Neuronal inwardly rectifying K+ channels differentially couple to PDZ proteins of the PSD-95/SAP90 family

Several signaling proteins clustered at the postsynaptic density specializa tion in neurons harbor a conserved C-terminal PDZ domain recognition sequen ce (X-S/T-X-V/I) that mediates binding to members of the PSD-95/SAP90 prote in family. This motif is also present in the C termini of some inwardly rec tifying K+ (Kir) channels. Constitutively active Kir2 channels as well as G protein-gated Kir3 channels, which are fundamental for neuronal excitabili ty, were analyzed as candidates for binding to PSD-95/SAP90 family members. Therefore C termini of Kir2.1(+), Kir2.3(+), Kir2.4(-), Kir3.1(-), Kir3.2( -), Kir3.3(-) and Kir3.4(-) subunits (+, motif present; -, motif absent) we re used as baits in the yeast two-hybrid assay to screen for in vivo intera ction with PDZ domains 1-3 of PSD-95/SAP90. In contrast to Kir2.1 and Kir2. 3, all Kir3 fragments failed to bind PSD-95 in this assay, which was suppor ted by the lack of coimmunoprecipitation and colocalization of the entire p roteins in mammalian cells. A detailed analysis of interaction domains demo nstrated that the C-terminal motif in Kir3 channels is insufficient for bin ding PDZ domains. Kir2.1 and Kir2.3 subunits on the other hand coprecipitat e with PSD-95. When coexpressed in a bicistronic internal ribosome entry si te expression vector in HEK-293 cells macroscopic and elementary current an alysis revealed that PSD-95 suppressed the activity of Kir2.3 channels by > 50%. This inhibitory action of PSD-95, which predominantly affects the sing le-channel conductance, is likely attributable to a molecular association w ith additional internal interaction sites in the Kir2.3 protein.