The isoforms generated by alternative translation initiation adopt similar conformation in the selectivity filter in TREK-2.
TREK-2 (TWIK-related K(+) channel-2), a member of two-pore domain potassium (K2P) channel family, tunes cellular excitability via conducting leak or background currents. In TREK-2, the isoforms generated by alternative translation initiation (ATI) mechanism exhibit large divergence in unitary conductance, but similar in selectivity to K(+). Up to now, the structural basis for this similarity in ion selectivity is unknown. Here, we report that externally applied Ba(2+) inhibits the currents of TREK-2 in a concentration- and time-dependent manner. The blocking effect is blunted by elevated extracellular K(+) or mutation of S4 K(+) binding site, which suggests that the inhibitory mechanism of Ba(2+) is due to its competitive docking properties within the selectivity filter (SF). Next, we demonstrate that all the ATI isoforms exhibit analogous behaviors upon the application of Ba(2+) and alteration of extracellular pH (pHo), which acts on the outer position of the SF. These results strongly support the notion that all the ATI isoforms of TREK-2 possess resembled SF conformation in S4 site and the position defined by pHo, which implicates that neither the role of N-terminus (Nt) nor the unitary conductance is associated with SF conformation. Our findings might help to understand the detail gating mechanism of TREK-2 and K2P channels.
State Key Laboratory of Toxicology and Medical Countermeasures, Department of Biochemical Pharmacology, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing, 100850, China.
Ren-Gong
RG
Zhuo
State Key Laboratory of Toxicology and Medical Countermeasures, Department of Biochemical Pharmacology, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing, 100850, China.|Chinese PLA General Hospital, 28 Fuxing Street, Beijing, 100853, China.
Peng
P
Peng
State Key Laboratory of Toxicology and Medical Countermeasures, Department of Biochemical Pharmacology, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing, 100850, China.
Xiao-Yan
XY
Liu
State Key Laboratory of Toxicology and Medical Countermeasures, Department of Biochemical Pharmacology, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing, 100850, China.
Shu-Zhuo
SZ
Zhang
Department of Neuropharmacology, School of Pharmaceutical Sciences, Southern Medical University, No.1838, North Guangzhou Avenue, Guangzhou, 510515, China.
Jiang-Ping
JP
Xu
State Key Laboratory of Toxicology and Medical Countermeasures, Department of Biochemical Pharmacology, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing, 100850, China.
Jian-Quan
JQ
Zheng
State Key Laboratory of Toxicology and Medical Countermeasures, Department of Biochemical Pharmacology, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing, 100850, China. weixl@bmi.ac.cn.
Xiao-Li
XL
Wei
State Key Laboratory of Toxicology and Medical Countermeasures, Department of Biochemical Pharmacology, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing, 100850, China. maxyer22@yahoo.com.
Xiao-Yun
XY
Ma
D000595:Amino Acid Sequence; D000818:Animals; D017609:Barium Compounds / Q000494:pharmacology; D001665:Binding Sites; D002478:Cells, Cultured; D002712:Chlorides / Q000494:pharmacology; D005260:Female; D006801:Humans; D020128:Inhibitory Concentration 50; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D011188:Potassium / Q000494:pharmacology; D024683:Potassium Channels, Tandem Pore Domain / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000502:physiology*; D011485:Protein Binding; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000502:physiology; D017434:Protein Structure, Tertiary; D014982:Xenopus laevis
D017609:Barium Compounds; D002712:Chlorides; C503438:KCNK10 protein, human; D024683:Potassium Channels, Tandem Pore Domain; D020033:Protein Isoforms; C024986:barium chloride; D011188:Potassium
Alternative translation initiation; Ba2+; S4 K+ binding site; Selectivity filter; TREK-2