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+.
O'Callaghan
Dermott W
DW
Physiological Laboratory, University of Liverpool, Liverpool L69 3BX, England, UK.
Tepikin
Alexei V
AV
Burgoyne
Robert D
RD
D001426:Bacterial Proteins; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D006367:HeLa Cells; D051597:Hippocalcin; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D009474:Neurons / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology*; D011993:Recombinant Fusion Proteins
D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D002614:Chelating Agents; C496691:HPCA protein, human; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D051597:Hippocalcin; D006632:Histamine; D002118:Calcium