Abstract
Two-pore domain K+ channels (K2P) display a characteristic extracellular cap structure formed by two M1-P1 linkers, the functional role of which is poorly understood. It has been proposed that the presence of the cap explains the insensitivity of K2P channels to several K+ channel blockers including tetraethylammonium (TEA). We have explored this hypothesis using mutagenesis and functional analysis, followed by molecular simulations. Our results show that the deletion of the cap structure of TASK-3 (TWIK-related acid-sensitive K+ channel) generates a TEA-sensitive channel with an IC50 of 11.8 ± 0.4 mM. The enhanced sensitivity to TEA displayed by the cap-less channel is also explained by the presence of an extra tyrosine residue at position 99. These results were corroborated by molecular simulation analysis, which shows an increased stability in the binding of TEA to the cap-less channel when a ring of four tyrosine is present at the external entrance of the permeation pathway. Consistently, Y99A or Y205A single-residue mutants generated in a cap-less channel backbone resulted in TASK-3 channels with low affinity to external TEA.
Highlights
Leak K+ channel family, known as K2P or two-pore domain K+ channels, are widely expressed among different cell types, where they play a critical role in setting the resting membrane potential [1,2]
Mutations of TASK-3 are associated with Birk Barel syndrome [9], and TASK-3 overexpression was found in human breast cancer tumors, where it has been proposed to act as a proto-oncogene [10]
TEA Is a PotenmtutBatleodctkoearlaonifnKe ivn2th.1e bCachkagrnounnedl obfuthteNTAoStKa-3n/2Elooffpe2c, tthive echBanlnoeclskedrispolafyTedAaSsKub-s3tanCtihalannel insensitivity to TEA similar to that observed in wild-type TASK-3 channels
Summary
Leak K+ channel family, known as K2P or two-pore domain K+ channels, are widely expressed among different cell types, where they play a critical role in setting the resting membrane potential [1,2]. The K2P family consists of fifteen different members divided into six subfamilies based on structural and functional properties [3,4,5,6]. Mutations of TASK-3 are associated with Birk Barel syndrome [9], and TASK-3 overexpression was found in human breast cancer tumors, where it has been proposed to act as a proto-oncogene [10]. Further study showed that TASK-3 gene knock down in breast cancer cells is associated with an induction of cellular senescence and cell cycle arrest [11]
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