Abstract
Rod photoreceptors are composed of a soma and an inner segment (IS) connected to an outer segment (OS) by a thin cilium. OSs are composed of a stack of ∼800 lipid discs surrounded by the plasma membrane where phototransduction takes place. Intracellular calcium plays a major role in phototransduction and is more concentrated in the discs, where it can be incorporated and released. To study calcium dynamics in rods, we used the fluorescent calcium dye CaSiR-1 AM working in the near-infrared (NIR) (excitation at 650 and emission at 664 nm), an advantage over previously used dyes. In this way, we investigated calcium dynamics with an unprecedented accuracy and most importantly in semidark-adapted conditions. We observed light-induced drops in [Ca(2+)](i) with kinetics similar to that of photoresponses recorded electrophysiologically. We show three properties of the rods. First, intracellular calcium and key proteins have concentrations that vary from the OS base to tip. At the OS base, [Ca(2+)](i) is ∼80 nM and increases up to ∼200 nM at the OS tip. Second, there are spontaneous calcium flares in healthy and functional rod OSs; these flares are highly localized and are more pronounced at the OS tip. Third, a bright flash of light at 488 nm induces a drop in [Ca(2+)](i) at the OS base but often a flare at the OS tip. Therefore, rod OSs are not homogenous structures but have a structural and functional gradient, which is a fundamental aspect of transduction in vertebrate photoreceptors.