Zinc Modulation of L-type Calcium Channels at Photoreceptor Terminals

Abstract

Purpose: : To determine whether the release of endogenous zinc acts to modulate calcium channels at the photoreceptor terminal.

Methods: : Photoreceptor cells were isolated from tiger salamander (Ambystoma tigrinium) retina. Light-adapted photoreceptors were loaded with the calcium indicator dye Fluo-4,AM. Calcium influx was initiated by depolarization with 30mM KCl. Changes in dye intensity were recorded with a Zeiss fluorescence microscope to reveal differences in [Ca ²⁺ ]i. Drugs were delivered via a pressure perfusion system with fast, valve-controlled solution changes. Images were taken every second (exposure time=150ms). An average decay curve of peak fluorescence intensity with time was obtained from repeated applications of 30mM KCl alone. Trials started with a control application of KCl alone and subsequent responses were normalized to the first peak.

Results: : Application of 2 mM Zn ²⁺ resulted in an 88% reduction in Ca ²⁺ influx and was reversible. The L-type Ca ²⁺ channel blockers nicardipine and verapamil (50-100uM) produced a similar reduction in Ca ²⁺ influx. Chelation of endogenously released Zn ²⁺ with the membrane-impermeable chelator histidine (10mM) increased the Ca ²⁺ influx by 51%. Application of Zn ²⁺ together with histidine recovered 23% of the Ca ²⁺ signal. The enhancing effect on the Ca ²⁺ current by the Zn ²⁺ chelator histidine was blocked with both nicardipine and verapamil.

Conclusions: : The co-release of ionic Zn ²⁺ and glutamate at rod photoreceptor terminals was shown previously. Here, we show that exogenous ionic Zn ²⁺ is a potent, but reversible blocker of L-type Ca ²⁺ channels at the cone photoreceptor terminal. The action of Zn ²⁺ at these channels was mimicked by the Ca ²⁺ channel blockers nicardipine and verapamil; their effect, however, was not as readily reversible. More importantly, chelation of endogenously released Zn ²⁺ by histidine resulted in the relief of inhibition on L-type Ca ²⁺ channels and led to an increase in Ca ²⁺ influx. Thus, the concentration of Zn ²⁺ co-released with glutamate, under normal physiological conditions, serves an important modulatory role at the photoreceptor terminal.

Keywords: calcium • photoreceptors • imaging/image analysis: non-clinical