Dissecting the Molecular Mechanism of Nucleotide-Dependent Activation of the KtrAB K+ Transporter

KtrAB belongs to the Trk/Ktr/HKT superfamily of monovalent cation (K⁺ and Na⁺) transport proteins that closely resemble K⁺ channels. These proteins underlie a plethora of cellular functions that are crucial for environmental adaptation in plants, fungi, archaea, and bacteria. The activation mechanism of the Trk/Ktr/HKT proteins remains unknown. It has been shown that ATP stimulates the activity of KtrAB while ADP does not. Here, we present X-ray structural information on the KtrAB complex with bound ADP. A comparison with the KtrAB-ATP structure reveals conformational changes in the ring and in the membrane protein. In combination with a biochemical and functional analysis, we uncover how ligand-dependent changes in the KtrA ring are propagated to the KtrB membrane protein and conclude that, despite their structural similarity, the activation mechanism of KtrAB is markedly different from the activation mechanism of K⁺ channels.     

KtrB, a member of the superfamily of K+ transporters

KtrB is the K(+)-translocating subunit of the K(+)-uptake system KtrAB from bacteria. It is a member of the superfamily of K(+)transporters (SKT proteins) with other sub-families occurring in archaea, bacteria, fungi, plants and trypanosomes. SKT proteins may have originated from small K(+) channels by at least two gene duplication and two gene fusion events. They contain four covalently linked M(1)PM(2) domains, in which M(1) and M(2) stand for transmembrane stretches, and P for a P-loop, which folds back from the external medium into the membrane. SKT proteins distinguish themselves in two important aspects from K(+) channels: first, with just one conserved glycine residue in their P-loops they contain a much simpler K(+)-selectivity filter sequence than K(+) channels with their conserved Thr-Val-Gly-Tyr-Gly sequence. Secondly, the middle part M(2C2) from the long transmembrane stretch M(2C) of KtrB from the bacterium Vibrio alginolyticus forms a gate inside the membrane, which prevents K(+) permeation to the cytoplasm. Beside the mechanism of K(+) transport via KtrB and other SKT proteins existing hypotheses of how the KtrA protein regulates the K(+)-transport activity of KtrB are discussed.     

All four putative selectivity filter glycine residues in KtrB are essential for high affinity and selective K+ uptake by the KtrAB system from Vibrio alginolyticus

The subunit KtrB of bacterial Na+-dependent K+-translocating KtrAB systems belongs to a superfamily of K+ transporters. These proteins contain four repeated domains, each composed of two transmembrane helices connected by a putative pore loop (p-loop). The four p-loops harbor a conserved glycine residue at a position equivalent to a glycine selectivity filter residue in K+ channels. We investigated whether these glycines also form a selectivity filter in KtrB. The single residues Gly70, Gly185, Gly290, and Gly402 from p-loops P(A) to P(D) of Vibrio alginolyticus KtrB were replaced with alanine, serine, or aspartate. The three alanine variants KtrB(A70), KtrB(A185), and KtrB(A290) maintained a substantial activity in KtrAB-mediated K+ uptake in Escherichia coli. This activity was associated with a decrease in the affinity for K+ by 2 orders of magnitude, with little effect on Vmax. Minor activities were also observed for three other variants: KtrB(A402), KtrB(S70), and KtrB(D185). With all of these variants, the property of Na+ dependence of K+ transport was preserved. Only the four serine variants mediated Na+ uptake, and these variants differed considerably in their K+/Na+ selectivity. Experiments on cloned ktrB in the pBAD18 vector showed that V. alginolyticus KtrB alone was still active in E. coli. It mediated Na+-independent, slow, high affinity, and mutation-specific K+ uptake as well as K+-independent Na+ uptake. These data demonstrate that KtrB contains a selectivity filter for K+ ions and that all four conserved p-loop glycine residues are part of this filter. They also indicate that the role of KtrA lies in conferring velocity and ion coupling to the Ktr complex.     

The RCK domain of the KtrAB K+ transporter: multiple conformations of an octameric ring

The KtrAB ion transporter is a complex of the KtrB membrane protein and KtrA, an RCK domain. RCK domains regulate eukaryotic and prokaryotic membrane proteins involved in K(+) transport. Conflicting functional models have proposed two different oligomeric arrangements for RCK domains, tetramer versus octamer. Our results for the KtrAB RCK domain clearly show an octamer in solution and in the crystal. We determined the structure of this protein in three different octameric ring conformations that resemble the RCK-domain octamer observed in the MthK potassium channel but show striking differences in size and symmetry. We present experimental evidence for the association between one RCK octameric ring and two KtrB membrane proteins. These results provide insights into the quaternary organization of the KtrAB transporter and its mechanism of activation and show that the RCK-domain octameric ring model is generally applicable to other ion-transport systems.     

Characterization of the molecular properties of KtrC,a second RCK domain that regulates a Ktr channel in Bacillus subtilis

RCK (regulating conductance of K⁺) domains are common regulatory domains that control the activity of eukaryotic and prokaryotic K⁺ channels and transporters. In bacteria these domains play roles in osmoregulation, regulation of turgor and membrane potential and in pH homeostasis. Whole-genome sequencing unveiled RCK gene redundancy, however the biological role of this redundancy is not well understood. In Bacillus subtilis, there are two closely related RCK domain proteins (KtrA and KtrC) that regulate the activity of the Ktr cation channels. KtrA has been well characterized but little is known about KtrC. We have characterized the structural and biochemical proprieties of KtrC and conclude that KtrC binds ATP and ADP, just like KtrA. However, in solution KtrC exist in a dynamic equilibrium between octamers and non-octameric species that is dependent on the bound ligand, with ATP destabilizing the octameric ring relative to ADP. Accordingly, KtrC-ADP crystal structures reveal closed octameric rings similar to those in KtrA, while KtrC-ATP adopts an open assembly with RCK domains forming a super-helix. In addition, both KtrC-ATP and -ADP octamers are stabilized by the signaling molecule cyclic-di-AMP, which binds to KtrC with high affinity. In contrast, c-di-AMP binds with 100-fold lower affinity to KtrA. Despite these differences we show with an E. coli complementation assay that KtrC and KtrA are interchangeable and able to form functional transporters with both KtrB and KtrD. The distinctive properties of KtrC, in particular ligand-dependent assembly/disassembly, suggest that this protein has a specific physiological role that is distinct from KtrA.     

Answers and questions from the KvAP structures

The recent landmark structures of KvAP, a voltage-gated potassium (Kv) channel, provide the first high-resolution experimental structural models of this class of proteins. Previous extensive studies of Kv channels provide a means to evaluate and interpret the KvAP structures. In this minireview, we survey different experimental approaches to Kv channels and map these findings to KvAP, showing that the relationship between the KvAP structures and other Kv channels is uncertain.     

1H, 13C and 15N chemical shift assignments for the N-terminal PAS domain of the KCNH channel from Zebrafish

The KCNH channels are voltage-gated potassium channels that play important roles in heart and nerve cells. The N-terminal region of the KCNH channel contains a Per-Arnt-Sim (PAS) domain which is important for the channel gating through interaction with other regions of the channel. To study the solution structure of the N-terminal PAS domain of the KCNH channel from Zebrafish (zNTD), we over-expressed and purified zNTD. We report the resonance assignments for zNTD. The data will allow us to perform structural studies for this domain, which will provide insight into its structural basis for the molecular interaction with other regions of the KCNH channel.     

A sodium-mediated structural switch that controls the sensitivity of Kir channels to PtdIns(4,5)P(2)

Inwardly rectifying potassium (Kir) channels are gated by the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)). Among them, Kir3 requires additional molecules, such as the betagamma subunits of G proteins or intracellular sodium, for channel gating. Using an interactive computational-experimental approach, we show that sodium sensitivity of Kir channels involves the side chains of an aspartate and a histidine located across from each other in a crucial loop in the cytosolic domain, as well as the backbone carbonyls of two more residues and a water molecule. The location of the coordination site in the vicinity of a conserved arginine shown to affect channel-PtdIns(4,5)P(2) interactions suggests that sodium triggers a structural switch that frees the crucial arginine. Mutations of the aspartate and the histidine that affect sodium sensitivity also enhance the channel's sensitivity to PtdIns(4,5)P(2). Furthermore, on the basis of the molecular characteristics of the coordination site, we identify and confirm experimentally a sodium-sensitive phenotype in Kir5.1.     

Structural and functional consequences of the presence of a fourth disulfide bridge in the scorpion short toxins: solution structure of the potassium channel inhibitor HsTX1

We have determined the three-dimensional structure of the potassium channel inhibitor HsTX1, using nuclear magnetic resonance and molecular modeling. This protein belongs to the scorpion short toxin family, which essentially contains potassium channel blockers of 29 to 39 amino acids and three disulfide bridges. It is highly active on voltage-gated Kv1.3 potassium channels. Furthermore, it has the particularity to possess a fourth disulfide bridge. We show that HsTX1 has a fold similar to that of the three-disulfide-bridged toxins and conserves the hydrophobic core found in the scorpion short toxins. Thus, the fourth bridge has no influence on the global conformation of HsTX1. Most residues spatially analogous to those interacting with voltage-gated potassium channels in the three-disulfide-bridged toxins are conserved in HsTX1. Thus, we propose that Tyr21, Lys23, Met25, and Asn26 are involved in the biological activity of HsTX1. As an additional positively charged residue is always spatially close to the aromatic residue in toxins blocking the voltage-gated potassium channels, and as previous mutagenesis experiments have shown the critical role played by the C-terminus in HsTX1, we suggest that Arg33 is also important for the activity of the four disulfide-bridged toxin. Docking calculations confirm that, if Lys23 and Met25 interact with the GYGDMH motif of Kv1.3, Arg33 can contact Asp386 and, thus, play the role of the additional positively charged residue of the toxin functional site. This original configuration of the binding site of HsTX1 for Kv1.3, if confirmed experimentally, offers new structural possibilities for the construction of a molecule blocking the voltage-gated potassium channels.     

Three-dimensional structure of the S4-S5 segment of the Shaker potassium channel

The propagation of action potentials during neuronal signal transduction in phospholipid membranes is mediated by ion channels, a diverse group of membrane proteins. The S4-S5 linker peptide (S4-S5), that connects the S4 and S5 transmembrane segments of voltage-gated potassium channels is an important region of the Shaker ion-channel protein. Despite its importance, very little is known about its structure. Here we provide evidence for an amphipathic alpha-helical conformation of a synthetic S4-S5 peptide of the voltage-gated Drosophila melanogaster Shaker potassium channel in water/trifluoroethanol and in aqueous phospholipid micelles. The three-dimensional solution structures of the S4-S5 peptide were obtained by high-resolution nuclear magnetic resonance spectroscopy and distance-geometry/simulated-annealing calculations. The detailed structural features are discussed with respect to model studies and available mutagenesis data on the mechanism and selectivity of the potassium channel.     

Functional diversity of potassium channel voltage-sensing domains

Voltage-gated potassium channels or Kv's are membrane proteins with fundamental physiological roles. They are composed of 2 main functional protein domains, the pore domain, which regulates ion permeation, and the voltage-sensing domain, which is in charge of sensing voltage and undergoing a conformational change that is later transduced into pore opening. The voltage-sensing domain or VSD is a highly conserved structural motif found in all voltage-gated ion channels and can also exist as an independent feature, giving rise to voltage sensitive enzymes and also sustaining proton fluxes in proton-permeable channels. In spite of the structural conservation of VSDs in potassium channels, there are several differences in the details of VSD function found across variants of Kvs. These differences are mainly reflected in variations in the electrostatic energy needed to open different potassium channels. In turn, the differences in detailed VSD functioning among voltage-gated potassium channels might have physiological consequences that have not been explored and which might reflect evolutionary adaptations to the different roles played by Kv channels in cell physiology.     

A helical region in the C terminus of small-conductance Ca2+-activated K+ channels controls assembly with apo-calmodulin.

Small conductance Ca(2+)-activated potassium (SK) channels underlie the afterhyperpolarization that follows the action potential in many types of central neurons. SK channels are voltage-independent and gated solely by intracellular Ca(2+) in the submicromolar range. This high affinity for Ca(2+) results from Ca(2+)-independent association of the SK alpha-subunit with calmodulin (CaM), a property unique among the large family of potassium channels. Here we report the solution structure of the calmodulin binding domain (CaMBD, residues 396-487 in rat SK2) of SK channels using NMR spectroscopy. The CaMBD exhibits a helical region between residues 423-437, whereas the rest of the molecule lacks stable overall folding. Disruption of the helical domain abolishes constitutive association of CaMBD with Ca(2+)-free CaM, and results in SK channels that are no longer gated by Ca(2+). The results show that the Ca(2+)-independent CaM-CaMBD interaction, which is crucial for channel function, is at least in part determined by a region different in sequence and structure from other CaM-interacting proteins.     

Reverse physiology in drosophila: identification of a novel allatostatin-like neuropeptide and its cognate receptor structurally related to the mammalian somatostatin/galanin/opioid receptor family.

By using degenerate oligonucleotide primers deduced from the conserved regions of the mammalian somatostatin receptors, a novel G-protein-coupled receptor from Drosophila melanogaster has been isolated exhibiting structural similarities to mammalian somatostatin/galanin/opioid receptors. To identify the bioactive ligand, a 'reverse physiology' strategy was used whereby orphan Drosophila receptor-expressing frog oocytes were screened against potential ligands. Agonistic activity was electrophysiologically recorded as inward potassium currents mediated through co-expressed G-protein-gated inwardly rectifying potassium channels (GIRK). Using this approach a novel peptide was purified from Drosophila head extracts. Mass spectrometry revealed an octapeptide of 925 Da with a sequence Ser-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH(2) reminiscent of insect allatostatin peptides known to control diverse functions such as juvenile hormone synthesis during metamorphosis or visceral muscle contractions. Picomolar concentrations of the synthesized octapeptide activated the cognate receptor response mediated through GIRK1, indicating that we have isolated the 394-amino-acid Drosophila allatostatin receptor which is coupled to the Gi/Go class of G proteins.     

Functional and structural conservation in the mechanosensitive channel MscL implicates elements crucial for mechanosensation

mscL encodes a channel in Escherichia coli that is opened by membrane stretch force, probably serving as an osmotic gauge. Sequences more or less similar to mscL are found in other bacteria, but the degree of conserved function has been unclear. We subcloned and expressed these putative homologues in E . coli and examined their products under patch clamp. Here, we show that each indeed encodes a conserved mechanosensitive channel activity, consistent with the interpretation that this is an important and primary function of the protein in a wide range of bacteria. Although similar, channels of different bacteria differ in kinetics and their degree of mechanosensitivity. Comparison of the primary sequence of these proteins reveals two highly conserved regions, corresponding to domains previously shown to be important for the function of the wild-type E . coli channel, and a C-terminal region that is not conserved in all species. This structural conservation is providing insight into regions of this molecule that are vital to its role as a mechanosensitive channel and may have broader implications for the understanding of other mechanosensitive systems.     

Structural Properties of PAS Domains from the KCNH Potassium Channels

KCNH channels form an important family of voltage gated potassium channels. These channels include a N-terminal Per-Arnt-Sim (PAS) domain with unknown function. In other proteins PAS domains are implicated in cellular responses to environmental queues through small molecule binding or involvement in signaling cascades. To better understand their role we characterized the structural properties of several channel PAS domains. We determined high resolution structures of PAS domains from the mouse EAG (mEAG), drosophila ELK (dELK) and human ERG (hERG) channels and also of the hERG domain without the first nine amino acids. We analyzed these structures for features connected to ligand binding and signaling in other PAS domains. In particular, we have found cavities in the hERG and mEAG structures that share similarities with the ligand binding sites from other PAS domains. These cavities are lined by polar and apolar chemical groups and display potential flexibility in their volume. We have also found that the hydrophobic patch on the domain β-sheet is a conserved feature and appears to drive the formation of protein-protein contacts. In addition, the structures of the dELK domain and of the truncated hERG domain revealed the presence of N-terminal helices. These helices are equivalent to the helix described in the hERG NMR structures and are known to be important for channel function. Overall, these channel domains retain many of the PAS domain characteristics known to be important for cell signaling.     

The roles of intracellular regions in the activation of voltage-dependent potassium channels

The involvement of the transmembrane regions S2, S3 and S4 in the activation of potassium channels by depolarization has been well clarified. However, a role of the intracellular regions in channel function is emerging. Here we review recent evidence for the roles of intracellular regions in the functioning of members of two families of channels. The Kv2.1 potassium channel, a member of the voltage activated Kv family, has long intracellular regions. By mutagenesis studies and expression in oocytes, we identify residues in both the N- and C-terminal regions that contribute to determining activation kinetics of this channel. It seems that the C-terminus wraps around the N-terminus and interacts with it functionally. The voltage-activated ether-a-go-go (eag) channels also have long intracellular regions. Despite considerable homology, eag1 and eag2 channels display different activation kinetics. By making chimeras between these channels and again expressing in oocytes, we show that residues in both the N-terminal region and the membrane-spanning region are involved in determining these differences in activation kinetics. The intracellular N- and C-terminal regions are likely to continue to prove fertile regions in future investigations into the functioning of ion channels.Presented at the Biophysical Society Meeting on Ion channels—from structure to disease held in May 2003, Rennes, France     

Molecular determinants of the hanatoxin binding in voltage-gated K+-channel drk1

The carboxyl terminus of S3 segment (S3(C)) in voltage-gated potassium channels was proposed to bear the binding site for gating modifier toxins like Hanatoxin and a helical secondary structural arrangement was suggested. Due to the lack of complete structure in high resolution for such a channel molecule, no further direct experimental data to elucidate the mechanism for their binding conformations could thus far be derived. In order to examine the putative three-dimensional structure of S3(C) and to illustrate the residues required for Hanatoxin binding, molecular simulation and docking were performed, based on the solution structure of Hanatoxin and the structural information from lysine-scanning results for S3(C) fragment. From our results, it is indicated that both hydrophobic and electrostatic interactions are utilized to stabilize the toxin binding. Detailed docking residues and appropriate orientation for binding regarding hydrophobic/-philic environments are also described. Compared with the functional data proposed by previous studies, the helical structural arrangement for the C-terminus of S3 segment in voltage-gated potassium channels can therefore be further emphasized.     

Binding of the anticonvulsant drug lamotrigine and the neurotoxin batrachotoxin to voltage-gated sodium channels induces conformational changes associated with block and steady-state activation

Voltage-gated sodium channels are dynamic membrane proteins characterized by rapid conformational changes that switch the molecule between closed resting, activated, and inactivated states. Sodium channels are specifically blocked by the anticonvulsant drug lamotrigine, which preferentially binds to the channel pore in the inactivated open state. Batrachotoxin is a lipid-soluble alkaloid that causes steady-state activation and binds in the inner pore of the sodium channel with overlapping but distinct molecular determinants from those of lamotrigine. Using circular dichroism spectroscopy on purified voltage-gated sodium channels from Electrophorus electricus, the secondary structures associated with the mixture of states present at equilibrium in the absence of these ligands were compared with specific stabilized states in their presence. As the channel shifts to open states, there appears to be a significant change in secondary structure to a more alpha-helical conformation. The observed changes are consistent with increased order involving the S6 segments that form the pore, the domain III-IV linker, and the P-loops that form the outer pore and selectivity filter. A molecular model has been constructed for the sodium channel based on its homology with the pore-forming regions of bacterial potassium channels, and automated docking of the crystal structure of lamotrigine with this model produces a structure in which the close contacts of the drug are with the residues previously identified by mutational studies as forming the binding site for this drug.