N-TERMINAL DOMAINS OF NEUROCALCIN 8 AND HIPPOCALCIN DETERMINE DIFFERENCE IN THEIR Ca²⁺-DEPENDENT SIGNALING

Hippocalcin (HPCA) and Neurocalcin 8 (NCALD) are the members of neuronal Ca²⁺-sensor (NCS) protein family. In spite of minor distinctions in AA sequence between HPCA and NCALD, these proteins demonstrate a substantial difference in their Ca²⁺-sensitivity and kinetic of translocation to the plasma membrane. Both proteins contain myristoilated N-terminal region responsible for interaction with cellular membrane and 3 EF-hand domains responsible for binding of Ca²⁺. Here we compared translocation of wild type NCALD with NCALD-HPCA chimera protein, NCHC, consisting of the first N-terminal region of NCALD and three other domains from HPCA (three Ca²⁺-binding EF-hands). We suggested that NCHC translocation to the plasma membrane would tend to be HPCA-like if cytosolic Ca²⁺ affinity is important for protein signaling, and NCALD-like if stabilization of Ca²⁺-bound form on the membrane plays a crucial role. NCALD and NCHC tagged by different fluorescent proteins were co-expressed in cultured rat hippocampal neurons. Protein translocation to the plasma membrane was induced by membrane depolarization resulting in fast [Ca²⁺]_i transients and was assayed by fluorescent measurements in dendrites of hippocampal neurons. We have demonstrated that NCALD and NCHC had very similar translocation kinetics and Ca²⁺-sensitivity, whereas NCALD and HPCA had substantial difference in these biophysical parameters. We conclude that the difference in signaling between NCALD and HPCA may be attributed to the distinctions of their N-terminal domains.