Endogenous Ca2+ buffer concentration and Ca2+ microdomains in hippocampal neurons.
Ca2+-binding proteins are ubiquitously expressed throughout the CNS and serve as valuable immunohistochemical markers for certain types of neurons. However, the functional role of most Ca2+-binding proteins has to date remained obscure because their concentration in central neurons is not known. In this study, we investigate the intracellular concentration of the widely expressed Ca2+-binding protein calbindin-D28k in adult hippocampal slices using patch-clamp recordings and immunohistochemistry. First, we show that calbindin-D28k freely exchanges between patch pipette and cytoplasm during whole cell patch-clamp recordings with a time constant of approximately 10 min. Substituting known concentrations of recombinant calbindin-D28k in patch pipettes enabled us to determine the endogenous calbindin-D28k concentration by postrecording immunohistochemistry. Using this calibration procedure, we find that mature granule cells (doublecortin-) contain approximately 40 microm, and newborn granule cells (doublecortin+) contain 0-20 microm calbindin-D28k. CA3 stratum radiatum interneurons and CA1 pyramidal cells enclose approximately 47 and approximately 45 microm calbindin-D28k, respectively. Numerical simulations showed that 40 microm calbindin-D28k is capable of tuning Ca2+ microdomains associated with action potentials at the mouth of single or clustered Ca2+ channels: calbindin-D28k reduces the increment in free Ca2+ at a distance of 100 and 200 nm by 20 and 35%, respectively, and strongly accelerates the collapse of the Ca2+ gradient after cessation of Ca2+ influx. These data suggest that calbindin-D28k equips hippocampal neurons with approximately 160 microm mobile, high-affinity Ca2+-binding sites (kappa(S) approximately 200) that slow and reduce global Ca2+ signals while they enhance the spatiotemporal fidelity of submicroscopic Ca2+ signals.
Müller
Andreas
A
Department of Neurosurgery, University Clinic Bonn, D-53105 Bonn, Germany.
Kukley
Maria
M
Stausberg
Pia
P
Beck
Heinz
H
Müller
Wolfgang
W
Dietrich
Dirk
D
D000375:Aging / Q000378:metabolism; D000818:Animals; D064092:Calbindin 1; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D003956:Dialysis; D000089662:Doublecortin Protein; D005455:Fluorescent Antibody Technique; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D066298:In Vitro Techniques; D007395:Interneurons / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000378:metabolism* / Q000502:physiology; D009474:Neurons / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D017966:Pyramidal Cells / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D064030:S100 Calcium Binding Protein G / Q000378:metabolism* / Q000502:physiology
C576499:Calb1 protein, mouse; C576500:Calb1 protein, rat; D064092:Calbindin 1; D064026:Calbindins; D015220:Calcium Channels; C000714848:Dcx protein, rat; D000089662:Doublecortin Protein; D009419:Nerve Tissue Proteins; D064030:S100 Calcium Binding Protein G; D002118:Calcium