Changing Val-76 towards Kir channels drastically influences the folding and gating properties of the bacterial potassium channel KcsA (original) (raw)
Related papers
Crystal Structure of the Potassium Channel KirBac1.1 in the Closed State
Science, 2003
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.
Febs Letters, 2005
The lack of a membrane environment in membrane protein crystals is considered one of the major limiting factors to fully imply X-ray structural data to explain functional properties of ion channels [Gulbis, J.M. and Doyle, D. (2004) Curr. Opin. Struct. Biol. 14, 440–446]. Here, we provide infrared spectroscopic evidence that the structure and stability of the potassium channel KcsA and its chymotryptic derivative 1–125 KcsA reconstituted into native-like membranes differ from those exhibited by these proteins in detergent solution, the latter taken as an approximation of the mixed detergent-protein crystal conditions.
Protonation state of E71 in KcsA and its role for channel collapse and inactivation
Proceedings of the National Academy of Sciences, 2012
The prototypical prokaryotic potassium channel KcsA alters its pore depending on the ambient potassium; at high potassium, it exists in a conductive form, and at low potassium, it collapses into a nonconductive structure with reduced ion occupancy. We present solid-state NMR studies of KcsA in which we test the hypothesis that an important channel-inactivation process, known as C-type inactivation, proceeds via a state similar to this collapsed state. We test this using an inactivation-resistant mutant E71A, and show that E71A is unable to collapse its pore at both low potassium and low pH, suggesting that the collapsed state is structurally similar to the inactivated state. We also show that E71A has a disordered selectivity filter. Using site-specific K+ titrations, we detect a local change at E71 that is coupled to channel collapse at low K+. To gain more insight into this change, we site specifically measure the chemical shift tensors of the side-chain carboxyls of E71 and its h...
EMBO reports, 2000
Very little is known about the biogenesis and assembly of oligomeric membrane proteins. In this study, the biogenesis of KcsA, a prokaryotic homotetrameric potassium channel, is investigated. Using in vivo pulse-chase experiments, both the monomeric and tetrameric form could be identified. The conversion of monomers into a tetramer is found to be a highly efficient process that occurs in the Escherichia coli inner membrane. KcsA does not require ATP hydrolysis by SecA for insertion or tetramerization. The presence of the protonmotive force (pmf) is not necessary for transmembrane insertion of KcsA; however, the pmf proved to be essential for the efficiency of oligomerization. From in vivo and in vitro experiments it is concluded that the electrical component, ∆ψ, is the main determinant for this effect. These results demonstrate a new role of the pmf in membrane protein biogenesis.
Functional Characterization of a Prokaryotic Kir Channel
Journal of Biological Chemistry, 2004
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, Kir-Bac1.1, 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 86 Rb ؉ uptake assay, we demonstrate that KirBac1.1 generates a K ؉-selective permeation path that is inhibited by extraliposomal Ba 2؉ and Ca 2؉ ions. In contrast to KcsA (an acid-activated bacterial potassium channel), KirBac1.1 is inhibited by extraliposomal acid (pK a ϳ 6). This characterization of KirBac1.1 activity now paves the way for further correlation of structure and function in this model Kir channel.
Structure, 2015
The ligand-gated potassium channels are stimulated by various kinds of messengers. Previous studies showed that ligand-gated potassium channels containing RCK domains (the regulator of the conductance of potassium ion) form a dimer of tetramer structure through the RCK octameric gating ring in the presence of detergent. Here, we have analyzed the structure of Kch, a channel of this type from Escherichia coli, in a lipid environment using electron crystallography. By combining information from the 3D map of the transmembrane part of the protein and docking of an atomic model of a potassium channel, we conclude that the RCK domains face the solution and that an RCK octameric gating ring arrangement does not form under our crystallization condition. Our findings may be applied to other potassium channels that have an RCK gating ring arrangement.
A distinct mechanism of C-type inactivation in the Kv-like KcsA mutant E71V
Nature Communications, 2022
C-type inactivation is of great physiological importance in voltage-activated K+ channels (Kv), but its structural basis remains unresolved. Knowledge about C-type inactivation has been largely deduced from the bacterial K+ channel KcsA, whose selectivity filter constricts under inactivating conditions. However, the filter is highly sensitive to its molecular environment, which is different in Kv channels than in KcsA. In particular, a glutamic acid residue at position 71 along the pore helix in KcsA is substituted by a valine conserved in most Kv channels, suggesting that this side chain is a molecular determinant of function. Here, a combination of X-ray crystallography, solid-state NMR and MD simulations of the E71V KcsA mutant is undertaken to explore inactivation in this Kv-like construct. X-ray and ssNMR data show that the filter of the Kv-like mutant does not constrict under inactivating conditions. Rather, the filter adopts a conformation that is slightly narrowed and rigidi...
Nature Structural & Molecular Biology, 2012
KirBac channels are prokaryotic homologs of mammalian inwardly-rectifying (Kir) potassium channels and recent 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 that control opening of their primary activation gate remain unknown. In this study, we engineered the inner pore-lining helix (TM2) of KirBac3.1 to trap the bundle-crossing in an apparently open conformation, and determined the crystal structure of this mutant channel to 3.05 Å resolution. Contrary to previous speculation, this novel structure suggests a mechanistic model in which rotational 'twist' of the cytoplasmic domain is coupled to opening of the bundle-crossing gate via a network of inter-and intra-subunit interactions that involves the TM2 C-linker, slide-helix, G-loop and the CD-loop.
Journal of Membrane Biology, 2009
Protein aggregation is a result of malfunction in protein folding, assembly, and transport, caused by protein mutation and/or changes in the cell environment, thus triggering many human diseases. We have shown that bacterial K ?-channel KcsA, which acts as a representative model for ion channels, forms salt-induced large conductive complexes in a particular environment. In the present study, we investigated the effects of point mutations in the selectivity filter of KcsA on intrinsic stability, aggregation, and channel blocking behavior. First, we found that a low sodium chloride concentration in potassium-containing media induced fast transfer of single channels to a planar lipid bilayer. Second, increasing the sodium chloride concentration drastically increased the total channel current, indicating enhanced vesicle fusion and transfer of multiple channels to a planar lipid bilayer. However, such complexes exhibited high conductance as well as higher open probability compared to the unmodified KcsA behavior shown previously. Interestingly, the affinity of aggregated complexes for larger symmetric quaternary alkylammonium ions (QAs) was found to be much higher than that for tetraethylammonium, a classical blocker of the K ? channel. Based on these findings, we propose that mutant channel complexes exhibit larger pore dimensions, thus resembling more the topological properties of voltagegated and inwardly rectifying K ? channels.