Extracellular K⁺ Concentration Dependence of Outward Currents through Kir2.1 Channels Is Regulated by Extracellular Na⁺ and Ca

It has been known for more than three decades that outward Kir currents (IK₁) increase with increasing extracellular K⁺ concentration ([K⁺]o). Although this increase in IK₁ can have significant impacts under pathophysiological cardiac conditions, where [K⁺]o can be as high as 18 mM and thus predispose the heart to re-entrant ventricular arrhythmias, the underlying mechanism has remained unclear. Here, we show that the steep [K⁺]o dependence of Kir2.1-mediated outward IK₁ was due to [K⁺]o-dependent inhibition of outward IK₁ by extracellular Na⁺ and Ca²⁺. This could be accounted for by Na⁺/Ca²⁺ inhibition of IK₁ through screening of local negative surface charges. Consistent with this, extracellular Na⁺ and Ca²⁺ reduced the outward single-channel current and did not increase open-state noise or decrease the mean open time. In addition, neutralizing negative surface charges with a carboxylate esterifying agent inhibited outward IK₁ in a similar [K⁺]o-dependent manner as Na⁺/Ca²⁺. Site-directed mutagenesis studies identified Asp¹¹⁴ and Glu¹⁵³ as the source of surface charges. Reducing K⁺ activation and surface electrostatic effects in an R148Y mutant mimicked the action of extracellular Na⁺ and Ca²⁺, suggesting that in addition to exerting a surface electrostatic effect, Na⁺ and Ca²⁺ might inhibit outward IK₁ by inhibiting K⁺ activation. This study identified interactions of K⁺ with Na⁺ and Ca²⁺ that are important for the [K⁺]o dependence of Kir2.1-mediated outward IK₁.