Molecular basis involved in the blocking effect of antidepressant metergoline on C-type inactivation of Kv1.4 channel

Abstract

Voltage-gated potassium channels (VGKCs) are transmembrane ion channels specific for potassium. Currently there are nine kinds of VGKCs. Kv1.4 is one of shaker-related potassium channels. It is a representative alpha subunit of potassium channels that can inactivate A type-currents, leading to N pattern inactivation. Inactivation of Kv channels plays an important role in shaping electrical signaling properties of neuronal and muscular cells. The shape of N pattern inactivation can be modified by removing the N-terminal (NT) domain which results in non-inactivated currents and C pattern inactivation. In a previous work, we have reported the regulatory effect of metergoline on Kv1.4 and Nav1.2 channel activity. In the present study, we constructed a mutant of deleted 61 residues from NT of Kv1.4 channels (Kv1.4 Δ2-61) and found that it induced an outward peak and steady-state currents We also studied the modulation effect of metergoline on the activity of this Kv1.4 Δ2-61 mutant channel without having the N-terminal quick inactivation domain. Our results revealed that treatment with metergoline inhibited NT deleted Kv1.4 mutant channel activity in a concentration-dependent manner which was reversible. Interestingly, metergoline treatment induced little effects on the outward peak current in the deleted Kv1.4 mutant channel. However, metergoline treatment conspicuously inhibited steady state currents of Kv1.4 Δ2-61 channels with acceleration current mode. The acceleration of steady-state current of deleted Kv1.4 mutant channel occurred in a concentration-dependent manner. This means that metergoline can accelerate C pattern inactivation of Kv1.4 Δ2-61 channel by acting as an open state dependent channel blocker. We also performed site-directed mutations in V561A and K532Y, also known as C-type inactivation sites. V561A, K532Y, and V561A + K532Y substitution mutants significantly attenuated the acceleration effect of metergoline on C pattern inactivation of hKv1.4 channel currents. In docking modeling study, predicted binding residues for metergoline were analyzed for six amino acids. Among them, the K532 residue known as the C-type inactivation site was analyzed to be a major site of action. Then various mutants were constructed. K532 substitution mutant significantly abolished the effect of metergoline on Kv1.4 currents among various mutants whereas other changes had slight inhibitory effects. Furthermore, we found that metergoline had specificity for Kv1.4, but not for Kv1.5 currents. In addition, the A type current in rat neuronal cell was inhibited and accelerated of inactivation. This result further shows that metergoline might interact with Lys532 residue and then accelerate C pattern inactivation of Kv1.4 channels with channel type specificity. Taken together, these results demonstrate the molecular basis involved in the effect of metergoline, an ergot alkaloid, on human Kv1.4 channel, providing a novel interaction ligand.