The Basic Property of Lys385 Is Important for Potentiation of the Human α1 Glycine Receptor by Ethanol

Abstract

Ethanol alters the function of several members of the Cys-loop ligand-gated ion channel superfamily. Recent studies have shown that the sensitivity of the ␣1 glycine receptor (GlyR) to ethanol can be affected by the state of G protein activation mediated by the interaction of G␤␥ with intracellular amino acids in the GlyR. Here, we evaluated the physicochemical property of Lys385 that contributes to ethanol modulation by using mutagenesis, patchclamp, and biochemical techniques. A conserved substitution (K385R) did not affect either the apparent glycine EC 50 (40 Ϯ 1 versus 41 Ϯ 0.5 M) or the ethanol-induced potentiation (53 Ϯ 5 versus 46 Ϯ 5%) of the human ␣1 GlyR. On the other hand, replacement of this residue with glutamic acid (K385E), an acidic amino acid, reduced the potentiation of the GlyR to 10 Ϯ 1%. Furthermore, mutations with a hydrophobic leucine (K385L), a hydrogen bond donor glutamine (K385Q), or a neutral residue (K385A) also reduced ethanol modulation. Finally, substitution by a large and hydrophobic residue (K385F) and deletion of 385 (Lys385_) reduced ethanol modulation to 10 Ϯ 4 and 17 Ϯ 0.4%, respectively. Experiments using dynamic cysteine substitution with a methanethiosulfonate reagent and homology modeling indicate that the basic property and the position of Lys385, probably because of its interaction with G␤␥, is critical for ethanol potentiation of the receptor.

Ethanol effects on glycinergic transmission: From molecular pharmacology to behavior responses

“…Taken together, the sensitivity to ethanol and intracellular modulation of the different isoforms of glycine receptors are related to residues in the extracellular region (A52), transmembrane domains (G254, S296), intracellular loop domain ( 316 RFRRK 320 , 385 KK 386 ) and the lack of the splice cassette of α3 [60,148,153,154,162,164]. Because ethanol sensitivity is related to intracellular modulation of glycine receptors by Gβγ from activation of G proteins, the results obtained using chimeric receptors, site-directed mutations and wild type receptors from different regions of the CNS confirmed a highly significant correlation between the sensitivity to ethanol (100 mM) and G protein modulation [Figure 2] and showed the role of Gβγ signaling pathway in the effects of ethanol in the CNS [87,97,123,148,154,162,165–169].…”

Mutation of a Zinc-Binding Residue in the Glycine Receptor α1 Subunit Changes Ethanol Sensitivity In Vitro and Alcohol Consumption In Vivo

“…This is consistent with mutational studies locating the high-affinity potentiating and lowaffinity inhibitory binding sites for zinc in the extracellular domain of GlyRa1 outside of the agonist binding site (Harvey et al, 1999;Laube et al, 2000;Miller et al, 2005a,b;Grudzinska at al., 2008), whereas ethanol potentiation is thought to involve an alcohol-binding pocket that likely extends from residues of loop 2 in the extracellular domain (Crawford et al, 2008;Perkins et al, 2009;Perkins et al, 2012) to the thoroughly investigated residues of the helical segments in the transmembrane domain (Mihic et al, 1997;Mascia et al, 2000;Lobo et al, 2004Lobo et al, , 2006Lobo et al, , 2008McCracken et al, 2010a). Additionally, there is evidence that residues of the intracellular loop of the a1 GlyR subunit may be important for ethanol action at GlyRs (Yevenes et al, 2011;Castro et al, 2012).…”

Evidence for α-Helices in the Large Intracellular Domain Mediating Modulation of the α1-Glycine Receptor by Ethanol and Gβγ

“…In particular, the sequence of g2 shows a proline at position 326 in the LIL, a residue that can prevent the formation of helical structures as described in the a1-GlyR subunit. Also, the C terminus of g2-LIL lacked the equivalent lysine 385, recognized as important for modulation by ethanol, and presents an alanine 400 in this position (Castro et al, 2012). Therefore, we mutated the segment near the TM3 ( 309 NRKPS 313 ) in the chimera a1-LIL-g2 to an a1-GlyR sequence ( 309 RQHKE 313 ), together with the mutation A385K (Fig.…”

“…Whole-cell recordings were performed as previously described (Castro et al, 2012). A holding potential of 260 mV was used.…”

“…The large extracellular aminoterminal domain provides the neurotransmitter binding site (Speranskiy et al, 2007;Pless and Lynch, 2009). Finally, the large intracellular loop domain (LIL) between TM3 and TM4 is important for more recently found regulation of channel activity (Deeb et al, 2007;Carland et al, 2009;O'Toole and Jenkins, 2011;Castro et al, 2012) and is the most variable region of these receptor subunits. Additionally, it presents consensus sites for phosphorylation, modulation by intracellular mechanisms, and interaction with cytosolic and cytoskeletal proteins (Moss and Smart, 2001;Sine and Engel, 2006).…”