The Cytosolic GH Loop Regulates the Phosphatidylinositol 4,5-Bisphosphate-induced Gating Kinetics of Kir2 Channels

Abstract

Background:The detailed mechanism of PIP 2 -induced Kir channel gating remains elusive. Results: Specific mutations increase the flexibility of the cytosolic GH loop and accelerate the PIP 2 -induced gating kinetics of Kir2 channels. Conclusion: Interactions of the GH loop with the N terminus regulate the PIP 2 -induced gating kinetics of Kir2 channels. Significance: We identify a novel region in Kir channels involved in the control of PIP 2 -induced gating.

Cholesterol sensitivity of KIR2.1 depends on functional inter-links between the N and C termini

“…36 Specifically, it has been shown that mutation of E272, which is located between N251 and Y280/ L282, affects both the on-and off-gating kinetics of the channel. Moreover, we have recently shown that in addition to reversing the effect of the L222I mutation on the sensitivity of Kir2.1 to cholesterol, N251D also restores the strength of the interaction of the L222I mutant with PI(4,5)P 2 to the levels exhibited by the WT Kir2.1 channel.…”

Insights into the structural nature of the transition state in the Kir channel gating pathway

“…Such rearrangements may also promote disengagement of the CTD from the TM/ pore domain, thereby allowing it to move down/away and into the closed state conformation. Interestingly, many previous studies have also suggested that the flexibility and movement of the G-loop is critical for gating in several different Kir channels 11,12 and may even act as a physical gate 13 (i.e. barrier to ion permeation).…”

Structural Basis for Differences in Dynamics Induced by Leu Versus Ile Residues in the CD Loop of Kir Channels

“…Additionally, the oocytes were held at −80 mV for the deactivation of Ci-VSP. Sweeps were applied until the resulting currents reached a steady state [22]. A low-potassium solution (ND96) containing 96 mM NaCl, 1 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , and 5 mM HEPES (pH 7.4 with NaOH) was used to inhibit most of the Kir currents at −80 mV.…”

“…Two bath solutions were used: an FVP solution containing 60 mM KCl, 5 mM EDTA-K, 5 mM KF, 0.1 mM Na 3 VO 4 , 10 mM K 4 P 2 O 7 , and 10 mM HEPES (pH 7.4 with KOH) and another FVP solution with diC 8 PIP 2 at 0–100 μM for dose-response measurements and at 30 μM for gating kinetics measurements. diC8 PIP 2 was purchased from Avanti Lipids and prepared as described previously [22]. All other chemicals were purchased from Sigma.…”

“…The positively charged K219 in the CD loop is proposed as one binding site of the nonspecific secondary anionic phospholipid, an important composite of native cell membranes; this binding increases the PIP 2 sensitivity of Kir2.1 channel gating [21]. The mutation of human Kir2.1 V223L in the CD loop increases both the on- and off-gating kinetics by affecting the GH loop flexibility [22]. Moreover, a conserved hydrophobic Leu/Ile residue difference in the CD loop is found in all identified human Kir channels, except Kir3.1 and Kir5.1, as indicated by the presence of Leu in the Kir1.1, Kir2.1, Kir2.4, Kir4.1, Kir4.2, and Kir7.1 channels and the presence of Ile in the Kir2.2, Kir2.3, Kir3.2, Kir3.3, Kir3.4, Kir6.1, and Kir6.2 channels [4].…”