Hetromerization of Kir Channels: Principles of Assembly and Physiological Significance

G-protein coupled K+ (Kir3.x) channels are unique channels that translate chemical neurotransmission via GPCR activation to electrical signals. It generates slow postsynaptic K+ currents, which hyperpolarize the membrane to depress excitability. Kir3.x channels belong to the inwardly rectifying K+ (Kir) channels superfamily, named after their preference for an inward directed potassium flow. The Kir gene superfamily consists of fifteen members classified into seven subfamilies (Kir1.x to Kir7.x). The canonical view describes the assembly of functional tetrameric Kir channels by members within the same subfamily, e.g. within Kir2.x or Kir3.x subfamilies but not between them, although some cases using heterologous expression systems have been reported to challenge this view. As part of our search for novel proteins that associate with Kir3.1, we have carried out immuno-precipitation of membrane lysates from mouse brains, followed by mass-spectrometry analysis, to identify novel associations of Kir3.1 with other membranal proteins. We found significant associations of Kir3.1 with few members of other Kir subfamilies. These associations were further investigated using the proximity ligation assay, both in cultured neurons and brain sections, whole cell recording from hippocampal neurons, and TIRF microscopy using FRET assays to support the initial observations. Functional measurements performed in Xenopus oocytes, using dominant negative form of Kir3.1, also demonstrated the propensity of Kir3.1 channel to selectively assemble with other subfamily members not identified in the mass-spectrometry assay. These results challenge the current dogma and sets new rules for Kir channel assembly in vivo. It may suggest of much wider functional diversity in neurotransmitter regulation of electrical activity.