Allosteric effects of external K+ ions mediated by the aspartate of the GYGD signature sequence in the Kv2.1 K+ channel.

K+ channels achieve exquisite ion selectivity without jeopardizing efficient permeation by employing multiple, interacting K+-binding sites. Introduction ofa cadmium (Cd2+)-binding site in the external vestibule of Kv2.1 (drk1), allowed us to functionally characterize a binding site for external monovalent cations. Permeant ions displayed higher affinity for this site than non-permeant monovalent cations, although the selectivity profile was different from that of the channel. Point mutations identified the highly conserved aspartate residue immediately following the selectivity filter as a critical determinant of the antagonism between external K+ and Cd2+ ions. A conservative mutation at this position (D378E) significantly affected the open-state stability. Moreover, the mean open time was found to be modulated by external K+ concentration, suggesting a coupling between channel closing and the permeation process. Reducing the Rb+ conductance by mutating the selectivity filter to the sequence found inKv4.1, also significantly reduced the effectiveness ofRb+ ions to antagonize Cd2+ inhibition, thereby implicating the selectivity filter as the site at which K+ions exert their antagonistic effect on Cd2+ block. The equivalent of D378 in KcsA, D80, takes part in an inter-subunit hydrogen-bond network that allows D80to functionally interact with the selectivity filter. The results suggest that external K+ ions antagonize Cd2+inhibition (in I379C) and modulate the mean open time(in the wild-type Kv2.1) by altering the occupancy profile of the K+-binding sites in the selectivity filter.

Duke Scholars

Author Antonius M. J. VanDongen Pharmacology & Cancer Biology