Calmodulin Mediates a Step in the Excitation Cascade of Limulus Ventral Photoreceptors

Abstract

Abstract: : Purpose: The excitation cascade in Limulus photoreceptors involves IP3-mediated release of Ca²⁺, the subsequent activation of guanylate cyclase (GC), and finally the opening of cGMP-gated channels. We suspected that calmodulin might be involved in excitation because it exists in very high concentration in the rhabdom and because calmodulin-activated GC has been described in other systems. To test the role of calmodulin we injected Ca²⁺/calmodulin (CaM) or its inhibitors. Methods: Photoreceptors were impaled with one or two microelectrodes to record membrane potential and inject drugs. Results: Injection of 25 uM CaM produced a robust (up to 20 mV) depolarization (N=15). This was observed when the electrode tip was in the light-sensitive R-lobe, but not when the tip was in the non-transducing A-lobe (N=4). Two controls indicate that the excitation was not due to Ca²⁺ released from the injected calmodulin (Ca²⁺ itself can excite). First, the response to CaM injection was measured before and after inhibition of the Ca²⁺-mediated light response by the Ca²⁺ buffer 5,5’-dibromo-BAPTA. After the light response was decreased by buffer more than 80% relative to the control response, the response to CaM injection was unaffected (N=3). In a second set of controls using double-barreled theta electrodes, we found that injection of CaM out of one barrel vigorously excited the cell whereas equal injection of CaM in combination with CaM inhibitors, 200 uM CaMKII(290-309) or 100 uM trifluoperazine (TFP), from the second barrel was much less effective (N=4). To test whether endogenous CaM participates in excitation we injected 250 uM trifluoperazine into the R-lobe during the depolarization produced by a sustained exposure to light. Injection of TFP led to a transient and reproducible decrease in the response to light. Conclusions: These experiments provide the first evidence that CaM itself can promote excitation and is required for light-induced excitation. This may serve to explain why so much calmodulin is concentrated in the transducing lobes of invertebrate photoreceptors. We are currently testing whether CaM is working at the GC level of the cascade.

Keywords: photoreceptors • signal transduction: pharmacology/physiology • second messengers: pharmacology/physiology