Scanning the Topography of Polyamine Blocker Binding in an Inwardly Rectifying Potassium Channel*

Harley T Kurata,Alejandro Akrouh,Jennyb.W Li,Laurence J Marton,Colin G Nichols

doi:10.1074/jbc.m112.383794

Abstract

Steeply voltage-dependent inward rectification of Kir (inwardly rectifying potassium) channels arises from blockade by cytoplasmic polyamines. These polycationic blockers traverse a long (>70 Å) pore, displacing multiple permeant ions, en route to a high affinity binding site that remains loosely defined. We have scanned the effects of cysteine modification at multiple pore-lining positions on the blocking properties of a library of polyamine analogs, demonstrating that the effects of cysteine modification are position- and blocker-dependent. Specifically, introduction of positively charged adducts results in two distinct phenotypes: either disruption of blocker binding or generation of a barrier to blocker migration, in a consistent pattern that depends on both the length of the polyamine blocker and the position of the modified cysteine. These findings reveal important details about the chemical basis and specific location of high affinity polyamine binding.

Highlights

Rectifying potassium channels preferentially conduct currents into cells due to polyamine block
We present a novel approach to identify the sites occupied by polyamines in Kir channels, combining chemical modification of channels with a library of synthetic polyamine analogs [29]
MTSEA and MTSET Modification of Tandem Dimer Kir6.2 Channels—We designed a series of experiments to examine high affinity spermine binding and test two contrasting structural models of the steeply voltage-dependent polyamine binding site in Kir channels (Fig. 1A, gray clouds)

Summary

Introduction

Rectifying potassium channels preferentially conduct currents into cells due to polyamine block. Voltage-dependent inward rectification of Kir (inwardly rectifying potassium) channels arises from blockade by cytoplasmic polyamines These polycationic blockers traverse a long (>70 Å) pore, displacing multiple permeant ions, en route to a high affinity binding site that remains loosely defined. Introduction of positively charged adducts results in two distinct phenotypes: either disruption of blocker binding or generation of a barrier to blocker migration, in a consistent pattern that depends on both the length of the polyamine blocker and the position of the modified cysteine. These findings reveal important details about the chemical basis and specific location of high affinity polyamine binding

Results

Discussion

Conclusion