An Intersubunit Salt Bridge near the Selectivity Filter Stabilizes the Active State of Kir1.1

Abstract

ROMK (Kir1.1) potassium channels are closed by internal acidification with a pKa of 6.7 +/- 0.01 in 100 mM external K and a pKa of 7.0 +/- 0.01 in 1 mM external K. Internal acidification in 1 mM K (but not 100 mM K) not only closed the pH gate but also inactivated Kir1.1, such that realkalization did not restore channel activity until high K was returned to the bath. We identified a new putative intersubunit salt bridge (R128-E132-Kir1.1b) in the P-loop of the channel near the selectivity filter that affected the K sensitivity of the inactivation process. Mutation of either R128-Kir1.1b or E132-Kir1.1b caused inactivation in both 1 mM and 100 mM external K during oocyte acidification. However, 300 mM external K (but not 200 mM Na + 100 mM K) protected both E132Q and R128Y from inactivation. External application of a modified honey-bee toxin, tertiapin Q (TPNQ), also protected Kir1.1 from inactivation in 1 mM K and protected E132Q and R128Y from inactivation in 100 mM K, which suggests that TPNQ binding to the outer mouth of the channel stabilizes the active state. Pretreatment of Kir1.1 with external Ba prevented Kir1.1 inactivation, similar to pretreatment with TPNQ. In addition, mutations that disrupted transmembrane helix H-bonding (K61M-Kir1.1b) or stabilized a selectivity filter to helix-pore linkage (V121T-Kir1.1b) also protected both E132Q and R128Y from inactivation in 1 mM K and 100 mM K. Our results are consistent with Kir inactivation arising from conformational changes near the selectivity filter, analogous to C-type inactivation.

A conserved arginine near the filter of Kir1.1 controls Rb/K selectivity

“…Between 2 and 3 hours of alkalization were required for R128Y oocytes to recover their original channel activity in 100 mM external K solutions. This inactivation of R128Y in K solutions has been previously reported,17 but its elimination by external Rb is a new observation and is consistent with the general finding that Rb is a preferred substrate for R128Y-Kir1.1b.…”

“…8) and only recovered between 2 and 3 hours after realkalization 17. However, in the present study, we found that 100 mM external Rb prevented this pH inactivation such that R128Y conductance returned rapidly to its original value after realkalization (black line, Fig.…”

“…In theory, R128Y and R128M should be equally effective at breaking the intra-inter subunit salt-bridge at E118-R128-E132-Kir1.1b 17. However, the greater increase in both G Rb /G K and G Cs /G K with R128Y compared to R128M implies that salt bridge disruption cannot be the sole cause of the increased Rb/K selectivity.…”

Mechanism for selectivity-inactivation coupling in KcsA potassium channels

“…Furthermore, alterations in the equivalent salt bridge residues that affect selectivity in the Kir channel family also lead to the induction of a phenomenon similar to C-type inactivation in voltage-gated K þ channels (7). There is also a correlation between C-type inactivation and ion selectivity in eukaryotic voltage-gated K þ channels: Shaker channels and other Kv channels such as Kv2.1 show decreased K þ and increased Na þ permeability when progressing to a C-type inactivated state (8,9).…”

A Highly Conserved Salt Bridge Stabilizes the Kinked Conformation of β2,3-Sheet Essential for Channel Function of P2X4 Receptors

“…This interaction is the most commonly observed interaction to contribute to the stability of the conformation of proteins (46 -48). Previous studies have demonstrated that a salt bridge is implicated in many protein functions, such as the ligand binding of the GABA A receptor (33), the stabilization of the activation state of Kir1.1 (37), the folding of the serotonin transporter (38), the surface expression of ASIC channels (34), and the protein thermostability of enzymes (36). Here, we suggest that the salt bridge formed between Arg-309 and Asp-85 played an essential role in the protein stability and channel gating of P2X4 receptors.…”