Abstract
Abstract: :
Purpose: Rod photoreceptors of the salamander retina possess a glutamate transporter coupled to a Cl- conductance (Grant & Werblin, 1996). ECl in rods is positive to the resting potential and Cl- efflux inhibits rod calcium currents (ICa) (Thoreson et al, 2000, 2002). We investigated whether activation of the Cl- conductance associated with the glutamate transporter might inhibit ICa in rods. Methods: Gramicidin or nystatin perforated patch whole cell recordings were obtained from isolated rods or rods in retinal slices from tiger salamander. For ICa recordings, cells were perfused with 1.8 mM Ca2+, 5 mM Cs+, and 10mM TEA. Drugs were bath applied for 2-4 min. ICa was measured every 30 sec using voltage ramps (0.5 mV/ms). Cl- flux was evaluated using the fluorescent dye MEQ in isolated rods. Results: Application of L-glutamate (L-Glu) inhibited ICa by 15-20% at 0.1 mM and 30-40 % at 1mM. Consistent with activation of a Cl- conductance, L-Glu produced an inward current in rods that reversed around -29 mV, not significantly different from ECl (Thoreson et al, 2002). D-aspartate (D-Asp) (1 mM), which is a substrate for glutamate transporters, inhibited ICa as well. L-Glu failed to inhibit ICa after lowering intraterminal Cl- by recording from rods with low Cl- pipette solution. The glutamate transport inhibitor, TBOA (0.1 mM), prevented L-Glu from inhibiting ICa. The metabotropic glutamate receptor agonists t-ADA (Group I), DCG-IV (Group II), and L-AP4 (Group III) had no effect on ICa . Using Cl- imaging, L-Glu (0.1-1 mM) and D-Asp (0.1-1 mM) were found to stimulate a Cl- efflux from terminals of isolated rods whereas the glutamate receptor agonists NMDA (0.1 mM), AMPA (10 µM), kainate (30 µM) and 1S,3R-ACPD (0.1 mM) did not. L-Glu-evoked Cl- efflux was blocked by the glutamate transporter inhibitors TBOA (0.5 mM) and DHKA (1 mM) but not by glutamate receptor antagonists kynurenic acid (1 mM), MK801 (50 µM) or GYKI52466 (50 µM). Conclusions: The electrophysiological results indicate that L-Glu inhibits ICa by acting through the glutamate transporter. The Cl- imaging results indicate that activation of the glutamate transporter generates a Cl- efflux from rod terminals. Cl- efflux inhibits Ca2+ channel activity in rod terminals (Thoreson et al, 2000) and preventing Cl- efflux blocked the inhibitory effect of L-Glu on ICa. Taken together, these results suggest that activation of glutamate transporters generates a Cl- efflux that inhibits ICa in rods. Through this mechanism, the neurotransmitter L-Glu released from rod terminals could provide a negative feedback signal to inhibit further L-Glu release.
Keywords: photoreceptors • synapse • excitatory neurotransmitters