A mutation in the extracellular domain of the α7 nAChR reduces calcium permeability

Abstract

The α7 neuronal nicotinic acetylcholine receptor (nAChR) displays the highest calcium permeability among the different subtypes of nAChRs expressed in the mammalian brain and can impact cellular events including neurotransmitter release, second messenger cascades, cell survival, and apoptosis. The selectivity for cations in nAChRs is thought to be achieved in part by anionic residues which are located on either side of the channel mouth and increase relative cationic concentration. Mutagenesis studies have improved our understanding of the role of the second transmembrane domain and the intracellular loop of the channel in ion selectivity. However, little is known about the influence that the extracellular domain (ECD) plays in ion permeation. In the α7 nAChR, it has been found that the ECD contains a ring of ten aspartates (two per subunit) that is believed to face the lumen of the pore and could attract cations for permeation. Using mutagenesis and a combination of electrophysiology and imaging techniques, we tested the possible involvement of these aspartate residues in the calcium permeability of the rat α7 nAChR. We found that one of these residues (the aspartate at position 44) appears to be essential since mutating it to alanine resulted in a decrease in amplitude for both whole cell and single-channel responses and in the complete disappearance of detectable calcium changes in most cells, which indicates that the ECD of the α7 nAChR plays a key role in calcium permeation.

Microtubule dynamics at the growth cone are mediated by α7 nicotinic receptor activation of a Gαq and IP3receptor pathway

“…In these experiments, an empty pcDNA3.1 vector was used as a transfection control. Consistent with findings on D44A function in hippocampal neurons (16), our calcium imaging data suggest that expression of this mutant significantly diminishes calcium flow through the α7. A confirmation of D44A expression in PC12 cells indicates that expression of D44A increases total fBgtx fluorescence (by 89±15%) without altering the overall cellular distribution of the signal (Supplemental Fig.…”

“…A new mutant of the α7 (D44A; ref. 16) was used to examine the role of calcium flow in growth. PC12 cells transfected with D44A presented a lowered calcium response (–67±11%) compared to controls following choline treatment (Fig.…”

Alpha 7 nicotinic receptor coupling to heterotrimeric G proteins modulates RhoA activation, cytoskeletal motility, and structural growth

“…Previous work has shown that a site‐specific mutation in the α7 subunit (α7 D44A ) decreases channel permeability to calcium (Colón‐Sáez and Yakel ). We have shown that α7 D44A successfully expresses in PC12 cells and mediates a weakened calcium transient response following choline stimulation (Nordman and Kabbani ).…”

“…; Liu et al . ; Nordman and Kabbani ), or α7 D44A (Colón‐Sáez and Yakel ) using Lipofectamine 2000 (Thermo Fisher). Construct DNA was purified using a maxi prep kit (Xymo Research, Irvine, CA, USA).…”

Regulation of nicotinic acetylcholine receptors in Alzheimer׳s disease: A possible role of chaperones

“…In addition, cholinergic system inhibits pro-inflammatory cytokines release through α7-nAChRmediated toll like receptor (TLR) expression on the neurons (Kim et al, 2014;Pavlov et al, 2007).Activation of the α7-nAChRs on astrocytes in the hippocampus increases cytoplasmic calcium levels and participates in various mechanisms of neuroprotection perhaps via the calcium-dependent phosphatase calcineurin (Colón-Sáez and Yakel, 2013). Furthermore, α7-nAChR activation exerts anti-apoptotic effects on neuronal cells which reduce cleaved caspase 3 and inflammation induced by Aβ (Boatright and Salvesen, 2003;Colón-Sáez and Yakel, 2013;Luan et al, 2013;Ramin et al, 2011). It has been found that, α7-nAChR can effectively scavenge superoxide, hydroxyl and other free radicalsthrough Ca 2+ -dependent mechanisms (Liu and Zhao, 2004) which protects against oxidant stress-induced damage resulting from Aβ toxicity (Li et al, 2014).…”