FUNCTIONAL-ROLE OF A CONSERVED ASPARTATE IN THE EXTERNAL MOUTH OF VOLTAGE-GATED POTASSIUM CHANNELS

Mutation of the glycines in a conserved Gly-Tyr-Gly-Asp sequence in th e P-region of voltage-gated K channels has identified determinants of Na/K selectivity. But the function of the negatively charged Asp is no t known because mutations at this position are not tolerated, owing to the fourfold replication of mutations in a tetrameric channel. We hav e successfully mutated Asp(378) --> Thr in a tandem dimer K(v)2.1 cons truct to yield a twofold neutralization of charge at this site. When e xpressed in Xenopus oocytes, the mutated channels showed markedly alte red ion conduction and blockade. Potassium conduction in the inward di rection was selectively reduced, so that the instantaneous current-vol tage relationship obtained in isotonic KCl became strongly outwardly r ectifying. The relative permeability to Na+, P-Na/P-K, increased from 0.02 to 0.10 without changing the ion selectivity sequence K > Rb >> C s >> Na. The IC50 for block by external tetraethylammonium (TEA) incre ased more than 100-fold without affecting block by internal TEA. We co nclude that Asp(378) is essential part of a potassium ion binding site associated with the Na/K selectivity filter at the external mouth of the pore.