Sheremet Y. Expression and Spatial Distribution of the Neuronal Calcium Sensor Protein Hippocalcin in Subcellular Compartments

Hippocalcin (HPCA) is a neuronal calcium sensor (NCS) protein that provides intracellular signaling via its Ca2+-dependent translocation from the cytosol to the plasma membrane. Although such translocation of HPCA and some NCS proteins is wellestablished, no quantitative estimates of HPCA distribution between the cytosol and plasma membrane at a basal level of free intracellular calcium concentration ([Ca2+]i) have been obtained. Thus, it is still unknown whether HPCA regulates its plasma membrane targets under resting conditions. Moreover, exogenously expressed fluorescently-labeled proteins, widely used for biophysical studies, could probably shift endogenous proteins signaling. That’s why the main issues in this study is the estimation of the expression level of the 23 exogenous HPCA construct, compared to that of the corresponding endogenous one, and quantitative estimates of HPCA distribution between the cytosol and plasma membrane at a basal level [Ca2+]i. In this work, we propose a simple and universal approaches for such estimations. The approach for measurement of expression level is based upon common knowledge that the dye fluorescence is directly proportional to its quantum yield and the number of its molecules and that a coefficient of proportionality is determined by spectral properties of the dye and optical equipment used to record fluorescent signals. If two fluorescent dyes are present in the same volume, then a ratio of their concentrations is equal to a ratio of their fluorescence multiplied by some dye- and equipment-dependent coefficient. Thus, if the coefficient and concentration of one dye is known then the concentration of another dye can be determined. Here we have demonstrated how to calculate this coefficient (called a ratio factor) and how to use it for concentration measurements of fluorescently tagged molecules. Using this approach, we estimated a concentration of exogenously expressed HPCA, tagged by a fluorescent protein in a dendritic tree of rat hippocampal neurons loaded via a patch pipette with Alexa Fluor dye of known concentration. We have demonstrated that HPCA expression level (10 μM) in the hippocampal neurons under study is at least 3 times lower compared to the concentration of endogenous one. Thus, the exogenous protein may be used as a tool to visualize HPCA distribution, translocation and target interaction without substantial perturbation of endogenous HPCA signaling. Next, we have evaluated HPCA spatial distribution in living cells at a basal level of [Ca2+]i comparing this distribution with one of exogenously expressed membrane protein, EYFP-Mem. Using fluorescence recovery after photobleaching, we showed that the diffusion coefficient of fluorescently tagged HPCA in the dendrites of the hippocampal neurons (~40 μm2 /s) is within a range of values typical for cytosolic proteins. The coefficient was about 10-fold higher than that for EYFP-Mem. Both results indicate that HPCA is mainly localized in the cytosol of the dendrites of hippocampal neurons. Subsequent direct calculations for HPCA fractions in the cytosol and plasma membrane at a basal level of [Ca2+]i demonstrated that the plasma membrane fraction of HPCA does not exceed 8%. Altogether, our results suggest that although being mainly cytosolic, HPCA may potentially signal to its plasma membrane targets under resting conditions. New approaches, used for obtaining such estimates, can be applied for quantitative evaluation expression level of NCS protein and its spatial distribution in living cells under different experimental conditions and for development of precise biophysical models of their signaling.