Properties of Potassium Channel Kv1.1 on the Basis of Fluorescent Dimer of Alpha-Subunits mKATE2-Kv1.1-Kv1.1 in Neuro-2a Cells
摘要
Objective: Studying heterotetrameric voltage-gated potassium channels and their blockers requires mastering the approach to create bioengineered protein constructs that will form channels of a given composition and stoichiometry in cells. It is also important to ensure that the covalent linkage of α-subunits does not hinder the proper functioning of the channel. Methods: Confocal microscopy and electrophysiological techniques were used to study the properties of the voltage-gated potassium Kv1.1 channel, formed in Neuro-2a cells from dimers of the human Kv1.1 α-subunits linked by the Lys-Leu dipeptide and fused at the N-terminus with mKate2 fluorescent protein (mKate2-(Kv1.1)2). Results and Discussion: It was found that the linking of Kv1.1 α-subunits into a dimer did not cause changes either in the membrane expression of the channel or in the features of its cellular distribution compared with mKate2-Kv1.1 monomers. No differences were found between the channels based on mKate2-(Kv1.1)2 dimers and mKate2-Kv1.1 monomers in the half-activation potential, channel activation constants, and the magnitude and nature of potassium ion currents. Conclusions: The data obtained suggest the possibility of creating bioengineered protein constructs by similarly linking two different α-subunits, which would form fluorescent heterotetrameric voltage-gated potassium channels with an α-subunit stoichiometry of 2 : 2 in mammalian cells.