Reciprocal modulation of Ca_v2.3 voltage‐gated calcium channels by copper(II) ions and kainic acid

Abstract

Kainic acid (KA) is a potent agonist at non-N-methyl-D-aspartate (non-NMDA) ionotropic glutamate receptors and commonly used to induce seizures and excitotoxicity in animal models of human temporal lobe epilepsy. Among other factors, Ca 2.3 voltage-gated calcium channels have been implicated in the pathogenesis of KA-induced seizures. At physiologically relevant concentrations, endogenous trace metal ions (Cu , Zn ) occupy an allosteric binding site on the domain I gating module of these channels and interfere with voltage-dependent gating. Using whole-cell patch-clamp recordings in human embryonic kidney (HEK-293) cells stably transfected with human Ca 2.3d and β -subunits, we identified a novel, glutamate receptor-independent mechanism by which KA can potently sensitize these channels. Our findings demonstrate that KA releases these channels from the tonic inhibition exerted by low nanomolar concentrations of Cu and produces a hyperpolarizing shift in channel voltage-dependence by about 10 mV, thereby reconciling the effects of Cu chelation with tricine. When tricine was used as a surrogate to study the receptor-independent action of KA in electroretinographic recordings from the isolated bovine retina, it selectively suppressed a late b-wave component, which we have previously shown to be enhanced by genetic or pharmacological ablation of Ca 2.3 channels. Although the pathophysiological relevance remains to be firmly established, we speculate that reversal of Cu -induced allosteric suppression, presumably via formation of stable kainate-Cu complexes, could contribute to the receptor-mediated excitatory effects of KA. In addition, we discuss experimental implications for the use of KA in vitro, with particular emphasis on the seemingly high incidence of trace metal contamination in common physiological solutions.

Submicromolar copper (II) ions stimulate transretinal signaling in the isolated retina from wild type but not from Cav2.3-deficient mice

“…In a previous study, the isolated bovine retina exhibited a similar sensitivity towards 100 nM CuCl 2 as the retina from wild type mice (for details [28]).…”

“…For the human Ca v 2.3 R-type Ca 2+ channel, a reciprocal modulation by Cu 2+ and kainic acid was found when stably co-expressed with human b 3 -subunit in HEK-293 cells. Moreover, when tricine was used as a surrogate of kainic acid, it produced effects consistent with Ca v 2.3 channel modulation both in transfected HEK cells and in the isolated bovine retina [28]. In the murine retina, tricine was ineffective in changing transretinal signaling, likely due to the trace metal chelation by glutamate which was present in the Ames medium.…”

“…The ERG b-wave amplitude of the bovine retina is known to be reversibly reduced in the presence of 5 mM tricine [28], which was shown to be caused by the chelation of trace metal cations as impurities in HEPES buffer or by chelation of 100 nM added CuCl 2 . In the bovine retina, submicromolar Cu 2+ [28] blocks the Ca v 2.3 R-type Ca 2+ channel even more effectively than comparable NiCl 2 [12] or ZnCl 2 concentrations [8]. However, tricine did not change significantly the b-wave amplitude of the murine retina during successive application of 0.1, 1 and 3 mM tricine in the superfusing solution ( Fig.…”

“…Since adding tricine to the perfusion solution had no effect on the ERG b-wave, we concluded that L-Glu from the Ames solution chelated any impurities from the nutrient solution additives, resulting in different signals when compared to recording of the bovine retina [28]. Next, the scotopic full ERG was recorded from six independent freshly isolated retinae either from control or from Ca v 2.…”

“…Cu 2+ occupy an allosteric binding site on the domain I gating module of Ca v 2.3 channels and may interfere with voltage-dependent-gating. Several studies have shown that even very nominal Cu 2+ concentrations are sufficient for significant suppression of these channels, as electroretinographically demonstrated on the ex-vivo bovine retina [14]. It was found that L-Glutamate, as excitatory amino acids, can stimulate Ca v 2.3-channels by being as trace metal chelators and inhibiting the suppressing effects of metal ions such as Zn 2+ and Cu 2+ [15].…”