Limulus retinal mRNA induces light-dependent currents in Xenopus oocytes.
The Biological Bulletin October 1, 1996 | Mole, E.J.; Schaefer, J.; Mathiesz, K.; Dionne, V.E.; Knox, B.E.; Barlow, R.B., Jr.
We are investigating the expression of Limulus retinal mRNA in Xenopus laevis oocytes as a means for examining the properties of Limulus rhodopsin that may influence photoreceptor sensitivity and noise (1,2). Xenopus oocytes were chosen because they contain G-protein-mediated ionic conductances (3) and can express mRNA from both vertebrate and invertebrate retinas (4,5). We report here that oocytes injected with Limulus retinal poly [A.sup.+]RNA and incubated with 11-cis retinal exhibit light-dependent ionic currents. This finding demonstrates that Limulus retinal mRNA is efficiently translated by Xenopus oocytes and is able to direct the synthesis of proteins necessary for light transduction.
RNA from retinas excised from Limulus lateral eyes was purified using an acid guanidium method (6), and poly [A.sup.+]RNA was selected using immobilized oligo dT (Promega Company). The RNA was ethanol-precipitated twice, quantitated by UV spectroscopy, and resuspended in water for microinjection.
To obtain oocytes, we anesthetized Xenopus frogs with 0.15% tricaine and surgically removed the ovarian lobes. Oocytes were dissociated from the surrounding epithelium and de-folliculated using 2 mg/ml collagenase (type 1A Sigma). Stage V and VI cells were microinjected with 50-100 ml of Limulus retinal poly A+ RNA (0.5 [mu]g/[mu]1 in water), and incubated for 2-6 days at 17[degree]C in Barth’s solution containing sodium pyruvate (5 mM) and gentamicin (10 [mu]g/ml). We localized injections to the animal pole of each oocyte. website test flash player
For electrophysiological recording, we placed single oocytes in a recording chamber that was constantly perfused with recording solution and impaled them with two glass microelectrodes (2-4 MOhms). If oocytes had stable resting potentials after about 30 min in darkness, we perfused them with 20[mu]M 11-cis retinal for 45 min and tested them for light sensitivity. Light from an unfiltered tungsten filament lamp was delivered to the oocyte with a fiber-optic light pipe (0.02 mW/[cm.sup.2] at the surface of the oocyte). Light responses after 3 days of incubation were small or non-existent, but after 4 days they were robust and readily recordable. Five of 10 injected oocytes responded to the light.
Figure 1A shows the current clamp response of an oocyte to a 25-s light flash. After a 6-s delay from light onset, the membrane depolarized to a level of -12 mV, and it returned to prestimulus baseline about 1 min after light offset. Responses of all other oocytes were similar to this, depolarizing to -12 to -23mV, with latencies that ranged from 6 to 24 s. Figure 1B shows the response of another oocyte to light while voltage clamped at various potentials. Light flashes evoked sustained inward currents at clamp potentials more negative than -25mV, and outward currents at potentials more positive than -20mV. The cause of the reduction in inward current before light offset at clamp potentials of -30 and -40mV is not known and was not observed in other oocytes. The reversal potential for the current for this oocyte was -23mV. Light-evoked currents for other oocytes reversed direction at holding potentials between -12 and -23mV, suggesting that this response is mediated by the endogenous calcium-activated chloride conductance (7, 8). Many other expressed receptor proteins also are known to couple into this pathway, and the long response latency we observed is typical of the activation of this chloride conductance (9). No light responses were detected in mRNA-injected oocytes before the application of 11-cis retinal. Other studies using the same expression system detected no responses in non-injected oocytes after incubation with 11-cis retinal (5).
Figure 1C shows that repetitive flashes of light can evoke repetitive responses from oocytes without the need for additional incubation with 11-cis retinal. This sustained fight sensitivity supports an earlier finding that Limulus metarhodopsin is a relatively stable and photoreversible photoproduct of Limulus rhodopsin (10). This is not the case for bovine rhodopsin expressed in Xenopus oocytes (5), which requires additional incubation with 11-cis retinal to maintain light sensitivity.
Note that the light-evoked currents in Figure 1C increased in amplitude in response to repeated light flashes of a constant intensity, while the response latencies decreased from 10 s for the first response to about 4 s for subsequent responses. Decreasing the intensity of the light flashes decreased both the response amplitude and the steady state level. Flashes of low intensity often failed to evoke any response to the first test flash, but not to subsequent ones (data not shown). Assuming that repetitive flashes of equal intensity generate equal levels of activated rhodopsin, the increasing response amplitude in Figure 1C may reflect the accumulation of an internal transmitter involved in the transduction cascade that yields the light-dependent currents we record. The occasional failure of the first test flash in a series to evoke a response points to the existence of a threshold for action for one or more internal constituents of this transduction pathway. We have not explored this facilitory response to subsequent light flashes in sufficient detail to determine either the exact threshold for action for the internal transmitter or the time course of its delay. web site test flash player
In conclusion, we have demonstrated that Xenopus oocytes efficiently translate Limulus retinal mRNA and provide a suitable system for studying the characteristics of light transduction. We will combine this technology with molecular biological techniques to study how the properties of Limulus rhodopsin influence photoreceptor sensitivity and noise.
Supported by grants from the National Institutes of Health, and the National Science Foundation.
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Mole, E.J.; Schaefer, J.; Mathiesz, K.; Dionne, V.E.; Knox, B.E.; Barlow, R.B., Jr.
