An N-Terminal Histidine Is the Primary Determinant of α Subunit-Dependent Cu²⁺Sensitivity of αβ3γ2L GABA_AReceptors

Abstract

Copper (Cu 2ϩ ) is a physiologically important cation and is released from nerve terminals. Cu 2ϩ modulates GABA A receptor currents in an ␣ subunit subtype-dependent manner; ␣1␤3␥2L receptors are more sensitive to Cu 2ϩ than ␣6␤3␥2L receptors. We compared the effect of Cu 2ϩ on ␣␤3␥2L receptors containing each of the six ␣ subtypes and generated ␣1/␣6 chimeras and mutants to determine the functional domain(s) and specific residues responsible for ␣ subtype-dependent differences in Cu 2ϩ sensitivity. Whole-cell GABA A receptor currents were obtained from L929 fibroblasts coexpressing wildtype, chimeric and mutant ␣ subunits with ␤3 and ␥2L subunits. Maximal Cu 2ϩ inhibition of ␣1␤3␥2L and ␣2␤3␥2L receptor currents was larger (52.2 Ϯ 3.0 and 59.0 Ϯ 2.5%, respectively) than maximal inhibition of ␣3␤3␥2L, ␣4␤3␥2L, ␣5␤3␥2L, and ␣6␤3␥2L receptor currents (22.6 Ϯ 3.1, 19.2 Ϯ 3.4, 20.2 Ϯ 4.8, and 21.2 Ϯ 3.6%, respectively). Receptors containing chimeric constructs with ␣1 subtype N-terminal sequence between residues 127 and 232 were inhibited by Cu 2ϩ to an extent similar to those with ␣1 subtypes, suggesting that this N-terminal region (127-232) contains a major determinant for high Cu 2ϩ sensitivity. ␣1 subtype residues V134, R135, and H141 in a VRAECPMH motif (VQAECPMH in the ␣2 subtype) conferred higher Cu 2ϩ sensitivity, and the H141 residue was the major determinant in the motif. The ␤3 subtype M2 domain residue H267, which is a major determinant of Zn 2ϩ inhibition, and ␣6 subtype M2-M3 loop residue H273, which is responsible for the increased Zn 2ϩ sensitivity of the ␣6 subtype, also seemed to contribute to Cu 2ϩ inhibition. These data suggest that the Nterminal VR(Q)AECPMH motif in ␣1 and ␣2 subtypes is the major determinant of increased subtype-dependent inhibition by Cu 2ϩ , that residue H141 is the major determinant in that motif, and that Cu 2ϩ may also interact with GABA A receptors at sites similar to or overlapping Zn 2ϩ sites.Fast inhibitory synaptic transmission in the mammalian central nervous system is mediated primarily by GABA A receptors, which are members of a superfamily of ligandgated ion channels including nicotinic acetylcholine, glycine, and 5-hydroxytryptamine type 3 receptors. GABA A receptors are composed of a pentameric combination of subunits that form an intrinsic chloride ion channel. Each GABA A receptor subunit has a putative membrane topology consisting of a large N-terminal extracellular domain, four membrane spanning domains (TM1-TM4) and an extracellular C terminus.

Acute and subchronic copper treatments alter extracellular nucleotide hydrolysis in zebrafish brain membranes

“…Copper can be found in different parts of CNS and it is accumulated in brain as other physiological metals, such as zinc, with relative importance for synaptic mechanisms (Kim and Macdonald, 2003). It has been known that copper concentrations in cerebrospinal fluid have been estimated to be around 70 М and the normal extracellular copper concentrations in brain ranged from 0.2 to 1.7 М (Mathie et al, 2006).…”

“…The genome of this fish shares many similarities with the human genome (Barbazuc et al, 2000). Ionotropic P2X receptors have already been identified and characterized in this specie (Kucenas et al, 2003) and GABAergic neurons were described in adult zebrafish brain (Kim and Macdonald, 2003). Studies have recently demonstrated the presence of NTPDases (Rico et al, 2003) and ecto-5 -nucleotidase (Senger et al, 2005) in brain membranes of zebrafish.…”

“…It can form a complex binding to AMPA/kainate receptors in cultured rat cortical neurons and, possibly, the co-release of copper with glutamate is important to modulate excitatory synapses (Weiser and Wienrich, 1996). In addition, copper also promotes the inhibition of GABA A receptor currents in a ␣ subunit-dependent manner controlling the fast inhibitory synaptic neurotransmission (Kim and Macdonald, 2003). Deficiency of copper levels in central neurons induces severe neurological disorders as Menkes disease (Kreuder et al, 1993).…”

In vivo and in vitro analyses of amygdalar function reveal a role for copper

“…Cu had no effect on the monosynaptic excitatory current but reversibly potentiated the disynaptic inhibitory current. This likely reflects disinhibition of interneurons, which express the highest levels of Cu-sensitive, ␣ 1 -subunit-containing GABA A receptors (Kim and Macdonald 2003;. Based on our previous electrophysiological study and the increased sensitivity of PAM ϩ/Ϫ mice to the anxiolytic effects of benzodiazepines (Gaier et al 2010), this result was particularly interesting.…”

Copper and Copper Proteins in Parkinson’s Disease

“…Further studies have determined that copper inhibits currents in GABA receptors with an IC50 = 2.4  μ M [48]. Copper seems to act in the gating system of the GABA receptor channel [36].…”