Role of polyphosphate in regulation of the Streptomyces lividans KcsA channel

We examine the hypotheses that the Streptomyces lividans potassium channel KcsA is gated at neutral pH by the electrochemical potential, and that its selectivity and conductance are governed at the cytoplasmic face by interactions between the KcsA polypeptides and a core molecule of inorganic polyphosphate (polyP). The four polypeptides of KcsA are postulated to surround the end unit of the polyP molecule with a collar of eight arginines, thereby modulating the negative charge of the polyP end unit and increasing its preference for binding monovalent cations. Here we show that KcsA channels can be activated in planar lipid bilayers at pH 7.4 by the chemical potential alone. Moreover, one or both of the C-terminal arginines are replaced with residues of progressively lower basicity-lysine, histidine, valine, asparagine-and the effects of these mutations on conductance and selectivity for K⁺ over Mg²⁺ is tested in planar bilayers as a function of Mg²⁺ concentration and pH. As the basicity of the C-terminal residues decreases, Mg²⁺ block increases, and Mg²⁺ becomes permeant when medium pH is greater than the pI of the C-terminal residues. The results uphold the premise that polyP and the C-terminal arginines are decisive elements in KcsA channel regulation.     

Streptomyces lividans potassium channel contains poly-(R)-3-hydroxybutyrate and inorganic polyphosphate

The Streptomyces lividans KcsA potassium channel, a homotetramer of 17.6 kDa subunits, was found to contain two nonproteinaceous polymers, namely, poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP). PHB and polyP are ubiquitous cellular constituents with a demonstrated capacity for cation selection and transport. PHB was detected in both tetramer and monomer species of KcsA by reaction to anti-PHB IgG on Western blots, and estimated as 28 monomer units of PHB per KcsA tetramer by a chemical assay in which PHB is converted to its unique degradation product, crotonic acid. PolyP was detected in KcsA tetramers, but not in monomers, by metachromatic reaction to o-toluidine blue stain on SDS-PAGE gels. A band of free polyP was also visible, suggesting that polyP is released when tetramers dissociate. The exopolyphosphatase of Saccharomyces cerevisiae degraded the free polyP, but tetramer-associated polyP was not affected, indicating it was inaccessible to the enzyme. PolyP in KcsA was estimated as 15 monomer units per tetramer by an enzymatic assay in which polyphosphate kinase is used to transfer phosphates from polyP to [(14)C]ADP, yielding [(14)C]ATP. The experimentally determined isoelectric point of KcsA tetramer was 6.5-7.5, substantially more acidic than the theoretical pI of 10.3, and consistent with the inclusion of a polyanion. The results suggest that PHB is covalently bound to KcsA subunits while polyP is held within tetramers by ionic forces. It is posited that KcsA protein creates an environment in which PHB/polyP is selective for K(+). The basic amino acids attenuate the negative charge density of polyP, thereby transforming the cation binding preference from multivalent to monovalent, and discrimination between K(+) and Na(+) is accomplished by adjusting the ligand geometry in cation binding cavities formed by PHB and polyP.     

pH regulates cation selectivity of poly-(R)-3-hydroxybutyrate/polyphosphate channels from E. coli in planar lipid bilayers

Poly-(R)-3-hydroxybutyrate/polyphosphate (PHB/polyP) complexes, whether isolated from the plasma membranes of bacteria or prepared from the synthetic polymers, form ion channels in planar lipid bilayers that are highly selective for Ca(2+) over Na(+) at physiological pH. This preference for divalent over monovalent cations is attributed to a high density of negative charge along the polyP backbone and the higher binding energies of divalent cations. Here we modify the charge density of polyP by varying the pH, and observe the effect on cation selectivity. PHB/polyP complexes, isolated from E. coli, were incorporated into planar lipid bilayers, and unitary current-voltage relations were determined as a function of pH. When Ca(2+) was the sole permeant cation, conductance diminished steadily from 97 +/- 6 pS at pH 7.4 to 47 +/- 3 pS at pH 5.5. However, in asymmetric solutions of Ca(2+) and Na(+), there was a moderate increase in conductance from 98 +/- 4 at pH 7.4 to 129 +/- 4 pS at pH 6.5, and a substantially larger increase to 178 +/- 6 pS at pH 5.6, signifying an increase in Na(+) permeability or disorganization of channel structure. Reversal potentials point to a sharp decrease in preference for Ca(2+) over Na(+) over a relatively small decrease in pH. Ca(2+) was strongly favored over Na(+) at physiological pH, but the channels became nonselective near the pK(2) of phosphate (approximately 6.8), and displayed weak selectivity for Na(+) over Ca(2+) at acidic pH. Evidently, PHB/polyP complexes are versatile ion carriers whose selectivity may be modulated by small adjustments of the local pH. The results may be relevant to the physiological function of PHB/polyP channels in bacteria and the role of PHB and polyP in the Streptomyces lividans potassium channel.     

Differential effects of temperature on E. coli and synthetic polyhydroxybutyrate/polyphosphate channels

Complexes of poly-(R)-3-hydroxybutyrate and inorganic polyphosphate (PHB/polyP), isolated from the plasma membranes of Escherichia coli or prepared synthetically (HB(128)/polyP(65)), form Ca(2+)-selective ion channels in planar lipid bilayers that exhibit indistinguishable gating and conductance characteristics at 22 degrees C. Here we examine the gating and conductance of E. coli and synthetic PHB/polyP complexes in planar lipid bilayers as a function of temperature from 15 to 45 degrees C. E. coli PHB/polyP channels remained effectively open throughout this range, with brief closures that became more rare at higher temperatures. Conversely, as temperatures were gradually increased, the open probability of HB(128)/polyP(65) channels progressively decreased. The effect was fully reversible. Channel conductance exhibited three distinct phases. Below 25 degrees C, as PHB approached its glass temperature (ca. 10 degrees C), the conductance of both E. coli and synthetic channels remained at about the same level (95-105 pS). Between 25 degrees C and ca. 40 degrees C, the conductance of E. coli and synthetic channels increased gradually with temperature coefficients (Q(10)) of 1.45 and 1.42, respectively. Above 40 degrees C, E. coli channel conductance increased sharply, whereas the conductance of HB(128)/polyP(65) channels leveled off. The discontinuities in the temperature curves for E. coli channels coincide with discontinuities in thermotropic fluorescence spectra and specific growth rates of E. coli cells. It is postulated that E. coli PHB/polyP complexes are associated with membrane components that inhibit their closure at elevated temperatures.     

Functional role and affinity of inorganic cations in stabilizing the tetrameric structure of the KcsA K+ channel

Crystal structures of the tetrameric KcsA K+ channel reveal seven distinct binding sites for K+ ions within the central pore formed at the fourfold rotational symmetry axis. Coordination of an individual K+ ion by eight protein oxygen atoms within the selectivity filter suggests that ion-subunit bridging by cation-oxygen interactions contributes to structural stability of the tetramer. To test this hypothesis, we examined the effect of inorganic cations on the temperature dependence of the KcsA tetramer as monitored by SDS-PAGE. Inorganic cations known to permeate or strongly block K+ channels (K+, Rb+, Cs+, Tl+, NH4+, Ba2+, and Sr2+) confer tetramer stability at higher temperatures (T0.5 range = 87 degrees C to >99 degrees C) than impermeant cations and weak blockers (Li+, Na+, Tris+, choline+; T0.5 range = 59 degrees C to 77 degrees C). Titration of K+, Ba2+, and other stabilizing cations protects against rapid loss of KcsA tetramer observed in 100 mM choline Cl at 90 degrees C. Tetramer protection titrations of K+, Rb+, Cs+, Tl+, and NH4+ at 85 degrees C or 90 degrees C exhibit apparent Hill coefficients (N) ranging from 1.7 to 3.3 and affinity constants (K0.5) ranging from 1.1 to 9.6 mM. Ba2+ and Sr2+ titrations exhibit apparent one-site behavior (N congruent with 1) with K0.5 values of 210 nM and 11 microM, respectively. At 95 degrees C in the presence of 5 mM K+, titration of Li+ or Na+ destabilizes the tetramer with K0.5 values of 57 mM and 109 mM, respectively. We conclude that specific binding interactions of inorganic cations with the selectivity filter are an important determinant of tetramer stability of KscA.     

Structural characterization and pH-induced conformational transition of full-length KcsA

The bacterial K+ channel KcsA from Streptomyces lividans was analyzed by neutron and x-ray small-angle solution scattering. The C-terminally truncated version of KcsA, amenable to crystallographic studies, was compared with the full-length channel. Analyzing the scattering data in terms of radius of gyration reveals differences between both KcsA species of up to 13.2 A. Equally, the real-space distance distribution identifies a 40 to 50 A extension of full-length KcsA compared to its C-terminally truncated counterpart. We show that the x-ray and neutron scattering data are amenable for molecular shape reconstruction of full-length KcsA. The molecular envelopes calculated display an hourglass-shaped structure within the C-terminal intracellular domain. The C-terminus extends the membrane spanning region of KcsA by 54-70 A, with a central constriction 10-30 A wide. Solution scattering techniques were further employed to characterize the KcsA channel under acidic conditions favoring its open conformation. The full-length KcsA at pH 5.0 shows the characteristics of a dumbbell-shaped macromolecular structure, originating from dimerization of the tetrameric K+ channel. Since C-terminally truncated KcsA measured under the same low pH conditions remains tetrameric, oligomerization of full-length KcsA seems to proceed via structurally changed C-terminal domains. The determined maximum dimensions of the newly formed complex increase by 50-60%. Shape reconstruction of the pseudooctameric complex indicates the pH-induced conformational reorganization of the intracellular C-terminal domain.     

Thermal stability of the K+ channel tetramer: cation interactions and the conserved threonine residue at the innermost site (S4) of the KcsA selectivity filter

The selectivity filter of most K+ channels contains a highly conserved Thr residue that uniquely forms the S4 binding site for K+ by dual coordination with the backbone carbonyl oxygen and side chain hydroxyl of the same residue. This study examines the effect of mutations of Thr75 in the S4 site of theKcsA K+ channel on the cation dependence of the thermal stability of the tetramer, a phenomenon that reflects the structural role of cations in the filter. Conservative mutations of Thr75 destabilize the tetramer and alter its temperature dependence. Replacement of Thr with Ala or Cys lowers the apparent affinity ofK+, Rb+, and Cs+ for tetramer stabilization by factors ranging from 4- to 14-fold. These same mutations lower the apparent affinity of Ba2+ by approximately 10(3)- or approximately 10(4)-fold for Ala and Cys substitution, respectively,consistent with the known preference of the S4 site for Ba2+. In contrast, substitution of Ala or Cys at T75 anomalously enhances the ability of Na+ to stabilize the tetramer, suggesting that the native Thr residue at S4 is important for ultrahigh K+/Na+ selectivity of K+ channel pores. Elevated temperature orCu2+ cation catalyzes formation of covalent dimers of the T75C mutant of KcsA via formation of disulfide bonds between Cys residues of adjacent subunits. Thiophilic cations such as Hg2+ and Ag+ specifically protect the T75C tetramer against heat-induced dimer formation, demonstrating the contribution of cation interactions to tetramer stability in a channel with a non-native S4 site engineered to bind foreign cations.     

Mechanism underlying slow kinetics of the OFF gating current in Shaker potassium channel

Based on the structure of the KcsA potassium channel, the Shaker K+ channel is thought to have, near the middle of the membrane, a cavity that can be occupied by a permeant or a blocking cation. We have studied the interaction between cations in the cavity and the activation gate of the channel, using a set of monovalent cations together with Shaker mutants that modify the structure of the cavity. Our results show that reducing the size of the side chain at position 470 makes it possible for the mutant channel, unlike native Shaker, to close with tetraethylammonium (TEA+) or the long-chain TEA-derivative C10+ trapped inside the channel. Neither I470 mutants nor Shaker can close when N-methyl-glucamine (NMG+) is in the channel, even though this ion is smaller than C10+. Apparently, the carbohydrate side chain of NMG+ prevents gate closing. Gating currents recorded from Shaker and I470C were measured in the presence of different intracellular cations to further analyze the interaction of cations with the gate. Our results suggest that the cavity in Shaker is so small that even permeant cations like Rb+ or Cs+ must leave the cavity before the channel gate can close.     

Transmembrane ion transport by polyphosphate/poly-(R)-3-hydroxybutyrate complexes

Transmembrane ion transport, a critical process in providing energy for cell functions, is carried out by pore-forming macromolecules capable of discriminating among very similar ions and responding to changes in membrane potential. It is widely regarded that ion channels are exclusively proteins, relatively late arrivals in cell evolution. Here we discuss the formation of ion-selective, voltage-activated channels by complexes of two simple homopolymers, namely, inorganic polyphosphates (polyPs) and poly-(R)-3-hydroxybutyrates (PHBs), derived from phosphate and acetate, respectively. Each has unique molecular characteristics that facilitate ion selection, solvation, and transport. Complexes of the two polymers, isolated from bacterial plasma membranes or prepared from the synthetic polymers, form voltage-dependent, Ca2+-selective channels in planar lipid bilayers that are selective for divalent over monovalent cations, permeant to Ca2+, Sr2+, and Ba2+, and blocked by transition metal cations in a concentration-dependent manner. Recently, both polyP and PHB have been found to be components of ion-conducting proteins: namely, the human erythrocyte Ca2+-ATPase pump and the Streptomyces lividans potassium channel. The contribution of polyP and PHB to ion selection and/or transport in these proteins is yet unknown, but their presence gives rise to the hypothesis that these and other ion transporters are supramolecular structures in which proteins, polyP, and PHB cooperate in forming well-regulated and specific cation transfer systems.     

Inwardly rectifying current-voltage relationship of small-conductance Ca2+-activated K+ channels rendered by intracellular divalent cation blockade

Small conductance Ca2+-activated K+ channels (SK(Ca) channels) are a group of K+-selective ion channels activated by submicromolar concentrations of intracellular Ca2+ independent of membrane voltages. We expressed a cloned SK(Ca) channel, rSK2, in Xenopus oocytes and investigated the effects of intracellular divalent cations on the current-voltage (I-V) relationship of the channels. Both Mg2+ and Ca2+ reduced the rSK2 channel currents in voltage-dependent manners from the intracellular side and thus rectified the I-V relationship at physiological concentration ranges. The apparent affinity of Mg2+ was changed as a function of both transmembrane voltage and intracellular Ca2+ concentration. Extracellular K+ altered the voltage dependence as well as the apparent affinities of Mg2+ binding from intracellular side. Thus, the inwardly rectifying I-V relationship of SK(Ca) channels is likely due to the voltage-dependent blockade of intracellular divalent cations and that the binding site is located within the ion-conducting pathway. Therefore, intracellular Ca2+ modulates the permeation characteristics of SK(Ca) channels by altering the I-V relationship as well as activates the channel by interacting with the gating machinery, calmodulin, and SK(Ca) channels can be considered as Ca2+-activated inward rectifier K+ channels.     

Genetic selection of activatory mutations in KcsA

The KcsA potassium channel from Streptomyces lividans is one of the most actively studied ion channels. However, there are still unresolved issues about its gating mechanism in vivo because the channel is only activated by highly acidic intracellular pH, meaning that it will be mostly inactive in its host environment. In this study we have used a genetic complementation assay of K+-auxotrophic E. coli (TK2420) and S. cerevisiae (SGY1528) to identify activatory or 'gain-of-function' mutations which allow functional activity of KcsA in the physiological environment of two markedly different expression systems. These mutations clustered at the helix-bundle-crossing in both TM1 and TM2 (residues H25, L105, A108, T112, W113, F114, E118 and Q119), and include residues previously implicated in the pH-gating mechanism. We discuss how these gain-of-function mutations may result in their activatory phenotype, the relative merits of the E. coli and S. cerevisiae genetic complementation approaches for the identification of gating mutations in prokaryotic K+ channels, and ways in which this assay may be improved for future use in screening protocols.     

The conserved potassium channel filter can have distinct ion binding profiles: Structural analysis of rubidium,cesium, and barium binding in NaK2K

Potassium channels are highly selective for K⁺ over the smaller Na⁺. Intriguingly, they are permeable to larger monovalent cations such as Rb⁺ and Cs⁺ but are specifically blocked by the similarly sized Ba²⁺. In this study, we used structural analysis to determine the binding profiles for these permeant and blocking ions in the selectivity filter of the potassium-selective NaK channel mutant NaK2K and also performed permeation experiments using single-channel recordings. Our data revealed that some ion binding properties of NaK2K are distinct from those of the canonical K⁺ channels KcsA and MthK. Rb⁺ bound at sites 1, 3, and 4 in NaK2K, as it does in KcsA. Cs⁺, however, bound predominantly at sites 1 and 3 in NaK2K, whereas it binds at sites 1, 3, and 4 in KcsA. Moreover, Ba²⁺ binding in NaK2K was distinct from that which has been observed in KcsA and MthK, even though all of these channels show similar Ba²⁺ block. In the presence of K⁺, Ba²⁺ bound to the NaK2K channel at site 3 in conjunction with a K⁺ at site 1; this led to a prolonged block of the channel (the external K⁺-dependent Ba²⁺ lock-in state). In the absence of K⁺, however, Ba²⁺ acts as a permeating blocker. We found that, under these conditions, Ba²⁺ bound at sites 1 or 0 as well as site 3, allowing it to enter the filter from the intracellular side and exit from the extracellular side. The difference in the Ba²⁺ binding profile in the presence and absence of K⁺ thus provides a structural explanation for the short and prolonged Ba²⁺ block observed in NaK2K.     

Evidence for a deep pore activation gate in small conductance Ca2+-activated K+ channels

Small conductance calcium-gated potassium (SK) channels share an overall topology with voltage-gated potassium (K(v)) channels, but are distinct in that they are gated solely by calcium (Ca(2+)), not voltage. For K(v) channels there is strong evidence for an activation gate at the intracellular end of the pore, which was not revealed by substituted cysteine accessibility of the homologous region in SK2 channels. In this study, the divalent ions cadmium (Cd(2+)) and barium (Ba(2+)), and 2-aminoethyl methanethiosulfonate (MTSEA) were used to probe three sites in the SK2 channel pore, each intracellular to (on the selectivity filter side of) the region that forms the intracellular activation gate of voltage-gated ion channels. We report that Cd(2+) applied to the intracellular side of the membrane can modify a cysteine introduced to a site (V391C) just intracellular to the putative activation gate whether channels are open or closed. Similarly, MTSEA applied to the intracellular side of the membrane can access a cysteine residue (A384C) that, based on homology to potassium (K) channel crystal structures (i.e., the KcsA/MthK model), resides one amino acid intracellular to the glycine gating hinge. Cd(2+) and MTSEA modify with similar rates whether the channels are open or closed. In contrast, Ba(2+) applied to the intracellular side of the membrane, which is believed to block at the intracellular end of the selectivity filter, blocks open but not closed channels when applied to the cytoplasmic face of rSK2 channels. Moreover, Ba(2+) is trapped in SK2 channels when applied to open channels that are subsequently closed. Ba(2+) pre-block slows MTSEA modification of A384C in open but not in closed (Ba(2+)-trapped) channels. The findings suggest that the SK channel activation gate resides deep in the vestibule of the channel, perhaps in the selectivity filter itself.     

Ions and blockers in potassium channels: insights from free energy simulations

Potassium ion channels enable efficient and selective permeation of K+ ions across nonpolar biological membranes. Here we review the results of recent free energy calculations related to the permeation of monovalent cations through K+ channels and to the channel inhibition by blocker compounds. In particular, the progress in computational studies of the bacterial KcsA channel is discussed.     

Genetic selection of activatory mutations in KcsA

The KcsA potassium channel from Streptomyces lividans is one of the most actively studied ion channels. However, there are still unresolved issues about its gating mechanism in vivo because the channel is only activated by highly acidic intracellular pH, meaning that it will be mostly inactive in its host environment. In this study we have used a genetic complementation assay of K+-auxotrophic E.coli (TK2420) and S.cerevisiae (SGY1528) to identify activatory or 'gain-of-function' mutations which allow functional activity of KcsA in the physiological environment of two markedly different expression systems. These mutations clustered at the helix-bundle-crossing in both TM1 and TM2 (residues H25, L105, A108, T112, W113, F114, E118 & Q119), and include residues previously implicated in the pH-gating mechanism. We discuss how these gain-of-function mutations may result in their activatory phenotype, the relative merits of the E.coli and S.cerevisiae genetic complementation approaches for the identification of gating mutations in prokaryotic K+ channels, and ways in which this assay may be improved for future use in screening protocols.     

Identification and characterization of the slowly exchanging pH-dependent conformational rearrangement in KcsA

Gating of ion channels is strictly regulated by physiological conditions as well as intra/extracellular ligands. To understand the underlying structures mediating ion channel gating, we investigated the pH-dependent gating of the K(+) channel KcsA under near-physiological conditions, using solution-state NMR. In a series of (1)H(15)N-TROSY HSQC (transverse relaxation optimized spectroscopy-heteronuclear single quantum coherence) spectra measured at various pH values, significant chemical shift changes were detected between pH 3.9 and 5.2, reflecting a conformational rearrangement associated with the gating. The pH-dependent chemical shift changes were mainly observed for the resonances from the residues near the intracellular helix bundle, which has been considered to form the primary gate in the K(+) channel, as well as the intracellular extension of the inner helix. The substitution of His-25 by Ala abolished this pH-dependent conformational rearrangement, indicating that the residue serves as a "pH-sensor" for the channel. Although the electrophysiological open probability of KcsA is less than 10%, the conformations of the intracellular helix bundle between the acidic and neutral conditions seem to be remarkably different. This supports the recently proposed "dual gating" properties of the K(+) channel, in which the activation-coupled inactivation at the selectivity filter determines the channel open probability of the channel. Indeed, a pH-dependent chemical shift change was also observed for the signal from the Trp-67 indole, which is involved in a hydrogen bond network related to the activation-coupled inactivation. The slow kinetic parameter obtained for the intracellular bundle seems to fit better into the time scale for burst duration than very fast fluctuations within a burst period, indicating the existence of another gating element with faster kinetic properties.     

Surface structure and its dynamic rearrangements of the KcsA potassium channel upon gating and tetrabutylammonium blocking

KcsA is the first potassium channel for which the molecular structure was revealed. However, the high resolution structural information is limited to the transmembrane domain, and the dynamic picture of the full KcsA channel remains unsolved. We have developed a new approach to investigate the surface structure of proteins, and we applied this method to investigate the full length of the KcsA channel. Single-cysteine substitution was introduced into 25 sites, and specific reaction of these mutated channels to a bare surface of a flat gold plate was evaluated by surface plasmon resonance measurements. The surface plasmon resonance signals revealed the highest exposure for the mutant of the C-terminal end. When the gate of the KcsA channel is kept closed at pH 7.5, the extent of exposure showed periodic patterns for the consecutive sites located in the cytoplasmic (CP) and N-terminal domain. This suggests that these stretches take the alpha-helical structure. When the channel was actively gated at pH 4.0, many sites in the CP domain became exposed. Compared with the rigid structure in pH 7.5, these results indicate that the CP domain became loosely packed upon active gating. The C-terminal end of the M2 helix is a moving part of the gate, and it is exposed to the outer surface slightly at pH 4.0. By adding a channel blocker, tetrabutylammonium, the gate is further exposed. This suggests that in the active gating tetrabutylammonium keeps the gate open rather than being trapped in the central cavity.     

Single streptomyces lividans K(+) channels: functional asymmetries and sidedness of proton activation.

Basic electrophysiological properties of the KcsA K(+) channel were examined in planar lipid bilayer membranes. The channel displays open-state rectification and weakly voltage-dependent gating. Tetraethylammonium blocking affinity depends on the side of the bilayer to which the blocker is added. Addition of Na(+) to the trans chamber causes block of open-channel current, while addition to the cis side has no effect. Most striking is the activation of KcsA by protons; channel activity is observed only when the trans bilayer chamber is at low pH. To ascertain which side of the channel faces which chamber, residues with structurally known locations were mapped to defined sides of the bilayer. Mutation of Y82, an external residue, results in changes in tetraethylammonium affinity exclusively from the cis side. Channels with cysteine residues substituted at externally exposed Y82 or internally exposed Q119 are functionally modified by methanethiosulfonate reagents from the cis or trans chambers, respectively. Block by charybdotoxin, known to bind to the channel's external mouth, is observed only when the toxin is added to the cis side of channels mutated to be toxin sensitive. These results demonstrate unambiguously that the protonation sites linked to gating are on the intracellular portion of the KcsA protein.     

Light at the end of the Ca(2+)-release channel tunnel: structures and mechanisms involved in ion translocation in ryanodine receptor channels

RyR and InsP3R are Ca(2+)-release channels. When induced to open by the appropriate stimulus, these channels allow Ca2+ to leave intracellular storage organelles at an astonishing rate. Investigations of the ion-handling properties of isolated RyR channels have demonstrated that, at least in comparison to voltage-gated channels of surface membranes, these channels display limited powers of discrimination between physiologically relevant cations and this relative lack of selectivity is likely to contribute to the ability of Ca(2+)-release channels to maintain high rates of cation translocation without compromising function. A range of ion-handling properties in RyR are consistent with the proposal that this channel functions as a single-ion channel and theoretical considerations indicate that the high rates of ion translocation monitored for RyR would require the pore of such a structure to be short and possess a large capture radius. Measurements of the dimensions of regions of RyR involved in ion conduction and discrimination indicate that this is likely to be the case. In each monomer of RyR/InsP3R, residues making up the last two trans-membrane spanning domains and a luminal loop linking these two helices contribute to the formation of the channel pore. The luminal loops of both RyR and InsP3R contain amino acid sequences similar to those known to form the selectivity filter of K+ channels. In addition the luminal loops of both Ca(2+)-release channels contain sequences that are likely to form helices that may be analogous to the pore helix visualised in KcsA. The correlation in structural elements of the luminal loops of RyR/InsP3R and KcsA has prompted us to speculate on the tertiary arrangement for this region of the Ca(2+)-release channels using the established structure of KcsA as a framework.     

Construction of a cyclic nucleotide-gated KcsA K+ channel

The ability of an ion channel to open in response to a defined stimulus is central to its function. In ligand-gated channels, pore opening is conferred through transduction of a conformational change in a gating domain to the helices of the pore. Here, we present the construction of a designed cyclic nucleotide-gated (CNG) channel, named KcsA-CNG, by addition of a prokaryotic cyclic nucleotide-binding domain to a KcsA-derived K+ channel. This channel is functional in lipid bilayers at physiological pH and has the combined properties of both of its parent-derived components. It conducts K+ and is blocked by the K+ channel inhibitors Na+ and agitoxin-2. Channel open times are increased by about two orders of magnitude compared to wild-type KcsA. The average number of open channels increases by approximately 50% upon addition of cAMP. Although the absolute open probabilities are somewhat variable from one channel to the next, the property of cyclic nucleotide sensitivity is very reproducible. An apparent Kd value of approximately 90 nM was estimated. The successful construction of a cyclic nucleotide-gated KcsA K+ channel suggests that it should be possible to produce channels that will respond to novel ligands.