Structural, Biochemical, and Functional Characterization of the Calcium Sensor Neurocalcin δ in the Inner Retinal Neurons and Its Linkage with the Rod Outer Segment Membrane Guanylate Cyclase Transduction System

Abstract

This study documents the detailed biochemical, structural, and functional identity of a novel Ca(2+)-modulated membrane guanylate cyclase transduction system in the inner retinal neurons. The guanylate cyclase is the previously characterized ROS-GC1 from the photoreceptor outer segments (PROS), and its new modulator is neurocalcin delta. At the membrane, the myristoylated form of neurocalcin delta senses submicromolar increments in free Ca(2+), binds to its specific ROS-GC1 domain, and stimulates the cyclase. Neurocalcin delta is not present in PROS, indicating the absence of the pathway in the outer segments and the dissociation of its linkage with phototransduction. Thus, the pathway is linked specifically with the visual transduction machinery in the secondary neurons of the retina. With the inclusion of this pathway, the findings broaden the understanding of the existing mechanisms showing how ROS-GC1 is able to receive and transduce diverse Ca(2+) signals into the cell-specific generation of second-messenger cyclic GMP in the retinal neurons.

S100B‐modulated Ca2+‐dependent ROS‐GC1 transduction machinery in the gustatory epithelium: a new mechanism in gustatory transduction

“…4B, lane ''-Ca 2þ ''). This is probably due to the presence of the residual Ca 2þ concentration in the membranes, as has been observed earlier for the neurocalcin d/ROS-GC1 complex in the inner plexiform layer of the retinal neurons [26] and for the neurocalcin d/ONE-GC complex in the cilia of the olfactory neurons [36]. Another possibility is that once S100B binds ROS-GC1, it remains bound to the cyclase even in the absence of Ca 2þ as has been observed with other intracellular targets of S100B [43,44].…”

“…Both ROS-GCs in the 10-50 nM free Ca 2þ range are stimulated and in the semimicro to the micromolar range are inhibited by GCAPs (reviewed in: [34,35]). In the semi to the micromolar range of free Ca 2þ , both are stimulated by S100B, and ROS-GC1 also by neurocalcin d (reviewed in: [26,28,34]). ONE-GC is only stimulated, and not inhibited, by free Ca 2þ when accompanied by neurocalcin d [36,37].…”

“…GCAP1, GCAP2, and neurocalcin d were cloned, expressed, and purified as described previously [25][26][27]. S100B was obtained commercially (Sigma Chemical Co).…”

“…Membranes were assayed for guanylate cyclase activity as described previously [26,28,[30][31][32]. The amount of cyclic GMP formed was determined by radioimmunoassay [33].…”

“…The experiments were done according to the previously developed protocol [26,28,31]. To validate the specificity of the antibody, Western blot analysis of COS cell membranes expressing ROS-GC1, ROS-GC2 and ONE-GC was performed.…”