Cherkas V. Hippocalcin cellular signaling in hippocampal neurons

Hippocalcin is a Ca2+-binding protein, which belongs to the family of neuronal Ca2+ sensors. It is highly expressed in the hippocampus but physiological role and molecular mechanisms underlying its action in this part of the brain have not been investigated in detail. To study whether intrinsic neuronal activity could result in hippocalcin-mediated signal transduction we examined spontaneous and action potential (AP)-dependent changes in fluorescence of Yellow Fluorescent Protein-tagged Hippocalcin (HPCA-YFP) in transiently transfected hippocampal cultured neurons. In 6-12 DIV neurons HPCA-YFP spontaneously translocates longitudinally both in dendrites and axons to certain sites of 1-5 ?m in length from neighboring diffusionally confined segments of neuronal processes. The translocation was expressed as a fast (1-5 sec) reversible increase in HPCA-YFP fluorescence in these sites coincided with a decrease in adjacent sites indicating genuine protein translocation. Both synchronous in remotely located sites, and asynchronous translocations of HPCA-YFP were observed in the same neuron. Physiologically relevant neuronal stimulation with short AP bursts also resulted in fast (1-3 sec), reversible, and [Ca2+]i-dependent translocation of HPCA-YFP to several sites synchronizing hippocalcin signalling in different segments of neuronal processes. The amount of translocated protein increased linearly with the number of action potentials in a burst, indicating that hippocalcin can serve as a local integrator of short-term neuronal activity.Hippocampal neurons within neuronal networks reveal network-driven activity leading to a complex spatio-temporal patterns of [Ca2+]i changes. We showed that these changes have to result in continuous hippocalcin transitions between Ca2+ free and bound states and, as a result, in its different partitioning between cytosol and membranes. To investigate this hypothesis we have examined spontaneous and stimulus-dependent translocation of EYFP-tagged hippocalcin to subcellular targets in transfected hippocampal neurons growing in low-density cultures. We have demonstrated that hippocalcin is a very fast and sensitive Ca2+ sensor, which is able to detect a few action potentials and may participate in local integration of neuronal activity. FRET approach was used to measure spatio-temporal pattern of hippocalcin insertion to the plasma membrane during translocation. We found some specific sites on the dendritic plasma membrane, sized from diffractionally limited to several microns, where local hippocalcin insertion to the plasma membrane was higher than in neighbouring sites. We checked whether translocation at these specific sites are associated with regions of higher intracellular free calcium concentration ([Ca2+]i). Creating spatially uniform [Ca2+]i transients in dendritic segments, we showed that hippocalcin translocation was significantly different in neighbouring sites having the same (in terms of kinetics and amplitude) patterns of [Ca2+]i changes. Producing long-lasting elevations of [Ca2+]i by activation of different Ca2+ mobilizing mechanisms, we also demonstrated that hippocalcin translocation was observed in the same set of sites independently of Ca2+ sources. These results indicate that [Ca2+]i is not the only determinant of hippocalcin translocation and that local differences in the plasma membrane affinity for hippocalcin are an important biophysical mechanism of hippocalcin signalling. Furthermore we have also developed original approaches for quantitative separate and simultaneous measurement of hippocalcin concentration in cytosolic and membrane cellular fractions of single living hippocampal neurons. Based on these approaches and simulation of Ca2+ and hippocalcin diffusion in the dendrites and spines of hippocampal neurons we have shown that hippocalcin concentration in dendritic membranes can undergo substantial local increase during intrinsic patterns of neuronal activity. We conclude that hippocalcin plays a very significant role in intracellular signaling as calcium buffer and sensor that is capable of local signal integration. Keywords:Са2+-signaling, NeuronalCalciumSensors, hippocalcin, fluorescent proteins, [Са2+]imeasurement, FRET.