The cone-specific calcium sensor guanylate cyclase activating protein 4 from the zebrafish retina

摘要

Guanylate cyclase activating proteins (GCAPs) serve as neuronal Ca2+-sensor proteins in vertebrate rod and cone photoreceptor cells. Zebrafish express in their retina a variety of six different GCAPs, of which four are specific for cone cells. One isoform, zGCAP4, is mainly expressed in double cones and long single cones. We cloned the zGCAP4 gene, purified non-myristoylated and myristoylated forms of the protein after heterologous expression in Escherichia coli and studied its properties: zGCAP4 was a strong activator of membrane-bound guanylate cyclases from bovine and zebrafish retina, showing half-maximal activation at 520-570 nM free Ca2+ concentration. Furthermore, the Ca2+-sensitive activation properties of non-myristoylated and myristoylated zGCAP4 were similar, indicating no influence of the myristoyl moiety on Ca2+-sensor function. Myristoylated zGCAP4 showed low affinity for membranes and did not exhibit a Ca2+-myristoyl switch, a feature typical of some but not all neuronal Ca2+-sensor proteins. However, tryptophan fluorescence studies and Ca2+-dependent differences in protease accessibility revealed Ca2+-induced conformational changes in myristoylated and non-myristoylated zGCAP4, indicating the operation as a Ca2+ sensor. Thus, expression and biochemical properties of zGCAP4 are in agreement with its function as an efficient Ca2+-sensitive regulator of guanylate cyclase activity in cone vision.