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Fogel and Hastings (1972, P.N.A.S. USA 69:690-693) first hypothesized the existence of voltage gated proton channels in bioluminescent dinoflagellates, where they were postulated to trigger the flash. The flash originates in discrete organelles termed scintillons (Nicolas et al., 1987 J. Cell Biol. 105: 723-735.) that contain luciferase and its substrate, luciferin. Luciferase is activated, and luciferin is released from a binding protein, at low pH. Proton channels in the membranes of the scintillon were postulated to conduct H+ from the acidic central flotation vacuole into the scintillon, activating luciferase and releasing luciferin, causing a bioluminescent flash. We have recently cloned a voltage gated proton channel (kHV1) from a non-bioluminescent dinoflagellate, Karlodinium veneficum (Smith et al., 2011 P.N.A.S.in-press). In heterologous expression systems, this channel exhibits a negative Vthreshold so that inward current is activated, consistent with the proposed role of allowing proton flux from the vacuole into the scintillon. The H+ current of heterologously expressed kHV1 is inhibited by Zn2+, but at much higher concentrations than in mammalian channels. We have developed a method to isolate functional scintillons from the bioluminescent species Noctiluca miliaris through gentle homogenization and sucrose density gradient centrifugation. The density of the scintillon fraction was found to be 1.213 g/cm3. Reducing the pH of the scintillon suspension to below pH 6 elicits a luminescent flash. Addition of divalent metals to the suspension diminishes the intensity of the flash. Zn2+, Ni2+, Cu2+, Co2+, and Cd2+ at millimolar concentrations abolished the luminescence from isolated scintillons. The sensitivity of scintillon luminescence to divalent metals is consistent with the idea that proton selective channels in the scintillon membrane mediate the activation of the flash. |
