Modulation of Kir1.1 Inactivation by External Divalents

Abstract

Kir1.1 inactivation, associated with transient internal acidification, was strongly dependent on external K, Ca and Mg. In 1mM K, a 15 min transient acidification (internal pH 8 to pH 6.3) produced 91±2% inactivation in 2mM Ca, but only 18±4% inactivation in the absence of external Ca and Mg. In 100mM external K, the same acidification protocol produced 29±5% inactivation in 10mM external Ca but no inactivation when extracellular Ca was reduced below 2mM (with 0 Mg). External Ca was more effective than external Mg at producing inactivation, but Mg caused a greater degree of open channel block than Ca, making it unlikely that Kir1.1 inactivation arises from divalent block. Since the Ca sensitivity of inactivation persisted in 100mM external K, it is also unlikely that Ca enhances Kir1.1 inactivation by reducing the local K concentration at the outer mouth of the channel. Removal of 4 surface, negative side-chains at E92, D97, E104, E132 (Kir1.1b) increased the sensitivity of inactivation to external Ca (and Mg); whereas adding a negative surface charge (N105E-Kir1.1b) decreased the sensitivity of inactivation to Ca and Mg. This result is consistent with negative surface charges stabilizing external K in the selectivity filter or at the S0-K binding site just outside the filter. In this model, extracellular Ca and Mg potentiate the slow, K-dependent, inactivation of Kir1.1 by allosterically destabilizing the conducting conformation of the selectivity filter, independent of divalent block in the permeation pathway.