Compartmentalization of calcium extrusion mechanisms in the outer and inner segments of photoreceptors - PubMed (original) (raw)
Compartmentalization of calcium extrusion mechanisms in the outer and inner segments of photoreceptors
D Krizaj et al. Neuron. 1998 Jul.
Abstract
Differential localization of calcium channel subtypes in divergent regions of individual neurons strongly suggests that calcium signaling and regulation could be compartmentalized. Region-specific expression of calcium extrusion transporters would serve also to partition calcium regulation within single cells. Little is known about selective localization of the calcium extrusion transporters, nor has compartmentalized calcium regulation within single neurons been studied in detail. Sensory neurons provide an experimentally tractable preparation to investigate this functional compartmentalization. We studied calcium regulation in the outer segment (OS) and inner segment/synaptic terminal (IS/ST) regions of rods and cones. We report these areas can function as separate compartments. Moreover, ionic, pharmacological, and immunolocalization results show that a Ca-ATPase, but not the Na+/K+, Ca2+ exchanger found in the OSs, extrudes calcium from the IS/ST region. The compartmentalization of calcium regulation in the photoreceptor outer and inner segments implies that transduction and synaptic signaling can be independently controlled. Similar separation of calcium-dependent functions is likely to apply in many types of neuron.
Figures
Figure 1. Calcium Extrusion Is Regulated Independently in Photoreceptor Inner and Outer Segments
Simultaneous measurements were made of the time courses of the spatially averaged [Ca2+]i in the IS and the OS of single rods. (A) A switch from control saline (2 mM KCl) to high KCl superfusate (90 mM KCl) raised [Ca2+]i in both segments. Immediately following high KCl, the superfusate was switched to Li+ saline (0 mM NaCl, 2 mM KCl, and 97 mM LiCl). The IS [Ca2+]i returned to baseline with a time course that was fitted with a single exponential of t = 29.9 s (upper trace). The OS [Ca2+]i remained at a plateau in LiCl saline; it returned to baseline upon return to control saline with t = 3.0 s (lower trace). (B) In another rod, control saline was replaced with Li+ saline. [Ca2+]i rose in the OS (lower trace) but remained unchanged in the IS. KCl elevated [Ca2+]i in both segments; again, the rise in the OS was slower. Upon switch to Li+, [Ca2+]i fell in the IS but rose further in the OS, an action we attribute to reversal of the Na+/K+, Ca2+ exchanger. Upon return to control saline, [Ca2+]i in the OS returned to baseline. [Ca2+]i was calibrated for both of these cells using ionomycin. Actual [Ca2+]i was estimated using Kd = 224 nM.
Figure 2. Spatiotemporal Dynamics of Calcium Changes in a Rod Photoreceptor
Sequential images of [Ca2+]i changes were recorded from a Fura 2–loaded rod. Between (a) and (b), the rod was surperfused with high KCl. The images in (b) and (c) were captured 3 and 21 s after KCl application, respectively. The image in (d) was captured 15 s after KCl was replaced by LiCl. The image in (e) was captured 7 s after the return to control saline. These images show that KCl-evoked increases in [Ca2+]i occurred most rapidly in the synaptic terminal region and then in the basal region of the inner segment. In LiCl, the IS and synaptic terminal returned to baseline while [Ca2+]i in the OS rose, most notably at the tip. The pseudocolor scale representing the 256 gray levels of the 340/380 ratios is shown on the bottom; red indicates the largest changes. Scale bar, 10 μm.
Figure 3. Antibodies to the Plasma Membrane Ca2+-ATPase Are Localized to the Inner Segments and Synaptic Terminals of Photoreceptors
(A) Immunostaining of radially cut section of tiger salamander retina. Nomarski DIC image (a) and anti-PMCA immunofluorescence (b) of retinal section are shown. The strongest labeling occurred at regions of synaptic contacts in the outer and inner plexiform layers. Photoreceptor inner segments were also stained, albeit not as strongly. Scale bar, 40 μm. (B) Example of an enzymatically dissociated cone labeled with a secondary antibody in the presence (a) and absence (b) of the primary PMCA antibody. The cells in (a) and (b) were plated and stained in parallel on concanavalin A–coated dishes. Scale bar, 10 μm.
Figure 4. Calcium Is Extruded from Photoreceptor Inner Segments by a Ca-ATPase
(A) The rod shown here was depolarized with sequential 1 s steps of 90 mM KCl. The cell was perfused with La3+-containing saline (1 mM) immediately following the second KCl step. After La3+, the superfusate was returned to control saline. That [Ca2+]i remained elevated during La3+ supports the idea that a Ca-ATPase is necessary for clearance of calcium following a calcium load. (B) The cone shown here was repeatedly stimulated by brief puffs of KCl. Trifluoperazine (4 μM), a calmodulin antagonist, was added to the superfusate after the second KCl puff. Subsequent transient calcium increases to KCl declined, and the resting [Ca2+]i rose in a step-like manner after each puff. (C) Effects of KCl on intracellular pH in a cone. The cell was loaded with 5 μM BCECF-AM and then stimulated with two sequential steps of KCl. The first step was applied in control superfusate containing 0 Ca/3 mM EGTA. There was no effect on baseline pH. A subsequent exposure to 90 mM KCl in control saline resulted in an acidification. pH changes were calibrated using nigericin.
References
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