Divalent Cations in Horizontal Cells of the Catfish Retina

GABA C responses were recorded in cultured cone-driven horizontal cells from the catfish retina using the patch clamp technique. At a holding potential of 2 49 mV, a bicuculline-resistant inward current (I GABA ) was observed when 10 m M GABA was applied. The amplitude of I GABA increased as the extracellular Ca 2 1 ([Ca 2 1 ] o ) was increased. Concentration–response curves of I GABA at 2.5 and 10 mM [Ca 2 1 ] o had similar EC 50 (3.0 and 3.1 m M) and Hill coefficients (1.54 and 1.24). However, the maximal response estimated at 10 mM [Ca 2 1 ] o was larger than the maximal response at 2.5 mM [Ca 2 1 ] o . Increasing Ca influx through voltage-gated Ca channels and the resulting rise in the intracellular Ca 2 1 concentration had no effects on I GABA . However, I GABA was inhibited by extracellular divalent cations, with the following order of the inhibitory potency: Zn 2 1 . Ni 2 1 . Cd 2 1 . Co 2 1 . The inhibitory action of Zn 2 1 on the [Ca 2 1 ] o -dependent I GABA increase was noncompetitive. The action of [Ca 2 1 ] o on I GABA was mimicked by Ba 2 1 or Sr 2 1 . These results demonstrate that the extracellular domain of GABA C receptors has two functionally distinct binding sites represented by Ca 2 1 (facilitation) and Zn 2 1 (inhibition). Since [Ca 2 1 ] o and [Zn 2 1 ] o change into the opposite direction by light, it seems likely that they modify cooperatively the efficacy of the positive feedback consisting of the GABA C receptor. key words: GABA • horizontal cell • retina • Ca • catfish i n t r o d u c t i o n The outer plexiform layer, the first synaptic layer of the vertebrate retina, is where signals transduced in photoreceptors are transmitted to bipolar cells. Photoreceptor-bipolar transmission is modulated by the feedback synapse from horizontal cells to photoreceptors. The input–output relation of these synapses has been studied in the tiger salamander and other animals (see Wu, 1994). The transmission at these synapses may be more complex than originally thought, based on the modulatory actions of divalent cations. For example, Zn 2 1 has been shown to be coreleased with transmitters from the synaptic vesicles of photoreceptors (Wu et al., 1993; Dong and Werblin, 1995). It has been reported that the Ca 2 1 concentrations in the inner and outer plexiform layers are changed by illumination (Livsey et al., 1990; Gallemore et al., 1994). These Ca 2 1 concentration changes are thought to result primarily from Ca influx accompanying transmitter release. We have shown that the extracellular Ca 2 1 concentration in the inner plexiform layer is important for determining the activities of nicotinic ACh receptors in retinal ganglion cells (Kaneda et al., 1995). Modulation of known chemical receptors by the extracellular Ca 2 1 concentration in the outer plexiform layer has not been reported. The GABA C receptor was initially identified in horizontal cells of the catfish retina (Qian and Dowling, 1993). Since the resting potential of the horizontal cell is z 2 80 mV ([K 1 ] o 5 5 mM), and the reversal potential of the GABA C response (E Cl ) of the intact horizontal cell is z 2 30 mV (Takahashi et al., 1995 a ), GABA induces a depolarization that enhances GABA release from horizontal cells (Schwartz, 1987). In this sense, GABA C receptors in catfish horizontal cells make a positive auto-feedback loop, the concept proposed by Stockton and Slaughter (1991) and Kamermans and Werblin (1992) for GABA A receptors of the amphibian horizontal cells. These GABA C auto-receptors are thought to contribute to accelerating light-evoked responses (Takahashi et al., 1995 a ). In the present experiments, we examined the actions of extracellular Ca 2 1 and other divalent cations on the GABA C receptor. We found that the extracellular domain of the GABA C receptor had two divalent cation binding sites with contrasting functions; i.e., a facilitative Ca 2 1 binding site and an inhibitory Zn 2 1 binding site. Based on the present findings, we speculate as to the functional role of GABA C receptors in horizontal cells. Address correspondence to M. Kaneda, MD, Ph.D., Department of Physiology, Keio University School of Medicine, Tokyo 160, Japan. Fax: 81-3-3359-0437; E-mail: [email protected] on Jne 9, 2017 D ow nladed fom Published December 1, 1997