Dynein light chains 1 and 2 are auxiliary proteins of pH-sensitive Kir4.1 channels.

Inward rectifier Kir4.1 potassium channels are abundantly expressed in cells that are important for electrolyte homeostasis. Dysregulation of Kir4.1 underlies various neurological disorders. Here, through biochemical and structural studies of full-length Kir4.1, we show that dynein light chain 1 and 2 proteins, also as known as LC8, copurify with Kir4.1 at stoichiometric levels. Direct interaction between Kir4.1 and LC8 is supported by in vitro binding assays and reiterated with native Kir4.1 proteins from mouse brain. Notably, we identify a LC8 binding motif in the unstructured N terminus of Kir4.1. Among Kir subtypes, the motif is unique to Kir4.1 and is highly conserved between Kir4.1 orthologs. Deletion of the predicted anchoring sequence (ΔTQT) resulted in loss of LC8 interaction with Kir4.1 N-terminal peptides as well as with full-length Kir4.1, suggesting that the binding site is necessary and sufficient for the interaction. Purified Kir4.1-ΔTQT mutant proteins exhibited normal channel activity in vitro, whereas WT proteins lost phosphoinositide-(4,5)-phosphate activation. Single-particle cryo-EM analysis of the full-length proteins revealed extremely heterogeneous particles, indicating deformation from the typical fourfold symmetric conformation. Additional electron density attached to the Kir4.1 tetramer, ascribed to an LC8 dimer, further supports the direct interaction between the two proteins. While the biological implications of this interaction await further elucidation, the strong conservation of the LC8 binding motif suggests its potential importance in the regulation of Kir4.1 channels.