Evidence for TRPM7 Channels in Rat Retinal Ganglion Cells

Abstract

Abstract: : Purpose: The M7 transient receptor potential (TRP) channel has been implicated as a mediator of anoxia–induced calcium influx and subsequent neuronal death in cortical neurons in vitro (Aarts et al., 2003, Cell 115:863–877). We sought to determine if TRPM7 channels might also be present in rat retinal ganglion cells (RGCs) and if anoxia produced currents or elevations of intracellular calcium concentration ([Ca²⁺]_i) consistent with activation of TRPM7 channels. Methods: RNA was extracted from adult mouse and rat cerebrum and retina, and from immunopurified cultures of RGCs from 7–8 day postnatal rats. The presence of TRPM7 mRNA was determined using RT–PCR with primers, 5'–TTGGTGATGCCCTCAAAG–3' (sense) and 5'–GAACTCCTTGCCCAATTC–3' (antisense), designed against the mouse TRPM7 sequence (GenBank Accession No. NM_021450). Putative TRPM7 protein was localized by immunohistochemistry in sections of adult (>5 weeks) rat retina using a goat polyclonal antibody against a synthetic peptide derived from human TRPM7 with sequence homology with rat (Abcam, ab729). Anoxia was produced chemically by incubating immunopanned RGCs in 3 mM cyanide for 45 min. Whole cell patch recording and fura–2 calcium imaging was used to investigate the effects of cyanide on RGCs. Results: RT–PCR revealed a product 300 bp in size in the cDNA from rat and mouse cerebrum and retina and from immunopurified RGCs. Putative TRPM7 immunoreactivity was identified within the ganglion cell layer and throughout the inner plexiform layer of rat retina. Treatment with 3 mM cyanide in Mg²⁺–free solution produced a delayed elevation of RGC [Ca²⁺]_i that recovered immediately following cyanide wash–out. The cyanide–induced increase of RGC [Ca²⁺]_i was not blocked by co–treatment with the non–specific TRP channel blockers Gd³⁺ (100 µM) and 2–amino–ethoxydiphenyl borate (2–APB; 100 µM) but was decreased by Mg²⁺. Cyanide treatment did not produce sustained TRP–like currents in RGCs but, in some cells, elicited repetitive transient inward currents (at –60 mV) that were reversibly blocked by 100 µM Gd³⁺. Conclusions: RT–PCR and immunohistochemistry indicated that rat RGCs contain TRPM7 channels. Chemical anoxia, by treatment with cyanide, did not produce clear evidence of TRPM7 activation.

Keywords: ganglion cells • ion channels • calcium