Selectivity filter gating in large-conductance Ca(2+)-activated K+ channels

Membrane voltage controls the passage of ions through voltage-gated K (K(v)) channels, and many studies have demonstrated that this is accomplished by a physical gate located at the cytoplasmic end of the pore. Critical to this determination were the findings that quaternary ammonium ions and certain peptides have access to their internal pore-blocking sites only when the channel gates are open, and that large blocking ions interfere with channel closing. Although an intracellular location for the physical gate of K(v) channels is well established, it is not clear if such a cytoplasmic gate exists in all K(+) channels. Some studies on large-conductance, voltage- and Ca(2+)-activated K(+) (BK) channels suggest a cytoplasmic location for the gate, but other findings question this conclusion and, instead, support the concept that BK channels are gated by the pore selectivity filter. If the BK channel is gated by the selectivity filter, the interactions between the blocking ions and channel gating should be influenced by the permeant ion. Thus, we tested tetrabutyl ammonium (TBA) and the Shaker "ball" peptide (BP) on BK channels with either K(+) or Rb(+) as the permeant ion. When tested in K(+) solutions, both TBA and the BP acted as open-channel blockers of BK channels, and the BP interfered with channel closing. In contrast, when Rb(+) replaced K(+) as the permeant ion, TBA and the BP blocked both closed and open BK channels, and the BP no longer interfered with channel closing. We also tested the cytoplasmically gated Shaker K channels and found the opposite behavior: the interactions of TBA and the BP with these K(v) channels were independent of the permeant ion. Our results add significantly to the evidence against a cytoplasmic gate in BK channels and represent a positive test for selectivity filter gating.     

Selection of Inhibitor-Resistant Viral Potassium Channels Identifies a Selectivity Filter Site that Affects Barium and Amantadine Block

Background

Understanding the interactions between ion channels and blockers remains an important goal that has implications for delineating the basic mechanisms of ion channel function and for the discovery and development of ion channel directed drugs.

Methodology/Principal Findings

We used genetic selection methods to probe the interaction of two ion channel blockers, barium and amantadine, with the miniature viral potassium channel Kcv. Selection for Kcv mutants that were resistant to either blocker identified a mutant bearing multiple changes that was resistant to both. Implementation of a PCR shuffling and backcrossing procedure uncovered that the blocker resistance could be attributed to a single change, T63S, at a position that is likely to form the binding site for the inner ion in the selectivity filter (site 4). A combination of electrophysiological and biochemical assays revealed a distinct difference in the ability of the mutant channel to interact with the blockers. Studies of the analogous mutation in the mammalian inward rectifier Kir2.1 show that the T→S mutation affects barium block as well as the stability of the conductive state. Comparison of the effects of similar barium resistant mutations in Kcv and Kir2.1 shows that neighboring amino acids in the Kcv selectivity filter affect blocker binding.

Conclusions/Significance

The data support the idea that permeant ions have an integral role in stabilizing potassium channel structure, suggest that both barium and amantadine act at a similar site, and demonstrate how genetic selections can be used to map blocker binding sites and reveal mechanistic features.     

Potassium channels as multi-ion single-file pores

A literature review reveals many lines of evidence that both delayed rectifier and inward rectifier potassium channels are multi-ion pores. These include unidirectional flux ratios given by the 2--2.5 power of the electrochemical activity ratio, very steeply voltage-dependent block with monovalent blocking ions, relief of block by permeant ions added to the side opposite from the blocking ion, rectification depending on E--EK, and a minimum in the reversal potential or conductance as external K+ ions are replaced by an equivalent concentration of T1+ ions. We consider a channel with a linear sequence of energy barriers and binding sites. The channel can be occupied by more than one ion at a time, and ions hop in single file into vacant sites with rate constants that depend on barrier heights, membrane potential, and interionic repulsion. Such multi-ion models reproduce qualitatively the special flux properties of potassium channels when the barriers for hopping out of the pore are larger than for hopping between sites within the pore and when there is repulsion between ions. These conditions also produce multiple maxima in the conductance-ion activity relationship. In agreement with Armstrong's hypothesis (1969. J. Gen. Physiol. 54:553--575), inward rectification may be understood in terms of block by an internal blocking cation. Potassium channels must have at least three sites and often contain at least two ions at a time.     

Pseudechetoxin binds to the pore turret of cyclic nucleotide-gated ion channels

Peptide toxins are invaluable tools for studying the structure and physiology of ion channels. Pseudechetoxin (PsTx) is the first known peptide toxin that targets cyclic nucleotide-gated (CNG) ion channels, which play a critical role in sensory transduction in the visual and olfactory systems. PsTx inhibited channel currents at low nM concentrations when applied to the extracellular face of membrane patches expressing olfactory CNGA2 subunits. Surprisingly, 500 nM PsTx did not inhibit currents through channels formed by the CNGA3 subunit from cone photoreceptors. We have exploited this difference to identify the PsTx-binding site on the extracellular face of CNG channels. Studies using chimeric channels revealed that transplantation of the pore domain from CNGA2 was sufficient to confer high affinity PsTx binding upon a CNGA3 background. To further define the binding site, reciprocal mutations were made at 10 nonidentical amino acid residues in this region. We found that two residues in CNGA2, D316 and Y321, were essential for high-affinity inhibition by PsTx. Furthermore, replacement of both residues was required to confer high-affinity PsTx inhibition upon CNGA3. Several other residues, including E325, also form favorable interactions with PsTx. In the CNGA2-E325K mutant, PsTx affinity was reduced by approximately 5-fold to 120 nM. An electrostatic interaction with D316 does not appear to be the primary determinant of PsTx affinity, as modification of the D316C mutant with a negatively charged methanethiosulfonate reagent did not restore high affinity inhibition. The residues involved in PsTx binding are found within the pore turret and helix, in similar positions to residues that form the receptor for pore-blocking toxins in voltage-gated potassium channels. Furthermore, biophysical properties of PsTx block, including an unfavorable interaction with permeant ions, also suggest that it acts as a pore blocker. In summary, PsTx seems to occlude the entrance to the pore by forming high-affinity contacts with the pore turret, which may be larger than that found in the KcsA structure.     

The pore structure and gating mechanism of K2P channels

Two-pore domain (K2P) potassium channels are important regulators of cellular electrical excitability. However, the structure of these channels and their gating mechanism, in particular the role of the bundle-crossing gate, are not well understood. Here, we report that quaternary ammonium (QA) ions bind with high-affinity deep within the pore of TREK-1 and have free access to their binding site before channel activation by intracellular pH or pressure. This demonstrates that, unlike most other K(+) channels, the bundle-crossing gate in this K2P channel is constitutively open. Furthermore, we used QA ions to probe the pore structure of TREK-1 by systematic scanning mutagenesis and comparison of these results with different possible structural models. This revealed that the TREK-1 pore most closely resembles the open-state structure of KvAP. We also found that mutations close to the selectivity filter and the nature of the permeant ion profoundly influence TREK-1 channel gating. These results demonstrate that the primary activation mechanisms in TREK-1 reside close to, or within the selectivity filter and do not involve gating at the cytoplasmic bundle crossing.     

Relationship between pore occupancy and gating in BK potassium channels

Permeant ions can have significant effects on ion channel conformational changes. To further understand the relationship between ion occupancy and gating conformational changes, we have studied macroscopic and single-channel gating of BK potassium channels with different permeant monovalent cations. While the slopes of the conductance-voltage curve were reduced with respect to potassium for all permeant ions, BK channels required stronger depolarization to open only when thallium was the permeant ion. Thallium also slowed the activation and deactivation kinetics. Both the change in kinetics and the shift in the GV curve were dependent on the thallium passing through the permeation pathway, as well as on the concentration of thallium. There was a decrease in the mean open time and an increase in the number of short flicker closing events with thallium as the permeating ion. Mean closed durations were unaffected. Application of previously established allosteric gating models indicated that thallium specifically alters the opening and closing transition of the channel and does not alter the calcium activation or voltage activation pathways. Addition of a closed flicker state into the allosteric model can account for the effect of thallium on gating. Consideration of the thallium concentration dependence of the gating effects suggests that the flicker state may correspond to the collapsed selectivity filter seen in crystal structures of the KcsA potassium channel under the condition of low permeant ion concentration.     

Functional Consequences of a Decreased Potassium Affinity in a Potassium Channel Pore : Ion Interactions and C-Type Inactivation

Ions bound near the external mouth of the potassium channel pore impede the C-type inactivation conformational change (Lopez-Barneo, J., T. Hoshi, S. Heinemann, and R. Aldrich. 1993. Receptors Channels. 1:61– 71; Baukrowitz, T., and G. Yellen. 1995. Neuron. 15:951–960). In this study, we present evidence that the occupancy of the C-type inactivation modulatory site by permeant ions is not solely dependent on its intrinsic affinity, but is also a function of the relative affinities of the neighboring sites in the potassium channel pore. The A463C mutation in the S6 region of Shaker decreases the affinity of an internal ion binding site in the pore (Ogielska, E.M., and R.W. Aldrich, 1998). However, we have found that this mutation also decreases the C-type inactivation rate of the channel. Our studies indicate that the C-type inactivation effects observed with substitutions at position A463 most likely result from changes in the pore occupancy of the channel, rather than a change in the C-type inactivation conformational change. We have found that a decrease in the potassium affinity of the internal ion binding site in the pore results in lowered (electrostatic) interactions among ions in the pore and as a result prolongs the time an ion remains bound at the external C-type inactivation site. We also present evidence that the C-type inactivation constriction is quite local and does not involve a general collapse of the selectivity filter. Our data indicate that in A463C potassium can bind within the selectivity filter without interfering with the process of C-type inactivation.     

External barium affects the gating of KCNQ1 potassium channels and produces a pore block via two discrete sites

The pore properties and the reciprocal interactions between permeant ions and the gating of KCNQ channels are poorly understood. Here we used external barium to investigate the permeation characteristics of homomeric KCNQ1 channels. We assessed the Ba(2+) binding kinetics and the concentration and voltage dependence of Ba(2+) steady-state block. Our results indicate that extracellular Ba(2+) exerts a series of complex effects, including a voltage-dependent pore blockade as well as unique gating alterations. External barium interacts with the permeation pathway of KCNQ1 at two discrete and nonsequential sites. (a) A slow deep Ba(2+) site that occludes the channel pore and could be simulated by a model of voltage-dependent block. (b) A fast superficial Ba(2+) site that barely contributes to channel block and mostly affects channel gating by shifting rightward the voltage dependence of activation, slowing activation, speeding up deactivation kinetics, and inhibiting channel inactivation. A model of voltage-dependent block cannot predict the complex impact of Ba(2+) on channel gating in low external K(+) solutions. Ba(2+) binding to this superficial site likely modifies the gating transitions states of KCNQ1. Both sites appear to reside in the permeation pathway as high external K(+) attenuates Ba(2+) inhibition of channel conductance and abolishes its impact on channel gating. Our data suggest that despite the high degree of homology of the pore region among the various K(+) channels, KCNQ1 channels display significant structural and functional uniqueness.     

Novel gating mechanism of polyamine block in the strong inward rectifier K channel Kir2.1

Inward rectifying K channels are essential for maintaining resting membrane potential and regulating excitability in many cell types. Previous studies have attributed the rectification properties of strong inward rectifiers such as Kir2.1 to voltage-dependent binding of intracellular polyamines or Mg to the pore (direct open channel block), thereby preventing outward passage of K ions. We have studied interactions between polyamines and the polyamine toxins philanthotoxin and argiotoxin on inward rectification in Kir2.1. We present evidence that high affinity polyamine block is not consistent with direct open channel block, but instead involves polyamines binding to another region of the channel (intrinsic gate) to form a blocking complex that occludes the pore. This interaction defines a novel mechanism of ion channel closure.     

Interaction between quaternary ammonium ions in the pore of potassium channels. Evidence against an electrostatic repulsion mechanism

We have examined the interaction between internal and external ions in the pore of potassium channels. We found that external tetraethylammonium was able to antagonize block of Shaker channels by internal TEA when the external and internal solutions contained K(+) ions. This antagonism was absent in solutions with Rb(+) as the only permeant ion. An externally applied trivalent TEA analogue, gallamine, was less effective than the monovalent TEA in inhibiting block by internal TEA. In addition, block by external TEA was little affected by changes in the concentration of internal K(+) ions, but was increased by the presence of internal Na(+) ions in the pore. These results demonstrate that external and internal TEA ions, likely located at opposite ends of the pore selectivity filter, do not experience a mutual electrostatic repulsion. We found that these results can be simulated by a simple 4-barrier-3-site permeation model in which ions compete for available binding sites without long-range electrostatic interactions.     

Binding of kappa-conotoxin PVIIA to Shaker K+ channels reveals different K+ and Rb+ occupancies within the ion channel pore

The x-ray structure of the KcsA channel at different [K(+)] and [Rb(+)] provided insight into how K(+) channels might achieve high selectivity and high K(+) transit rates and showed marked differences between the occupancies of the two ions within the ion channel pore. In this study, the binding of kappa-conotoxin PVIIA (kappa-PVIIA) to Shaker K(+) channel in the presence of K(+) and Rb(+) was investigated. It is demonstrated that the complex results obtained were largely rationalized by differences in selectivity filter occupancy of this 6TM channels as predicted from the structural work on KcsA. kappa-PVIIA inhibition of the Shaker K(+) channel differs in the closed and open state. When K(+) is the only permeant ion, increasing extracellular [K(+)] decreases kappa-PVIIA affinity for closed channels by decreasing the "on" binding rate, but has no effect on the block of open channels, which is influenced only by the intracellular [K(+)]. In contrast, extracellular [Rb(+)] affects both closed- and open-channel binding. As extracellular [Rb(+)] increases, (a) binding to the closed channel is slightly destabilized and acquires faster kinetics, and (b) open channel block is also destabilized and the lowest block seems to occur when the pore is likely filled only by Rb(+). These results suggest that the nature of the permeant ions determines both the occupancy and the location of the pore site from which they interact with kappa-PVIIA binding. Thus, our results suggest that the permeant ion(s) within a channel pore can determine its functional and pharmacological properties.     

Dynamic ion-ion and water-ion interactions in ion channels.

The dynamic interactions among ions and water molecules in ion channels are treated based on an assumption that ions at binding sites can be knocked off by both transient entering ions and local water molecules. The theory, when applied to a single-site model K+ channel, provides solutions for super- and subsaturations, flux-ratio exponent (n') greater than 1, osmotic streaming current, activity-dependent reversal potentials, and anomalous mole-fraction behavior. The analysis predicts that: (a) the saturation may but, in general, does not follow the Michaelis-Menten relation; (b) streaming current results from imbalanced water-ion knock-off interactions; (c) n' greater than 1 even for single-site channels, but it is unlikely to exceed 1.4 unless the pore is occupied by one or more ion(s); (d) in the calculation involving two permeant ion species with similar radii, the heavier ions show higher affinity; the ion-ion knock-off dissociation from the site is more effective when two interacting ions are identical. Therefore, the "multi-ion behaviors" found in most ion channels are the consequences of dynamic ion-ion and water-ion interactions. The presence of these interactions does not require two or more binding sites in channels.     

Tracing the roots of ion channels.

Two sets of recent findings draw our attention to questions concerning the origin of ion channels. First, there is sequence similarity among five classes of channels: voltage-gated channels, a putative Ca(2+)-activated K+ channel, cyclic nucleotide-gated cation channels, a putative Ca2+ channel for phosphoinositide-mediated Ca2+ entry, and a plant K+ channel/transporter. Like voltage-gated K+ channels, the most recently identified members of the superfamily share the basic design of one set of six potential membrane-spanning segments plus the H5 sequence; as such, they may resemble more closely the ancestral channel, which is likely to predate the separation of the animal and plant kingdoms. Second, several members of the ABC superfamily function as ion channels, even though they were previously known as transporters or enzymes. Did some ancestral enzymes subsequently acquire channel/transporter function? Or could it be the other way around? Aside from evolutionary considerations, enzymes and ion channels can no longer be treated as separate and nonoverlapping groups of proteins. When one molecule exhibits both functions, there are interesting mechanistic questions: How might the enzyme activity such as ATP hydrolysis be coupled to activation/regulation of the intrinsic channel activity? How might interactions between the permeant ions and the channel pore in turn regulate the enzymatic function of the same molecule? It seems possible that the latter is an extension of the observed coupling between permeant ions and the gating machinery of an ion channel (Swenson and Armstrong, 1981). Finally, the potential cross-regulation between channel activity and enzyme activity within the same molecule offers many intriguing possibilities for the integration of different cellular functions.     

Characterization and Functional Restoration of a Potassium Channel Kir6.2 Pore Mutation Identified in Congenital Hyperinsulinism

The inwardly rectifying potassium channel Kir6.2 assembles with sulfonylurea receptor 1 to form the ATP-sensitive potassium (K_ATP) channels that regulate insulin secretion in pancreatic β-cells. Mutations in K_ATP channels underlie insulin secretion disease. Here, we report the characterization of a heterozygous missense Kir6.2 mutation, G156R, identified in congenital hyperinsulinism. Homomeric mutant channels reconstituted in COS cells show similar surface expression as wild-type channels but fail to conduct potassium currents. The mutated glycine is in the pore-lining transmembrane helix of Kir6.2; an equivalent glycine in other potassium channels has been proposed to serve as a hinge to allow helix bending during gating. We found that mutation of an adjacent asparagine, Asn-160, to aspartate, which converts the channel from a weak to a strong inward rectifier, on the G156R background restored ion conduction in the mutant channel. Unlike N160D channels, however, G156R/N160D channels are not blocked by intracellular polyamines at positive membrane potential and exhibit wild-type-like nucleotide sensitivities, suggesting the aspartate introduced at position 160 interacts with arginine at 156 to restore ion conduction and gating. Using tandem Kir6.2 tetramers containing G156R and/or N160D in designated positions, we show that one mutant subunit in the tetramer is insufficient to abolish conductance and that G156R and N160D can interact in the same or adjacent subunits to restore conduction. We conclude that the glycine at 156 is not essential for K_ATP channel gating and that the Kir6.2 gating defect caused by the G156R mutation could be rescued by manipulating chemical interactions between pore residues.     

Potassium Channel Gating in the Absence of the Highly Conserved Glycine of the Inner Transmembrane Helix

Potassium channel activation regulates cellular excitability, such as in neuronal and cardiac cells. Regulation of ion channel activity relies on a switching mechanism between two major conformations, the open and closed states, known as gating. It has been suggested that potassium channels are generally gated via a pivoted mechanism of the pore-lining helix (TM2) in the proximity of a glycine that is conserved in about 80% of potassium channels, even though about 20% of the channels lack a glycine at this position. Yet, as we show in G-protein gated potassium (Kir3) channels that lack a glycine at this position, the βγ subunits of G-proteins can still stimulate channel activity. Our results suggest that the effect of mutation of the central glycine (at position 175 in Kir3.4) on βγ-induced whole-cell currents is related to the extent of the interaction between residues located at the position of the central glycine and two residues, one located in the signature sequence of the selectivity filter (T149 in Kir3.4) and the other in the pore helix (E147 in Kir3.4). Our results also suggest that interactions with position 149 are more detrimental to channel function than interactions with position 147. The ability of Gβγ to overcome such restraining interactions is likely to depend on a combination of characteristics specific to each residue.     

Potassium channel gating in the absence of the highly conserved glycine of the inner transmembrane helix

Potassium channel activation regulates cellular excitability, such as in neuronal and cardiac cells. Regulation of ion channel activity relies on a switching mechanism between two major conformations, the open and closed states, known as gating. It has been suggested that potassium channels are generally gated via a pivoted mechanism the pore-lining helix (TM2) in the proximity of a glycine that is conserved in about 80% of potassium channels, even though about 20% of the channels lack a glycine at this position. Yet, as we show in G-protein gated potassium (Kir3) channels that lack a glycine at this position, the betagamma subunits of G-proteins can still stimulate channel activity. Our results suggest that the effect of mutation of the central glycine (at position 175 in Kir3.4) on betagamma-induced whole-cell currents is related to the extent of the interaction between residues located at the position of the central glycine and two residues, one located in the signature sequence of the selectivity filter (T149 in Kir3.4) and the other in the pore helix (E147 in Kir3.4). Our results also suggest that interactions with position 149 are more detrimental to channel function than interactions with position 147. The ability of Gbetagamma to overcome such restraining interactions is likely to depend on a combination of characteristics specific to each residue.     

Effects of pore mutations and permeant ion concentration on the spontaneous gating activity of OmpC porin

Porins are trimers of beta-barrels that form channels for ions and other hydrophilic solutes in the outer membrane of Gram-negative bacteria. The X-ray structures of OmpF and PhoE show that each monomeric pore is constricted by an extracellular loop that folds into the channel vestibule, a motif that is highly conserved among bacterial porins. Electrostatic calculations have suggested that the distribution of ionizable groups at the constriction zone (or eyelet) may establish an intrinsic transverse electrostatic field across the pore, that is perpendicular to the pore axis. In order to study the role that electrostatic interactions between pore residues may have in porin function, we used spontaneous mutants and engineered site-directed mutants that have an altered charge distribution at the eyelet and compared their electrophysiological behavior with that of wild-type OmpC. We found that some mutations lead to changes in the spontaneous gating activity of OmpC porin channels. Changes in the concentration of permeant ions also altered this activity. These results suggest that the ionic interactions that exist between charged residues at the constriction zone of porin may play a role in the transitions between the channel's closed and open states.     

Interactions of divalent cations with single calcium channels from rat brain synaptosomes

Voltage-dependent calcium channels from a rat brain membrane preparation ("synaptosomes") were incorporated into planar lipid bilayers. The effects of calcium, barium, strontium, manganese, and cadmium ions on the amplitudes and kinetics of single channel currents were examined. The order of single channel conductances was gBa greater than gSr greater than gMn, which was the inverse of the order of the mean channel open times: TMn greater than TCa = TSr greater than TBa. In contrast, the identity of the charge carrier had little or no effect on the mean closed times of the channel. Manganese, in the absence of other permeant ions, can pass through single channels (gMn = 4 pS). However, when added to a solution that contained another type of permeant divalent cation, manganese reduced the single channel current in a voltage-dependent manner. Cadmium, a potent blocker of macroscopic "ensemble" calcium currents in many preparations, reduced the current through an open channel in a manner consistent with Cd ions both not being measurably permeant and interacting with a single site. The permeant ions competed with cadmium for this site with the following order: Mn greater than Sr = Ca greater than Ba. These results are consistent with the existence of no less than one divalent cation binding site in the channel that regulates ion permeation.     

Ionic permeation and conduction properties of neuronal KCNQ2/KCNQ3 potassium channels

Heteromeric KCNQ2/3 potassium channels are thought to underlie the M-current, a subthreshold potassium current involved in the regulation of neuronal excitability. KCNQ channel subunits are structurally unique, but it is unknown whether these structural differences result in unique conduction properties. Heterologously expressed KCNQ2/3 channels showed a permeation sequence of while showing a conduction sequence of A differential contribution of component subunits to the properties of heteromeric KCNQ2/3 channels was demonstrated by studying homomeric KCNQ2 and KCNQ3 channels, which displayed contrasting ionic selectivities. KCNQ2/3 channels did not exhibit an anomalous mole-fraction effect in mixtures of K(+) and Rb(+). However, extreme voltage-dependence of block by external Cs(+) was indicative of multi-ion pore behavior. Block of KCNQ2/3 channels by external Ba(2+) ions was voltage-independent, demonstrating unusual ionic occupation of the outer pore. Selectivity properties and block of KCNQ2 were altered by mutation of outer pore residues in a manner consistent with the presence of multiple ion-binding sites. KCNQ2/3 channel deactivation kinetics were slowed exclusively by Rb(+), whereas activation of KCNQ2/3 channels was altered by a variety of external permeant ions. These data indicate that KCNQ2/3 channels are multi-ion pores which exhibit distinctive mechanisms of ion conduction and gating.     

Energy barriers for passage of ions through channels. Exact solution of two electrostatic problems

Exact solutions are given to two electrostatic problems relevant to ion permeation through pores in membranes. The first assesses the importance of the pore forming molecule as a dielectric shield. It is shown on the basis of structural and dielectric considerations alone (neglecting effects attributable to possible charge distribution at the interior surface of the pre-former) that the minimum electrostatic barrier for monovalent ion passage through a gramicidin-like channel is 11 kT. It is further shown that given favorable circumstances, dielectric shielding might dramatically reduce the barrier to ion passage through potassium channels. The second problem considers the error introduced by treating ions as point charges. It is shown that for structureless pores the point charge approximation introduces no meaningful error, even if the ratio of ion radius to pore radius is as great as 0.95.