Lijun Shang

H Bonding at the Helix-Bundle Crossing Controls Gating in Kir Potassium Channels

Summary 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.

Structural and functional analysis of the putative pH sensor in the Kir1.1 (ROMK) potassium channel

The pH‐sensitive renal potassium channel Kir1.1 is important for K+ homeostasis. Disruption of the pH‐sensing mechanism causes type II Bartter syndrome. The pH sensor is thought to be an anomalously titrated lysine residue (K80) that interacts with two arginine residues as part of an ‘RKR triad’. We show that a Kir1.1 orthologue from Fugu rubripes lacks this lysine and yet is still highly pH sensitive, indicating that K80 is not the H+ sensor. Instead, K80 functionally interacts with A177 on transmembrane domain 2 at the ‘helix‐bundle crossing’ and controls the ability of pH‐dependent conformational changes to induce pore closure. Although not required for pH inhibition, K80 is indispensable for the coupling of pH gating to the extracellular K+ concentration, explaining its conservation in most Kir1.1 orthologues. Furthermore, we demonstrate that instead of interacting with K80, the RKR arginine residues form highly conserved inter‐ and intra‐subunit interactions that are important for Kir channel gating and influence pH sensitivity indirectly.

Functional analysis of missense variants in the TRESK ( KCNK18 ) K + channel

A loss of function mutation in the TRESK K2P potassium channel (KCNK18), has recently been linked with typical familial migraine with aura. We now report the functional characterisation of additional TRESK channel missense variants identified in unrelated patients. Several variants either had no apparent functional effect, or they caused a reduction in channel activity. However, the C110R variant was found to cause a complete loss of TRESK function, yet is present in both sporadic migraine and control cohorts, and no variation in KCNK18 copy number was found. Thus despite the previously identified association between loss of TRESK channel activity and migraine in a large multigenerational pedigree, this finding indicates that a single non-functional TRESK variant is not alone sufficient to cause typical migraine and highlights the genetic complexity of this disorder.

Control of pH and PIP2 gating in heteromeric Kir4.1/Kir5.1 channels by H-Bonding at the helix-bundle crossing.

Inhibition by intracellular H+ (pH gating) and activation by phosphoinositides such as PIP2 (PIP2 gating) are key regulatory mechanisms in the physiology of inwardly-rectifying potassium (Kir) channels. Our recent findings suggest that PIP2 gating and pH gating are controlled by an intrasubunit H-bond at the helix-bundle crossing between a lysine in TM1 and a backbone carbonyl group in TM2. This interaction only occurs in the closed state and channel opening requires this H-bond to be broken, thereby influencing the kinetics of PIP2- and pH-gating in Kir channels. In this addendum, we explore the role of H-bonding in heteromeric Kir4.1/Kir5.1 channels. Kir5.1 subunits do not possess a TM1 lysine. However, homology modelling and molecular dynamics simulations demonstrate that the TM1 lysine in Kir4.1 is capable of H-bonding at the helix-bundle crossing. Consistent with this, the rates of pH and PIP2 gating in Kir4.1/Kir5.1 channels (two H-bonds) were intermediate between those of wild-type homomeric Kir4.1 (four H-bonds) and Kir4.1(K67M) channels (no H-bonds) suggesting that the number of H-bonds in the tetrameric channel complex determines the gating kinetics. Furthermore, in heteromeric Kir4.1(K67M)/Kir5.1 channels, where the two remaining H-bonds are disrupted, we found that the gating kinetics were similar to Kir4.1(K67M) homomeric channels despite the fact that these two channels differ considerably in their PIP2 affinities. This indicates that Kir channel PIP2 affinity has little impact on either the PIP2- or pH-gating kinetics.

Genetic inactivation of KCNJ16 identifies Kir5.1 as an important determinant of neuronal PCO2/pH sensitivity

The molecular identity of ion channels which confer PCO2/pH sensitivity in the brain is unclear. Heteromeric Kir4.1/Kir5.1 channels are highly sensitive to inhibition by intracellular pH and are widely expressed in several brainstem nuclei involved in cardiorespiratory control, including the locus coeruleus. This has therefore led to a proposed role for these channels in neuronal CO2 chemosensitivity. To examine this, we generated mutant mice lacking the Kir5.1 (Kcnj16) gene. We show that although locus coeruleus neurons from Kcnj16(+/+) mice rapidly respond to cytoplasmic alkalinization and acidification, those from Kcnj16(−/−) mice display a dramatically reduced and delayed response. These results identify Kir5.1 as an important determinant of PCO2/pH sensitivity in locus coeruleus neurons and suggest that Kir5.1 may be involved in the response to hypercapnic acidosis.

Random mutagenesis screening indicates the absence of a separate H + -sensor in the pH-sensitive Kir channels

Several inwardly-rectifying (Kir) potassium channels (Kir1.1, Kir4.1 and Kir4.2) are characterised by their sensitivity to inhibition by intracellular H+ within the physiological range. The mechanism by which these channels are regulated by intracellular pH has been the subject of intense scrutiny for over a decade, yet the molecular identity of the titratable pH-sensor remains elusive. In this study we have taken advantage of the acidic intracellular environment of S. cerevisiae and used a K+-auxotrophic strain to screen for mutants of Kir1.1 with impaired pH-sensitivity. In addition to the previously identified K80M mutation, this unbiased screening approach identified a novel mutation (S172T) in the second transmembrane domain (TM2) that also produces a marked reduction in pH-sensitivity through destabilization of the closed-state. However, despite this extensive mutagenic approach, no mutations could be identified which removed channel pH-sensitivity or which were likely to act as a separate H+-sensor unique to the pH-sensitive Kir channels. In order to explain these results we propose a model in which the pH-sensing mechanism is part of an intrinsic gating mechanism common to all Kir channels, not just the pH-sensitive Kir channels. In this model, mutations which disrupt this pH-sensor would result in an increase, not reduction, in pH-sensitivity. This has major implications for any future studies of Kir channel pH-sensitivity and explains why formal identification of these pH-sensing residues still represents a major challenge.

Non-equivalent role of TM2 gating hinges in heteromeric Kir4.1/Kir5.1 potassium channels

Comparison of the crystal structures of the KcsA and MthK potassium channels suggests that the process of opening a K+ channel involves pivoted bending of the inner pore-lining helices at a highly conserved glycine residue. This bending motion is proposed to splay the transmembrane domains outwards to widen the gate at the “helix-bundle crossing”. However, in the inwardly rectifying (Kir) potassium channel family, the role of this “hinge” residue in the second transmembrane domain (TM2) and that of another putative glycine gating hinge at the base of TM2 remain controversial. We investigated the role of these two positions in heteromeric Kir4.1/Kir5.1 channels, which are unique amongst Kir channels in that both subunits lack a conserved glycine at the upper hinge position. Contrary to the effect seen in other channels, increasing the potential flexibility of TM2 by glycine substitutions at the upper hinge position decreases channel opening. Furthermore, the contribution of the Kir4.1 subunit to this process is dominant compared to Kir5.1, demonstrating a non-equivalent contribution of these two subunits to the gating process. A homology model of heteromeric Kir4.1/Kir5.1 shows that these upper “hinge” residues are in close contact with the base of the pore α-helix that supports the selectivity filter. Our results also indicate that the highly conserved glycine at the “lower” gating hinge position is required for tight packing of the TM2 helices at the helix-bundle crossing, rather than acting as a hinge residue.

Peptide Backbone Mutagenesis of Putative Gating Hinges in a Potassium Ion Channel

The dynamic movements that underlie the transitions between the closed and open states of a potassium ion channel are still not fully understood. Most potassium channels contain a highly conserved glycine residue in the second transmembrane domain (TM2) that is thought to act as a flexible gating hinge. However, inwardly rectifying (Kir) potassium channels also possess an additional invariant glycine at the base of TM2 near the helix-bundle crossing that has been proposed to contribute to TM2 flexibility during channel gating similar to the PVP motif in voltage-gated potassium channels. In this study we have addressed the relative contribution of these putative glycine gating hinges by using unnatural amino acid mutagenesis to introduce a-hydroxyacetic acid (aG) and thereby an amideto-ester mutation into the backbone of Kir2.1 at the upper (G168) and lower (G177) glycine positions. This mutation is predicted to increase the flexibility of the TM2 backbone at these positions without altering the side chain. We show that introduction of aG at the upper gating hinge position produces channels that open more slowly at hyperpolarized potentials whereas aG at the lower glycine position does not affect channel gating. These results are consistent with a structural model in which K channel gating involves bending of the inner pore helix (TM2) at or near the upper glycine, but where the lower glycine found in Kir channels is more likely to be required for tight packing of the TM2 helices at the helix-bundle crossing rather than acting as a gating hinge. Understanding the conformational changes that occur as a potassium channel undergoes the reversible transition between the open and closed states still represents one of the major challenges in ion channel structural biology. Comparison of the X-ray crystal structures of the KcsA K channel in the closed state with that of the MthK channel in the open state has led to a model of K channel gating in which the physical gate is formed by the four pore-lining TM2 helices crossing over each other near its intracellular entrance to constrict (i.e. , close) the pore. During channel opening it is proposed that the TM2 helices bend in the middle and splay outwards so that the gate at the lower “helix bundle crossing” widens thus forming an open pathway from the cytoplasm to the selectivity filter. 2] In the open-state MthK crystal structure the TM2 helix is bent at a highly conserved glycine residue. This glycine residue is conserved in >80% of K channel sequences, and functional studies of the KCNQ1 and NaChBac channels support a model in which pivoted bending of this glycine hinge occurs during channel gating. However, functional studies of the Kv Shaker family and the structure of Kv1.2 suggest that the helices are rigid at this point and instead bend at a highly conserved PVP motif lower down in S6 ACHTUNGTRENNUNG(TM2) within the helix bundle crossing. Understanding the role of the putative TM2 hinge in Kir channels is yet more complex because these channels have two highly conserved glycine residues in TM2; one at the higher MthK glycine hinge position, the other lower down closer to the helix bundle crossing in a position which aligns with the Shaker PVP motif (Figure 1). 8, 9]

State-dependent network connectivity determines gating in a K + channel

Summary X-ray crystallography has provided tremendous insight into the different structural states of membrane proteins and, in particular, of ion channels. However, the molecular forces that determine the thermodynamic stability of a particular state are poorly understood. Here we analyze the different X-ray structures of an inwardly rectifying potassium channel (Kir1.1) in relation to functional data we obtained for over 190 mutants in Kir1.1. This mutagenic perturbation analysis uncovered an extensive, state-dependent network of physically interacting residues that stabilizes the pre-open and open states of the channel, but fragments upon channel closure. We demonstrate that this gating network is an important structural determinant of the thermodynamic stability of these different gating states and determines the impact of individual mutations on channel function. These results have important implications for our understanding of not only K+ channel gating but also the more general nature of conformational transitions that occur in other allosteric proteins.

Kir5.1 underlies long-lived subconductance levels in heteromeric Kir4.1/Kir5.1 channels from Xenopus tropicalis.

The inwardly-rectifying potassium channel subunit Kir5.1 selectively co-assembles with members of the Kir4.0 subfamily to form novel pH-sensitive heteromeric channels with unique single channel properties. In this study, we have cloned orthologs of Kir4.1 and Kir5.1 from the genome of the amphibian, Xenopus tropicalis (Xt). Heteromeric XtKir4.1/XtKir5.1 channels exhibit similar macroscopic current properties to rat Kir4.1/Kir5.1 with a faster time-dependent rate of activation. However, single channel analysis of heteromeric XtKir4.1/XtKir5.1 channels reveals that they have markedly different long-lived, multi-level subconductance states. Furthermore, we demonstrate that the XtKir5.1 subunit is responsible for these prominent subconductance levels. These results are consistent with a model in which the slow transitions between sublevel states represent the movement of individual subunits. These novel channels now provide an excellent model system to determine the structural basis of subconductance levels and contribution of heteromeric pore architecture to this process.