Direct Gating of ATP-activated Ion Channels (P2X2 Receptors) by Lipophilic Attachment at the Outer End of the Second

tions suggest that lipids enter the interstices between the outer ends of the TM domains. The P2X2(I328C) receptor was acti- vated by propyl-methanethiosulfonate (MTS) as effectively as by ATP, but cysteine substitutions elsewhere in TM2 had no such effect. Other lipophilic MTS compounds (methyl, ethyl, and tert-butylethyl) had a similar effect but not polar MTS. The properties of the conducting pathway opened by covalent attachment of propyl-MTS were the same as those opened by ATP, with respect to unitary conductance, rectification, and permeability of N-methyl-D-glucamine. The ATP-binding resi- due Lys 69 was not required for the action of propyl-MTS, although propyl-MTS did not open P2X2(K308A/I328C) recep- tors. The propyl-MTS did not open P2X2 receptors in which the Val 48 side chain was removed (P2X2(V48G/I328C)). The results suggest that an interaction between Val 48 and Ile 328 stabilizes the closed channel and that this is broken by covalent attach- ment of a larger lipophilic moiety at the I328C receptors. Lipid intercalation between the separating TM domains during chan- nel opening would be facilitated in P2X2(I328C) receptors with attached propyl-MTS. The results are consistent with the chan- nel opening mechanism proposed on the basis of closed and open crystal structures and permit the refinement of the posi- tion of the TMs within the bilayer. The ion permeation pathway of P2X receptors is a central channel formed by six -helical transmembrane domains (TM), 2 two provided by each of the three subunits (1-3). Homology models based on structures solved for the crystal- lized zebrafish P2X4 receptor (2, 3) indicate that the TM2 helix, which in rat P2X2 receptors extends from Ile 327 to Leu 353 , pro- vides a face of side chains (Ile 332 , Thr 336 , Thr 339 , Val 343 , and Leu 347 ) that allows water and ions to permeate. The three TM2 helices are angled at nearly 45° from the membrane normal. The intersection at Thr 336 and Thr 339 forms the narrowest part