Dynamics and calcium sensitivity of the Ca²⁺/myristoyl switch protein hippocalcin in living cells

Hippocalcin is a neuronal calcium sensor protein that possesses a Ca²⁺/myristoyl switch allowing it to translocate to membranes. Translocation of hippocalcin in response to increased cytosolic [Ca²⁺] was examined in HeLa cells expressing hippocalcin–enhanced yellow fluorescent protein (EYFP) to determine the dynamics and Ca²⁺ affinity of the Ca²⁺/myristoyl switch in living cells. Ca²⁺-free hippocalcin was freely diffusible, as shown by photobleaching and use of a photoactivable GFP construct. The translocation was dependent on binding of Ca²⁺ by EF-hands 2 and 3. Using photolysis of NP-EGTA, the maximal kinetics of translocation was determined (t_1/2 = 0.9 s), and this was consistent with a diffusion driven process. Low intensity photolysis of NP-EGTA produced a slow [Ca²⁺] ramp and revealed that translocation of hippocalcin–EYFP initiated at around 180 nM and was half maximal at 290 nM. Histamine induced a reversible translocation of hippocalcin–EYFP. The data show that hippocalcin is a sensitive Ca²⁺ sensor capable of responding to increases in intracellular Ca²⁺ concentration over the narrow dynamic range of 200–800 nM free Ca²⁺.