage-gated calcium channels (VGCCs). Our results show that the distance between sites of calcium influx and vesicles ready to be released is longer when calcium influx is triggered by LiGluR instead of native VGCCs. Conclusion LiGluR activation directly and reversibly increases the intracellular calcium concentration. Light-gated calcium influx allows for the first time to control calcium-regulated exocytosis without the need of applying depolarizing solutions or voltage clamping in chromaffin cells. General significance LiGluR is a useful tool to study the secretory mechanisms and their spatiotemporal patterns in neurotransmission, and opens a window to study other calcium-dependent processes such as muscular contraction or cell migration.
AB - Background Neurons signal to each other and to non-neuronal cells as those in muscle or glands, by means of the secretion of neurotransmitters at chemical synapses. In order to dissect the molecular mechanisms of neurotransmission, new methods for directly and reversibly triggering neurosecretion at the presynaptic terminal are necessary. Here we exploit the calcium permeability of the light-gated channel LiGluR in order to reversibly manipulate cytosolic calcium concentration, thus controlling calcium-regulated exocytosis. Methods Bovine chromaffin cells expressing LiGluR were stimulated with light. Exocytic events were detected by amperometry or by whole-cell patch-clamp to quantify membrane capacitance and calcium influx. Results Amperometry reveals that optical stimulation consistently triggers exocytosis in chromaffin cells. Secretion of catecholamines can be adjusted between zero and several Hz by changing the wavelength of illumination. Differences in secretion efficacy are found between the activation of LiGluR and native voltage-gated calcium channels (VGCCs). Our results show that the distance between sites of calcium influx and vesicles ready to be released is longer when calcium influx is triggered by LiGluR instead of native VGCCs. Conclusion LiGluR activation directly and reversibly increases the intracellular calcium concentration. Light-gated calcium influx allows for the first time to control calcium-regulated exocytosis without the need of applying depolarizing solutions or voltage clamping in chromaffin cells. General significance LiGluR is a useful tool to study the secretory mechanisms and their spatiotemporal patterns in neurotransmission, and opens a window to study other calcium-dependent processes such as muscular contraction or cell migration.
KW - Calcium
KW - Exocytosis
KW - Light-gated glutamate receptor (LiGluR)
KW - Neurotransmission
KW - Optical control
KW - Optogenetics
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U2 - 10.1016/j.bbagen.2012.11.003
DO - 10.1016/j.bbagen.2012.11.003
M3 - Article
C2 - 23178861
AN - SCOPUS:84873043621
SN - 0304-4165
VL - 1830
SP - 2853
EP - 2860
JO - Biochimica et Biophysica Acta - General Subjects
JF - Biochimica et Biophysica Acta - General Subjects
IS - 3
ER -