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D.–Q. Zhang, G.–X. Ruan, D.G. McMahon; Characterization of Voltage–Gated Calcium Channels in the Genetically Labeled Catecholamine Amacrine Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2282.
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© ARVO (1962-2015); The Authors (2016-present)
A tyrosine hydroxylase (TH)–driven red fluorescent protein (RFP) transgenic mouse has been generated for morphological, functional and molecular analysis of catecholamine amacrine (CA) cells (Zhang et al., NeuroReport, 2004). Using this model in combination with electrophysiological, calcium imaging and single–cell RT–PCR techniques, we sought to characterize expression of calcium channels in CA cells.
Acute cultured TH::RFP expressing cells were visualized by the fluoresecence microscopy using a DsRed filter set. Whole–cell patch clamp was applied for the calcium current recording. Cultures of retinal cells were incubated in 1 µM Fluo–3 for 30 min and then calcium images were collected using confocal microscopy. Individual Type 2 CA cells were harvested using glass pipettes, and transcripts of nine different calcium channel α1 subunits were tested for those cells using single–cell RT–PCR.
When Type 2 CA cells were held at –60 mV and then depolarized, inward currents activated at approximately –40 mV, and peaked between –20 mV and +10 mV with a mean current of –50 pA (n=3), suggesting that high–voltage activated calcium channels are expressed in Type 2 CA cells. Next, the cell was held at –90 mV and the voltage steps repeated. The low–voltage activated calcium currents, which were obtained by subtracting the currents measured at holding potentials of –60 mV from those at –90 mV, activated at approximately –65 mV, and peaked at around –50 mV. Calcium imaging of Type 2 CA cells demonstrated that a depolarization induced by 45 mM KCl yielded an average increase in intracellular calcium of 160% (n=4), whereas an increase of 79% evoked by KCl was observed in the presence of 100 µM cadmium(n=4). Single–cell RT–PCR analysis of calcium channel α subunits revealed an expression of α1–A subunit in all 12 cells tested, α1–B subunit in 9 of 12 cells, and α1–E subunit in 1 of 12 cells, indicating type 2 CA cells possess P/Q–type, N–type, and R–type calcium channels. In 1 of 12 cells, mRNAs for the α1–C subunit and α1–D subunit of L–type calcium channels were also detected. The α1–G subunit of T–type calcium channels was detected in 8 of 12 cells.
The results suggest that type 2 CA cells express both high–voltage activated calcium channels and low–voltage activated calcium channels, which mediate an influx of calcium during the cell depolarization. Single–cell RT–PCR data indicate that calcium channels of Type 2 CA cells are mainly composed of P/Q–type, N–type and T–type calcium channels, which need to be further confirmed by studying pharmacological properties of those channels.
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