Prokaryotic Kir Channel Research Articles

Conformational changes upon gating of KirBac1.1 into an open-activated state revealed by solid-state NMR and functional assays

The conformational changes required for activation and K+ conduction in inward-rectifier K+ (Kir) channels are still debated. These structural changes are brought about by lipid binding. It is unclear how this process relates to fast gating or if the intracellular and extracellular regions of the protein are coupled. Here, we examine the structural details of KirBac1.1 reconstituted into both POPC and an activating lipid mixture of 3:2 POPC:POPG (wt/wt). KirBac1.1 is a prokaryotic Kir channel that shares homology with human Kir channels. We establish that KirBac1.1 is in a constitutively active state in POPC:POPG bilayers through the use of real-time fluorescence quenching assays and Förster resonance energy transfer (FRET) distance measurements. Multidimensional solid-state NMR (SSNMR) spectroscopy experiments reveal two different conformers within the transmembrane regions of the protein in this activating lipid environment, which are distinct from the conformation of the channel in POPC bilayers. The differences between these three distinct channel states highlight conformational changes associated with an open activation gate and suggest a unique allosteric pathway that ties the selectivity filter to the activation gate through interactions between both transmembrane helices, the turret, selectivity filter loop, and the pore helix. We also identify specific residues involved in this conformational exchange that are highly conserved among human Kir channels.

Conformational changes at cytoplasmic intersubunit interactions control Kir channel gating

The defining structural feature of inward-rectifier potassium (Kir) channels is the unique Kir cytoplasmic domain. Recently we showed that salt bridges located at the cytoplasmic domain subunit interfaces (CD-Is) of eukaryotic Kir channels control channel gating via stability of a novel inactivated closed state. The cytoplasmic domains of prokaryotic and eukaryotic Kir channels show similar conformational rearrangements to the common gating ligand, phosphatidylinositol bisphosphate (PIP2), although these exhibit opposite coupling to opening and closing transitions. In Kir2.1, mutation of one of these CD-I salt bridge residues (R204A) reduces apparent PIP2 sensitivity of channel activity, and here we show that Ala or Cys substitutions of the functionally equivalent residue (Arg-165) in the prokaryotic Kir channel KirBac1.1 also significantly decrease sensitivity of the channel to PIP2 (by 5-30-fold). To further understand the structural basis of CD-I control of Kir channel gating, we examined the effect of the R165A mutation on PIP2-induced changes in channel function and conformation. Single-channel analyses indicated that the R165A mutation disrupts the characteristic long interburst closed state of reconstituted KirBac1.1 in giant liposomes, resulting in a higher open probability due to more frequent opening bursts. Intramolecular FRET measurements indicate that, relative to wild-type channels, the R165A mutation results in splaying of the cytoplasmic domains away from the central axis and that PIP2 essentially induces opposite motions of the major β-sheet in this channel mutant. We conclude that the removal of stabilizing CD-I salt bridges results in a collapsed state of the Kir domain.

Cholesterol Regulates Prokaryotic Kir Channel by Direct Binding to Channel Protein

Cholesterol is known to play a major role in regulation of variety of ion channels but the mechanisms underlying cholesterol sensitivity of ion channels are still poorly understood. In this study, we address the key question of whether cholesterol-induced regulation of ion channel function can be attributed to direct binding of the lipid to the channel protein and our observations provide the first direct evidence that cholesterol binds to an ion channel. Specifically, we show that cholesterol binds to a prokaryotic Kir channel, KirBac1.1 in a saturable way and that there is clear competition between radioactive and non-radioactive cholesterol for the binding site. There is no competition, however, between cholesterol and 5-Androsten 3β-17 β-diol, a sterol that we showed previously to have no effect on KirBac1.1 function. Furthermore, inhibition of cholesterol-KirBac1.1 binding by trifluoperazine, known to inhibit cholesterol binding to other proteins results in full recovery of the channel activity. Taken together, results from this study indicate that cholesterol-induced suppression of KirBac1.1 activity is mediated by direct interaction between cholesterol and the channel protein.

Crystal Structure of a Prokaryotic Kir Channel in an Open Conformation

KirBac channels are prokaryotic homologs of mammalian inwardly-rectifying (Kir) potassium channels and recent X-ray crystal structures of both Kir and KirBac channels have provided a major insight into their unique structural architecture. However, all of the available structures are closed at the helix bundle-crossing and therefore the structural mechanisms which control opening of their primary activation gate remain unknown. Previous studies suggest that the open state of the Kir channel state is energetically unstable and will thus be difficult to trap in a crystal form. To circumvent this problem we have engineered KirBac3.1 to trap the bundle-crossing in an apparently open conformation, and we present the crystal structure of this mutant channel at 3.05 A resolution. Contrary to previous speculation, this novel structure now suggests a mechanistic model in which rotational ‘twist’ of the intracellular assembly is converted into opening of the bundle-crossing gate via a network of inter- and intra-subunit interactions that couples the TM2 C-linker to the slide-helix, the G-loop and the CD-loop.

Comparison of the Structure of a Bacterial Potassium Channel in Both 2D and 3D Crystals

Inwardly-rectifying potassium (Kir) channels regulate membrane electrical excitability and K+ transport in many cell types where they control such diverse processes as heart rate, vascular tone, insulin secretion and salt/fluid balance. Their physiological importance is highlighted by the fact that genetically inherited defects in Kir channels are responsible for a wide-range of channelopathies. To elucidate how channel function becomes defective in the disease state requires a detailed understanding of channel structure in both the open and closed states, but to date detailed information about the open state structure of the Kir channel is lacking. In this work, we have used EM analysis of 2D crystals of a prokaryotic Kir channel trapped in an open state and compared these results with an open state structure of the same channel that our lab recently determined by X-ray crystallography at 3A resolution. Intriguingly, the projection maps from the EM experiments suggest a larger opening of the pore in the 2D crystal form compared to that observed in the 3D crystal structure. The organization of these two crystal forms is different and suggests that the 2D crystals may permit stabilisation of an open state structure that is not compatible with 3D crystallisation. These results not only have major implications for our understanding of the open state structure of the Kir channel, but more importantly they demonstrate the general utility and importance of methods such as electron microscopy and 2D crystallography for the study of membrane protein structure.This work has been supported by the European Commission under the Marie Curie Programme, the Wellcome Trust and the BBSRC.

Cholesterol regulates prokaryotic Kir channel by direct binding to channel protein

Cholesterol is a major regulator of a variety of ion channels but the mechanisms underlying cholesterol sensitivity of ion channels are still poorly understood. The key question is whether cholesterol regulates ion channels by direct binding to the channel protein or by altering the physical environment of lipid bilayer. In this study, we provide the first direct evidence that cholesterol binds to prokaryotic Kir channels, KirBac1.1, and that cholesterol binding is essential for its regulatory effect. Specifically, we show that cholesterol is eluted together with the KirBac1.1 protein when separated on an affinity column and that the amount of bound cholesterol is proportional to the amount of the protein. We also show that cholesterol binding to KirBac1.1 is saturable with a KD of 390μM. Moreover, there is clear competition between radioactive and non-radioactive cholesterol for the binding site. There is no competition, however, between cholesterol and 5-Androsten 3β-17 β-diol, a sterol that we showed previously to have no effect on KirBac1.1 function. Finally, we show that cholesterol–KirBac1.1 binding is significantly inhibited by trifluoperazine, known to inhibit cholesterol binding to other proteins, and that inhibition of cholesterol–KirBac1.1 binding results in full recovery of the channel activity. Collectively, results from this study indicate that cholesterol-induced suppression of KirBac1.1 activity is mediated by direct interaction between cholesterol and the channel protein.

Cholesterol Binding Regulates Prokaryotic Kir Channels

Cholesterol is a major regulator of a variety of ion channels but the mechanisms underlying cholesterol sensitivity of ion channels are still poorly understood. The key question is whether cholesterol regulates ion channels by specific binding to the channel protein or by altering the physical environment of lipid bilayer. In this study, we provide the first direct evidence that cholesterol specifically binds to prokaryotic Kir channels, KirBac1.1, and that cholesterol binding is essential for its regulatory effect. Specifically, we show that cholesterol is eluted together with the KirBac1.1 protein when separated on an affinity column and that the amount of bound cholesterol is proportional to the amount of the protein. We also show that cholesterol binding to KirBac1.1 is saturable with a Km of 390 μM. Moreover, there is clear competition between radioactive and non-radioactive cholesterol for the binding site. There is no competition, however, between cholesterol and 5-Androsten 3β-17[]β-diol, a sterol that we showed previously to have no effect on KirBac1.1 function. Finally, we show that cholesterol-KirBac1.1 binding is significantly inhibited by trifluoperazine, known to inhibit cholesterol binding to other proteins, and that inhibition of cholesterol-KirBac1.1 binding results in full recovery of the channel function. We conclude, therefore, that cholesterol-induced suppression of KirBac1.1 activity is mediated by direct interaction between cholesterol and the channel protein.

Gating of a G protein-sensitive Mammalian Kir3.1 Prokaryotic Kir Channel Chimera in Planar Lipid Bilayers

Kir3 channels control heart rate and neuronal excitability through GTP-binding (G) protein and phosphoinositide signaling pathways. These channels were the first characterized effectors of the βγ subunits of G proteins. Because we currently lack structures of complexes between G proteins and Kir3 channels, their interactions leading to modulation of channel function are not well understood. The recent crystal structure of a chimera between the cytosolic domain of a mammalian Kir3.1 and the transmembrane region of a prokaryotic KirBac1.3 (Kir3.1 chimera) has provided invaluable structural insight. However, it was not known whether this chimera could form functional K(+) channels. Here, we achieved the functional reconstitution of purified Kir3.1 chimera in planar lipid bilayers. The chimera behaved like a bona fide Kir channel displaying an absolute requirement for PIP(2) and Mg(2+)-dependent inward rectification. The channel could also be blocked by external tertiapin Q. The three-dimensional reconstruction of the chimera by single particle electron microscopy revealed a structure consistent with the crystal structure. Channel activity could be stimulated by ethanol and activated G proteins. Remarkably, the presence of both activated Gα and Gβγ subunits was required for gating of the channel. These results confirm the Kir3.1 chimera as a valid structural and functional model of Kir3 channels.

Direct Regulation of Prokaryotic Kir Channel by Cholesterol

Our earlier studies have shown that channel activity of Kir2 sub-family of inward rectifiers is strongly suppressed by the elevation of cellular cholesterol. The goal of this study is to determine whether cholesterol suppresses Kir channels directly. To achieve this goal, purified prokaryotic Kir (KirBac1.1) channels were incorporated into liposomes of defined lipid composition and channel activity was assayed by 86Rb+ uptake. Our results show that 86Rb+ flux through KirBac1.1 is strongly inhibited by cholesterol.

Direct Regulation of Prokaryotic Kir Channel by Cholesterol

Our earlier studies have shown that channel activity of Kir2 subfamily of inward rectifiers is strongly suppressed by the elevation of cellular cholesterol. The goal of this study is to determine whether cholesterol suppresses Kir channels directly. To achieve this goal, purified prokaryotic Kir (KirBac1.1) channels were incorporated into liposomes of defined lipid composition, and channel activity was assayed by (86)Rb(+) uptake. Our results show that (86)Rb(+) flux through KirBac1.1 is strongly inhibited by cholesterol. Incorporation of 5% (mass cholesterol/phospholipid) cholesterol into the liposome suppresses (86)Rb(+) flux by >50%, and activity is completely inhibited at 12-15%. However, epicholesterol, a stereoisomer of cholesterol with similar physical properties, has significantly less effect on KirBac-mediated (86)Rb(+) uptake than cholesterol. Furthermore, analysis of multiple sterols suggests that cholesterol-induced inhibition of KirBac1.1 channels is mediated by specific interactions rather than by changes in the physical properties of the lipid bilayer. In contrast to the inhibition of KirBac1.1 activity, cholesterol had no effect on the activity of reconstituted KscA channels (at up to 250 microg/mg of phospholipid). Taken together, these observations demonstrate that cholesterol suppresses Kir channels in a pure protein-lipid environment and suggest that the interaction is direct and specific.

Structural Reorganisation of a K+ Channel Pore During Gating

While single channel recordings readily discern whether a K+ pore is in a conducting or non-conducting state, the same does not apply to crystal structures of K+ channels. There is at present no cogent means for demarcation of physiological state, or for pinpointing non-native structure arising when a channel is removed from the natural environment of a bilayer. The nature of molecular reconfiguration during gating remains indistinct (other than significant variability in width of the intracellular aperture). Defining a consistent set of molecular indicators of physiological state would lay a solid foundation for interpretation of K+ channel structure, and enhance its usefulness to the biophysics community.The architecture of the potassium selective pore is conserved in all known K+ channels indicating that the core of the gating process may be universal, even though the means by which opening is instigated differs between families. We have targeted a single type of potassium channel for structure determination in conducting, intermediate, and non-conducting states. Our rationale is that excluding effects due to familial differences will pin down structural features reflecting the physiological gating transition. This initial study uses prokaryotic Kir channels, which are a good structural model for eukaryotic inward rectifiers. Our data suggest that discrete structural fingerprints characterise open and closed states.An earlier study in collaboration with D.A. Doyle yielded the structure of a prokaryotic inward rectifier (KirBac1.1). Complete obstruction of the pore by phenylalanine side chains denoted an unambiguously non-conductive state, providing a structural reference point. A close homologue, KirBac3.1, has now yielded several further structures that collectively reveal a pattern of molecular rearrangement. A consistent sketch is emerging. Additionally, a reduction to 2-fold symmetry in some structures, particularly pronounced in the intracellular regions, indicates that Kir channel opening occurs stepwise.

Crystal structure of a Kir3.1-prokaryotic Kir channel chimera

The Kir3.1 K(+) channel participates in heart rate control and neuronal excitability through G-protein and lipid signaling pathways. Expression in Escherichia coli has been achieved by replacing three fourths of the transmembrane pore with the pore of a prokaryotic Kir channel, leaving the cytoplasmic pore and membrane interfacial regions of Kir3.1 origin. Two structures were determined at 2.2 A. The selectivity filter is identical to the Streptomyces lividans K(+) channel within error of measurement (r.m.s.d.<0.2 A), suggesting that K(+) selectivity requires extreme conservation of three-dimensional structure. Multiple K(+) ions reside within the pore and help to explain voltage-dependent Mg(2+) and polyamine blockade and strong rectification. Two constrictions, at the inner helix bundle and at the apex of the cytoplasmic pore, may function as gates: in one structure the apex is open and in the other, it is closed. Gating of the apex is mediated by rigid-body movements of the cytoplasmic pore subunits. Phosphatidylinositol 4,5-biphosphate-interacting residues suggest a possible mechanism by which the signaling lipid regulates the cytoplasmic pore.

Functional Characterization of a Prokaryotic Kir Channel

The Kir gene family encodes inward rectifying K+ (Kir) channels that are widespread and critical regulators of excitability in eukaryotic cells. A related gene family (KirBac) has recently been identified in prokaryotes. While a crystal structure of one member, KirBac1.1, has been solved, there has been no functional characterization of any KirBac gene products. Here we present functional characterization of KirBac1.1 reconstituted in liposomes. Utilizing a 86Rb+ uptake assay, we demonstrate that KirBac1.1 generates a K+-selective permeation path that is inhibited by extraliposomal Ba2+ and Ca2+ ions. In contrast to KcsA (an acid-activated bacterial potassium channel), KirBac1.1 is inhibited by extraliposomal acid (pKa ~ 6). This characterization of KirBac1.1 activity now paves the way for further correlation of structure and function in this model Kir channel.

Crystal structure of the potassium channel KirBac1.1 in the closed state.

The KirBac1.1 channel belongs to the inward-rectifier family of potassium channels. Here we report the structure of the entire prokaryotic Kir channel assembly, in the closed state, refined to a resolution of 3.65 angstroms. We identify the main activation gate and structural elements involved in gating. On the basis of structural evidence presented here, we suggest that gating involves coupling between the intracellular and membrane domains. This further suggests that initiation of gating by membrane or intracellular signals represents different entry points to a common mechanistic pathway.