The M2 transmembrane segment as a molecular determinant of the ion permeation properties in the superfamily of ligand-gated ion channels.

Ligand-gated ion channels are crucial for synaptic transmission in the nervous system (1.2). After binding of the ligand to its binding site, the protein undergoes a conformational change from a closed nonconductive to an open conductive state that allows the selective flux of ions Structurally, the superfamily of ligand-gated ions channels display the presence of three major domains (l,2): i) a large extracellular N-terminus, that exposes the ligand binding site ii) a long hydrophilic segment containing protein kinases consensus sequences and is, presumably, located intracellularly. iii) Four putative transmembrane segments, Ml-M4 (1-3). Among them, M2 has been implicated in forming the aqueous pore of the ionic channel (1-3). M2 is amphipathic and predicted to be ahelical ( 1 -3) Mutations in M2 residues affect the ionic selectivity and the efficacy of blockade by open channel blockers (2-10). Furthermore, NMR studies show that M2 peptides are indeed a-helical in lipid bilayers and are oriented perpendicular to the membrane plane ( 1 1). Together, these results suggest that the M2 transmembrane segment is an important component of the aqueous pore of this superfamily of receptors. However, the molecular determinants that define their ion permeation properties are not yet known in detail The neuronal a 7 acetylcholine receptor (AChRa7) and the neuronal kainate/AMPA glutamate receptor subunit 1 (HBGRI) represent ideal receptor models because they express hnctional homomeric receptors, presumably, with pentameric stoichiometry. To determine if negatively -charged residues exposed to the lumen of the aqueous pore increase permeability to divalent cations, we introduced an acidic residue (D) at position T244 of AChRa7 (AChRa7T244D) ( 5 ) . in the N-terminus of the M2 Wild type and mutant receptors were assayed for functional expression in , Y L ' I I O ~ I I S oocytes. Unexpectedly, the AChRa7T244D displayed a higher sensitivity to ACh and a slower desensitization rate than the AChRa7wt These results along with those reported for other AChRa7 mutants in nearby residues (610). indicate the existence of a tight interplay between the binding domain and the pore domain of this channel protein An acidic residue in the M2 segment remarkably modulated the ion permeation properties of the AChRa7. Reversal potential measurements under different extracellular ionic conditions indicated that AChRa7T244D had a higher permeability to divalent over monovalent cations Furthermore, the mutant receptor was permeable to Mg2+, a cation that blocks AChRa7wt. Quantitation of the relative ionic permeability, using the Goldman-Hodgkin-Katz model, showed that the AChRa7T244D exhibited ~3fo ld increase in the relative permeability to Ba2+ over Na', without significantly affecting the relative permeability among monovalent cations In muscular and 7ixpedo AChRs the equivalent position of T244 is a molecular determinant of the sensitivity to QX-222 blockade (2.3), an analog of the local anesthetic lidocaine. Since QX-222 has a positively-charged quaternary ammonium group, it is expected that a negative residue in T244 would increase the efficacy of QX-222 blockade. Our results indeed show that the AChRa7T244D displays a higher sensitivity to QX-222 blockade than the AChRa7wt, namely, higher affinity and stronger voltage dependence. Collectively, these results support the notion that T244 in the M2 transmembrane segment of the AChRa7 is a structural determinant of its ion permeation properties and its sensitivity to open channel blockers. At variance with cholinergic receptors, mutagenesis experiments on glutamate receptors suggested that the ion permeation properties are determined by the N612 in NMDA receptors and the 4582 in kainate/AMPA receptors, near the C-terminus of M2 (l2,13). Our results indeed suggest that Q582 in HBGRl is a molecular determinant of its ionic permeability and its sensitivity to open channel blockers such as Mg2+ of HBGRI, in agreement with other reports (1 2). Mutation of residues nearby the equivalent position of T244 in AChRa7 did not affected the permeation properties of HBGRl nor of other glutamate receptors (12). Taken together, these results suggest that in acetylcholine receptors the selectivity filter is located near the N-terminus of M2 which lies in the cytoplasmic side of the pore, whereas in glutamate receptors it is close to the C-terminus, which faces the synaptic side of the pore. In conclusion, the M 2 transmembrane segment in the superfamily of neurotransmitter-activated ion channels contains the structure determinants of the ionic selectivity and, therefore. this segment is an essential component of the hydrophilic pore.