Electrostatic Interactions Between Polyamines And Charged Adducts In The Kir Inner Cavity

Abstract

Physiological regulation of conductance in Kir channels is accomplished by voltage-dependent block by intracellular cations (Mg2+, polyamines). We have investigated the fine details of polyamine binding in Kir channels, and report unique and unexpected effects of cysteine modification in the inner cavity. Introduction of positive charges near the spermine binding site in Kir6.2[N160D] channels alters the kinetic and steady-state properties of spermine block, although specific effects depend dramatically on both the modified position, and the properties of the modifying agent. MTSEA or MTSET modification of L157C, one helical turn above residue 160, dramatically reduces spermine affinity, and accelerates spermine unbinding. However, effects of MTSEA vs. MTSET modification of L164C, one helical turn below residue 160, are significantly divergent. MTSEA modification again reduces spermine affinity, whereas MTSET has no effect on steady-state spermine block, and slows spermine block and unblock. This stark difference between MTSEA and MTSET is attributed to interactions of the carboxylate sidechain at residue 160 with the primary amine of the ethylamine adduct (MTSEA), which are lost with the quaternarized ethyl-trimethylamine adduct introduced by MTSET modification. Thus, MTSEA modification of L164C reduces spermine block indirectly, by neutralization of the nearby â€∼rectification controller’ residue, rather than a direct interaction with spermine. In contrast, the chemistry of MTSET precludes this interaction, leaving spermine affinity unaltered. Importantly, MTSET modification of L164C reduces affinity for extended spermine analogs, whereas incorporation at a shallower site (S212C) again slows dissociation of extended blockers, shedding light on the localization of the trailing ends of polyamine analogs in the pore. Collectively, the data demonstrate the subtle effects of charge modification in the inner cavity on polyamine-mediated inward rectification, and confirm stable spermine binding between the rectification controller and the selectivity filter.