Rapedius M et al. (2007), H bonding at the helix-bundle crossing controls...

XB-ART-36626

Neuron 2007 Aug 16;554:602-14. doi: 10.1016/j.neuron.2007.07.026.

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H bonding at the helix-bundle crossing controls gating in Kir potassium channels.

Rapedius M , Fowler PW , Shang L , Sansom MS , Tucker SJ , Baukrowitz T .

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Specific stimuli such as intracellular H+ and phosphoinositides (e.g., PIP2) gate inwardly rectifying potassium (Kir) channels by controlling the reversible transition between the closed and open states. This gating mechanism underlies many aspects of Kir channel physiology and pathophysiology; however, its structural basis is not well understood. Here, we demonstrate that H+ and PIP2 use a conserved gating mechanism defined by similar structural changes in the transmembrane (TM) helices and the selectivity filter. Our data support a model in which the gating motion of the TM helices is controlled by an intrasubunit hydrogen bond between TM1 and TM2 at the helix-bundle crossing, and we show that this defines a common gating motif in the Kir channel superfamily. Furthermore, we show that this proposed H-bonding interaction determines Kir channel pH sensitivity, pH and PIP2 gating kinetics, as well as a K+-dependent inactivation process at the selectivity filter and therefore many of the key regulatory mechanisms of Kir channel physiology.

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Species referenced: Xenopus
Genes referenced: kcnj1 kcnj10 kcnj2 mapt

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	Figure 1. Assay to Measure the Kinetics of PIP2 Gating in Kir ChannelsKir channels were expressed in Xenopus oocytes and currents measured in excised inside-out patches.(A) Application of neomycin (Neo) to Kir1.1 channels as indicated; red line represents a monoexponential fit to the time course of recovery from neomycin inhibition; tauNeo value represents mean ± SEM, n = 5.(B) Channel rundown was induced by 1 mM Mg2+ and the omission of pyrophosphate and channels were reactivated with diC8-PIP2; red line represents a monoexponential fit to the time course of diC8-PIP2 reactivation; tauPIP2 value represents mean ± SEM, n = 11.(C) Time course of channel reactivation subsequent to complete rundown induced by 3 μM diC8-PIP2 (tauPIP2 = 16 ± 2 s, n = 5) and 30 μM diC8-PIP2 (tauPIP2 = 15 ± 3, n = 5); the rates were obtained in the same patch and not significantly different (p > 0.05); red lines represents monoexponential fits to the data.(D) Cartoon indicating that binding of PIP2 to the channel is fast and induces a slow conformational change subsequent to PIP2 binding that leads to pore opening. The latter process represents the measured diC8-PIP2 activation rate.
	Figure 2. A Residue at the Base of TM1 Controls the Speed of PIP2 Activation(A) Side view of the structural model of Kir1.1 (see Experimental Procedures for details) with one of the four subunits highlighted. The highlighted residues correspond to K80 and A177. TM1 is shown in green and TM2 in yellow with the slide helix in red. The expanded right panel shows the proximity and orientation of the side chain of K80 and the backbone carbonyl of A177.(B) Dose-response curves for neomycin inhibition of WT Kir1.1 and K80V channels fitted to a standard Hill function with a IC50 = 0.15 ± 0.05 mM and Hill coefficient of 3.7 ± 0.3 (WT) and IC50 = 0.16 ± 0.06 mM and Hill coefficient 2.8 ± 0.2 (K80V). Data points represent mean ± SEM of five experiments.(C) Time course of channel reactivation upon removal of 5 mM neomycin for WT and K80V channels.(D) Relative currents (IdiC8-PIP2/Ibefore run down) in response to diC8-PIP2 application subsequent to rundown for WT and K80V channels fitted to a standard Hill function with an EC50 and Hill coefficient of 0.9 ± 0.1 μM and 0.9 ± 0.1 for WT; 0.7 ± 0.1 μM and 1.1 ± 0.1 for K80V. Data points represent mean ± SEM of seven experiments.(E) Time course of channel reactivation upon application of 3 μM diC8-PIP2 for WT and K80V channels.
	Figure 3. TM1-TM2 H Bonding Determines the Rate of PIP2 Activation(A) Amino acid polarity (Engelman et al., 1986) plotted against the tau of channel activation (mean ± SEM of at least eight experiments) obtained with 3 μM diC8-PIP2 subsequent to complete rundown of Kir1.1 channels and the indicated mutants at the K80 position; note that there is no correlation between polarity and PIP2 activation rate. The right-hand panels depict the side chain of A177 with K80, K80Q, K80N, and K80S as well as the calculated minimum distance between the carbonyl oxygen of A177 and the terminal heavy atom of the residue at position 80. TM1 is highlighted in green and TM2 in yellow.(B) Time course of channel activation by 3 μM diC8-PIP2 for Kir1.1 WT and mutant channels as indicated.
	Figure 4. Conserved Role of TM1-TM2 H Bonding for Kir Channel PIP2 Gating(A) Time course for Kir4.1 channel rundown (induced by poly-lysine, only WT shown) and reactivation by 30 μM diC8-PIP2 for WT and indicated mutants. Right panel: bars represent tauPIP2 (mean ± SEM of at least five experiments) for diC8-PIP2 activation (30 μM) for WT and Kir4.1 mutants.(B) Time course for Kir2.1 channel rundown induced by 1 mM Mg2+ and the omission of pyrophosphate (only WT shown) and channel reactivation with 3 μM diC8-PIP2 for WT and with 30 μM diC8-PIP2 for M84K. Right panel: bars represent tauPIP2 (mean ± SEM of at least five experiments) for diC8-PIP2 activation (3 μM) for WT and M84K (30 μM).
	Figure 5. TM1-TM2 H Bonding Determines the pH Sensitivity of Kir ChannelsIC50 for pH inhibition (pH0.5) plotted for Kir1.1 WT (Lys-80) and indicated mutants and also Kir4.1 WT (Lys-67) and indicated mutants against the respective tauPIP2 for diC8-PIP2 activation as determined in Figures 3 and 4. The IC50 values for the Kir4.1 mutants represent an estimation because the low pH sensitivity of these mutants meant a complete dose-response curve could not be obtained (see Figure S2).
	Figure 6. TM1-TM2 H Bonding Controls pH Gating in Kir Channels(A) Time course of pH gating in Kir1.1 channels induced by changes in the intracellular pH established by a fast piezo-driven application system. Currents for the different pH values were equalized for better comparison (i.e., at pH 6.3 only 60% of the current was inhibited).(B) The inhibition and recovery time course from experiments as shown in (A) were fitted with monoexponential functions and tau values for pH inhibition (τon) and recovery from pH inhibition upon alkalization pH 8.0 (τoff) are plotted. Data points represent mean ± SEM of at least six experiments.(C) Time course of Kir1.1 currents upon K+ exchange (replacement with Na+ measured at +40 mV) are shown in gray and superimposed on the pH gating time course obtained in the same patch; red lines represent monoexponential fits for pH inhibition (τon) (mean ± SEM, n = 6) and recovery (τoff) (mean ± SEM, n = 6).(D) Time course of Kir1.1-K80V currents upon K+ exchange (replacement with Na+ measured at +40 mV) are shown in gray and superimposed on the pH gating time course obtained in the same patch; red lines represent monoexponential fits for pH inhibition (τon) (mean ± SEM, n = 7) and recovery (τoff) (mean ± SEM, n = 7).(E) Time course of pH inhibition (pH 4.5) for Kir1.1-K80 mutants and WT.(F) Time course of recovery from pH inhibition (pH 4.5) upon alkalization (pH 8.0) for Kir1.1-K80 mutants and WT.(G) From experiments such as in (E) and (F), the tau values for pH inhibition at pH 4.5 (τon) and recovery at pH 8.0 (τoff) are plotted. Data points represent mean ± SEM of at least five experiments.(H) Time course of pH inhibition and recovery for Kir2.1 and Kir2.1-M84K channels.
	Figure 7. PIP2 Depletion Induces K+-Dependent Inactivation Process at the Selectivity Filter(A) Rundown of Kir1.1 channels and lack of reactivation by 25 μM PIP2 in the absence of extracellular K+ (120 mM NMG+ solution), gray dotted line represents the time course of PIP2 reactivation typically obtained with 120 mM extracellular K+.(B) Bars represent normalized channel recovery induced by heparin (removal of poly-lysine, see Figure S3) subsequent to 45 s of channel inhibition by poly-lysine with extracellular 10 mM Ba2+ (added to a NMG+ solution), 120 mM K+, 120 mM Rb+, 120 mM Cs+, 120 mM Na+, and 120 mM NMG+. Data points represent mean ± SEM of at least four experiments.(C) Bars represent normalized channel recovery induced by heparin subsequent to 45 s of channel inhibition by poly-lysine with extracellular 120 mM NMG+ (zero K+ solution) for Kir1.1 WT, L136I, and V140T channels. Data points represent mean ± SEM of at least five experiments.(D) Dark bars represent normalized channel recovery induced by heparin (removal of poly-lysine) subsequent to 45 s of channel inhibition by poly-lysine with extracellular 120 mM NMG (zero K+ solution) for Kir1.1 WT, K80Q, K80N, and K80M channels; white bars represent normalized channel recovery induced by pH 8.0 subsequent to 45 s of H+ inhibition with extracellular 120 mM NMG+ (zero K+ solution) for Kir1.1 WT (pH 6.0), K80Q (pH 5.5), and K80V (pH 4.5). Data points represent mean ± SEM of at least four experiments.
	Figure 8. Gating Models of Kir1.1(A) Bottom-up view through the pore of the closed-state structure of Kir1.1. TM1 is shown in green, TM2 in yellow, and the slide helix in red. Residues Lys-80 and Ala-177 are shown as sticks and colored CPK. The H bond between the ɛ-nitrogen of Lys-80 and the backbone carbonyl oxygen of Ala-177 is thought to stabilize the closed state of the channel.(B) The predicted open-state structure of the pore of Kir1.1. This model is based upon the open-state structure of KirBac3.1 and highlights the relative movement between the TM helices. The minimum distance between Lys-80 and Ala-177 increases from <3 Å to >12 Å in the open state and would therefore rupture any H bond between these residues upon channel opening. The relative movement between the helices can be better visualized in the accompanying movie (see Supplemental Data, Movie S1).(C) Cartoon depicting the contribution of the TM1-TM2 H bond to collapse of the pore. TM1 is shown in green, TM2 in yellow, and the slide helix in red. Either PIP2 depletion or channel protonation induces channel closure at the helix-bundle crossing. With extracellular K+ removed (zero [K+]ext.), closed channels enter an inactivated state (pore collapse at the selectivity filter) in channels with TM1-TM2 H bonding (e.g., WT Kir1.1 or K80Q). In this situation the channel remains in an “inactivated state,” even when PIP2 is reapplied or the channel is deprotonated (upper gating pathway). However, in channels with no TM1-TM2 H bonding (e.g., K80V), inactivation is not possible and channels can open again upon the rebinding of PIP2 or channel deprotonation even in the absence of extracellular K+ (lower gating pathway). Note: the gate at the helix bundle crossing in the inactivated state (upper gating pathway) is shown to be open; however, this is just an assumption and requires further investigation.

References [+] :

Baukrowitz, PIP2 and PIP as determinants for ATP inhibition of KATP channels. 1998, Pubmed, Xenbase