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.
Chapman
Mark L
ML
Department of Pharmacology, Duke University, Durham, NC, USA. vando005@mc.duke.edu
Blanke
Marie L
ML
Krovetz
Howard S
HS
VanDongen
Antonius M J
AM
D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D002104:Cadmium; D020134:Catalytic Domain; D005786:Gene Expression Regulation; D015640:Ion Channel Gating / Q000502:physiology; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D017354:Point Mutation; D011188:Potassium / Q000378:metabolism*; D011485:Protein Binding; D012413:Rubidium; D051669:Shab Potassium Channels / Q000737:chemistry* / Q000378:metabolism*; D014982:Xenopus laevis
D009842:Oligopeptides; D051669:Shab Potassium Channels; C420882:glycyl-tyrosyl-glycyl-aspartate; D002104:Cadmium; D001224:Aspartic Acid; D012413:Rubidium; D011188:Potassium