A NEGATIVE CHARGE IN THE M2 TRANSMEMBRANE SEGMENT OF THE NEURONAL-ALPHA-7 ACETYLCHOLINE-RECEPTOR INCREASES PERMEABILITY TO DIVALENT-CATIONS

Threonine-244 (T244) in the putative channel-forming M2 segment of the neuronal alpha7 acetylcholine receptor (AChR), a residue proposed to form part of the selectivity filter, was mutated to aspartic acid to examine the influence of a negative charge on AChR ion permeation properties. Wild type (AChRalpha7wt) and mutant (AChRalpha7D244) acetylcholine receptors expressed in Xenopus oocytes give rise to acetylcholine (ACh)-activated, alpha-bungarotoxin-sensitive, cation-selective ionic currents. AChRalpha7D244 exhibited larger currents than AChRalpha7wt that, in addition, activated at lower ACh concentrations. The relative ionic permeability (P(x)/P(Na)) of AChRalpha7wt to K+ was P(K)/P(Na) = 1.2, and to Ba2+, P'(Ba)/P(Na) = 1.4. In contrast, AChR alpha7D244 was less selective in discriminating between K+ and Na+, P(K)/P(Na) = 0.95, but exhibited a remarkable increase in permeability to Ba2+, P'(Ba)/P(Na) = 3.7. Furthermore, only mutant receptors were permeable to Mg2+. Hence, a ring of negatively charged residues in the putative pore-forming segment of the receptor increases the permeability to divalent cations. Our results substantiate the notion that T244, or its equivalent, in the M2 transmembrane segment of cholinergic receptor channels is a key structural determinant of the selectivity filter.