Sigma-1 receptor stimulation attenuates calcium influx through activated L-type Voltage Gated Calcium Channels in purified retinal ganglion cells

Brett H. Mueller, Yong Park, Donald R. Daudt, Hai Ying Ma, Irina Akopova, Dorota L. Stankowska, Abbot F. Clark, Thomas Yorio

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Abstract

Sigma-1 receptors (σ-1rs) exert neuroprotective effects on retinal ganglion cells (RGCs) both in vivo and in vitro. This receptor has unique properties through its actions on several voltage-gated and ligand-gated channels. The purpose of this study was to investigate the role that σ-1rs play in regulating cell calcium dynamics through activated L-type Voltage Gated Calcium Channels (L-type VGCCs) in purified RGCs. RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using a Thy1.1 antibody. Calcium imaging was used to measure changes in intracellular calcium after depolarizing the cells with potassium chloride (KCl) in the presence or absence of two σ-1r agonists [(+)-SKF10047 and (+)-Pentazocine], one σ-1r antagonist (BD1047), and one L-type VGCC antagonist (Verapamil). Finally, co-localization studies were completed to assess the proximity of σ-1r with L-type VGCCs in purified RGCs. VGCCs were activated using KCl (20 mM). Pre-treatment with a known L-type VGCC blocker demonstrated a 57% decrease of calcium ion influx through activated VGCCs. Calcium imaging results also demonstrated that σ-1r agonists, (+)-N-allylnormetazocine hydrochloride [(+)-SKF10047] and (+)-Pentazocine, inhibited calcium ion influx through activated VGCCs. Antagonist treatment using BD1047 demonstrated a potentiation of calcium ion influx through activated VGCCs and abolished all inhibitory effects of the σ-1r agonists on VGCCs, implying that these ligands were acting through the σ-1r. An L-type VGCC blocker (Verapamil) also inhibited KCl activated VGCCs and when combined with the σ-1r agonists there was not a further decline in calcium entry suggesting similar mechanisms. Lastly, co-localization studies demonstrated that σ-1rs and L-type VGCCs are co-localized in purified RGCs. Taken together, these results indicated that σ-1r agonists can inhibit KCl induced calcium ion influx through activated L-type VGCCs in purified RGCs. This is the first report of attenuation of L-type VGCC signaling through the activation of σ-1rs in purified RGCs. The ability of σ-1rs to co-localize with L-type VGCCs in purified RGCs implied that these two proteins are in close proximity to each other and that such interactions regulate L-type VGCCs.

Original languageEnglish
Pages (from-to)21-31
Number of pages11
JournalExperimental eye research
Volume107
DOIs
StatePublished - 1 Feb 2013

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Keywords

  • Calcium signaling
  • L-type Voltage Gated Calcium Channel
  • Purified retinal ganglion cells
  • Sigma-1 receptor

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