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 (t1/2 = 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+

High-affinity interaction of the N-terminal myristoylation motif of the neuronal calcium sensor protein hippocalcin with phosphatidylinositol 4,5-bisphosphate

Many proteins are associated with intracellular membranes due to their N-terminal myristoylation. Not all myristoylated proteins have the same localization within cells, indicating that other factors must determine their membrane targeting. The NCS (neuronal calcium sensor) proteins are a family of Ca(2+)-binding proteins with diverse functions. Most members of the family are N-terminally myristoylated and are either constitutively membrane-bound or have a Ca(2+)/myristoyl switch that allows their reversible membrane association in response to Ca(2+) signals. In the case of hippocalcin and NCS-1, or alternatively KChIP1 (K(+) channel-interacting protein 1), their N-terminal myristoylation motifs are sufficient for targeting to distinct organelles. We have shown that an N-terminal myristoylated hippocalcin peptide is able to specifically reproduce the membrane targeting of hippocalcin/NCS-1 when introduced into permeabilized cells. The peptide binds to liposomes containing phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] with high affinity (K(d) 50 nM). Full-length hippocalcin also bound preferentially to liposomes supplemented with PtdIns(4,5)P(2). Co-expression of hippocalcin-(1–14)–ECFP (enhanced cyan fluorescent protein) or NCS-1–ECFP partially displaced the expressed PH (pleckstrin homology) domain of phospholipase δ1 from the plasma membrane in live cells, indicating that they have a higher affinity for PtdIns(4,5)P(2) than does this PH domain. The Golgi localization of the PH domain of FAPP1 (four-phosphate-adaptor protein 1), which binds to phosphatidylinositol 4-phosphate, was unaffected. The localization of NCS-1 and hippocalcin is likely to be determined, therefore, by their interaction with PtdIns(4,5)P(2)