Influx into Fly Photoreceptors

Ion-selective calcium microelectrodes were inserted into the compound eyes of the wild-type sheep blowfly Lucilia or into the retina of the no steady state (ms) mutant of Lucilia. These electrodes monitored light-induced changes in the extracellular concentration of calcium (A[Ca2+]o) together with the extracellularly recorded receptor potential. Prolonged dim lights induced a steady reduction in [Ca2+]o during light in the retina of normal Lucilia, while relatively litde change in [Ca2+]o was observed in the retina of the nss mutant. Prolonged intense light induced a multiphasic change in [Ca~+]o: the [Ca~+]o signal became transient, reaching a minimum within 6 s after light onset, and then rose to a nearly steady-state phase below the dark concentration. When lights were turned off, a rapid increase in [Ca2+]o was observed, reaching a peak above the dark level and then declining again to the dark level within 1 rain. In analogy to similar studies conduced in the honeybee drone, we suggest that the reduction in [Ca2÷]o reflects light-induced Ca 2÷ influx into the photoreceptors, while the subsequent increase in [Ca~÷]o reflects the activation of the Na-Ca exchange which extrudes Ca ~+ from the cells. In the ms mutant in response to intense prolonged light, the receptor potential declines to baseline during light while the Ca 2+ signal is almost abolished, revealing only a short transient reduction in [Ca~+]o. Application of lanthanum (LaSt), but not nickel (Ni2+), into the retinal extracellular space of normal Lucilia mimicked the effect of the ms mutation on the receptor potential, while complete elimination of the Ca ~+ signal in a reversible manner was observed. The results suggest that La 3+ and the ms mutation inhibit light-induced Ca ~+ influx into the photoreceptor in a manner similar to the action of the trp mutation in Drosophila, which has been shown to block specifically a light-activated Ca 2+ channel necessary to maintain light excitation. I N T R O D U C T I O N T he c o m p o u n d eye o f the fly has been an impor tant model system for studies of invertebrate phototransduct ion, particularly because of the available phototransducAddress reprint requests to Dr. Baruch Minke, Department of Physiology, The Hebrew UniversityHadassah Medical School, Jerusalem 91010, Israel. J. GEN. PHYSIOL. © The Rockefeller University Press 0022-1295/92/I 1/0767/15 $2.00 Volume 100 November 1992 767-781 767 on N ovem er 6, 2017 jgp.rress.org D ow nladed fom 768 T H E JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 . 1992 tion-defective mutants (for reviews, see Pak, 1979, 1991; Selinger and Minke, 1988; Montell, 1989; Ranganathan, Harris, and Zuker, 1991b; Minke and Selinger, 1992a). In both the transient receptor potential (trp) mutant of Drosophila (Cosens and Manning, 1969; Minke, Wu, and Pak, 1975; Lo and Pak, 1981; Minke, 1982; Montell, Jones, Hafen, and Rubin, 1985; Montell and Rubin, 1989; Wong, Schaefer, Roop, La Mendola, Johnson-Seaton, and Shao, 1989) and the no steady state (nss) mutant of Lucilia (Howard, 1982, 1984; Barash, Suss, Stavenga, Rubinstein, and Minke, 1988; Suss, Barash, Stavenga, Stieve, Selinger, and Minke, 1989), the receptor potential, which appears normal in response to dim light, declines to baseline within a few seconds of illumination with intense light. Several lines of evidence suggest that the trp and nss mutations affect the same gene product (Howard, 1982; Suss-Toby, Selinger, and Minke, 1991). The trp mutant lacks a protein whose sequence has been determined by Montell and Rubin (1989) and by Wong et al. (1989). Recent studies (Hochstrate, 1989; Suss-Toby et al., 1991) have demonstrated that when lanthanum (La~+), a blocker of Ca 2+ binding proteins, is applied into the retinal extracellular space of the fly, the electrophysiological properties of the photoreceptors become very similar to those of the trp or nss mutant, while La 5+ has very little effect on the light response of the nss mutant (Suss-Toby et al., 1991). The close similarity in the properties of the receptor potential of the La3+-treated photoreceptor of the wild-type and of the nss mutants, together with existing evidence for the highly reduced intracellular Ca ~+ ([Ca2+]i) level in nss photoreceptors, led Suss-Toby et al. (1991) to suggest that both La 3+ and the mutation cause a severe reduction in [Ca2+]i. It was further suggested that this effect may arise from an inhibition of a C a 2+ channel/transporter protein located in the surface membrane that normally replenishes Ca 2+ pools in the photoreceptors, and that this process is essential for a maintained light excitation. Direct evidence that insect photoreceptors have a Ca 2+ transport system to introduce Ca 2÷ from outside the cell during illumination comes from measurements of [Ca2+]o in the extracellular space during and after illumination in the honeybee drone retina (Minke and Tsacopoulos, 1986; Ziegler and Walz, 1989) and the blowfly Catliphora (Sandier and Kirschfeld, 1988, 1991; Ziegler and Walz, 1989). A large influx of Ca e+ during light is observed accompanied by a large Na÷-dependent Ca z+ efflux during and after bright light via the Na-Ca exchanger. Additional evidence for influx of Ca 2+ into Drosophila photoreceptors during light is provided by voltage clamp measurements of light-induced current using the whole cell recording technique (Hardie, 1991; Ranganathan, Harris, Stevens, and Zuker, 1991a). These studies indicate that the light-sensitive channels are primarily permeable to Ca 2+ (Hardie, 1991 ; Ranganathan et al., 199 lb). The suggestion of Minke and Selinger (1992a) and Suss-Toby et al. (1991) that the trp and nss mutations or La 3+ abolish the activity of a light-activated C a 2+ channel/ transporter protein located at the surface membrane is strongly supported by recent measurements in Drosophila photoreceptors. Using whole cell recordings from isolated Drosophila ommatidia, Hardie and Minke (1992) have recently demonstrated that the wild-type light response is mediated by at least two classes of light-sensitive channels. One type of channel, which is highly permeable t o C a 2+, is selectively on N ovem er 6, 2017 jgp.rress.org D ow nladed fom RoM-GLAS ElAL. The ms Mutation Inhibits Light-induced Ca 2+ Influx 769 blocked by the trp muta t ion or ex te rna l La z+. This f inding strongly suggests that the block o f visual exci ta t ion and adap ta t i on dur ing l ight by the trp muta t ion and by La 3+ results from blocking Ca 2+ influx into the pho to recep to r s . T o test directly, by an i n d e p e n d e n t me thod , whe the r the ms muta t ion o r La 3+ blocks Ca 2+ influx into fly pho to recep to r s , we app l i ed Ca2+-selective microe lec t rodes to no rma l Lucilia (untreat ed o r t rea ted with La 3+) and to the nss re t ina to measure changes in l igh t induced ext race l lu lar Ca ~+ concent ra t ion . T h e pho to recep to r s are the only cells in the insect re t ina capable o f absorb ing o r re leas ing Ca ~+ in response to l ight (Coles and Orkand , 1985; Ziegler and Walz, 1989). T h e o the r class o f cells loca ted in the reg ion o f Ca 2+ measu remen t s is the gila (p igment) cells and they do not change their [Ca2+]i du r ing l ight (Coles and Orkand , 1985). Therefore , l igh t induced ext racel lu lar Ca z+ concent ra t ion changes reflect Ca z+ movements across the pho to recep to r s ' m e m b r a n e (see Discussion). M A T E R I A L S AND M E T H O D S