The bacterial K+ channel structure and its implications for neuronal channels.

Voltage-gated K+ (Kv) channels play a central role in generating action potentials and rhythmic patterns, as well as in dendritic signal processing in neurons. Recently, the first structure of a member of the K+ channel family was solved. Although this channel is from bacteria and has a streamlined body plan with no voltage gating, it establishes the architecture of the functional core of the voltage-gated (K+) channels and their relatives. This architecture explains the crucial features of ion permeation and blockade, and gives some strong hints about gating. The bacterial K+ channel structure is the central piece in a puzzle; it remains to be seen how it will fit together with other domains of the Kv channels, with auxiliary subunits, and with other signal transduction molecules.     

Ca2+ permeation in cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) channels conduct Na+, K+ and Ca2+ currents under the control of cGMP and cAMP. Activation of CNG channels leads to depolarization of the membrane voltage and to a concomitant increase of the cytosolic Ca2+ concentration. Several polypeptides were identified that constitute principal and modulatory subunits of CNG channels in both neurons and non-excitable cells, co-assembling to form a variety of heteromeric proteins with distinct biophysical properties. Since the contribution of each channel type to Ca2+ signaling depends on its specific Ca2+ conductance, it is necessary to analyze Ca2+ permeation for each individual channel type. We have analyzed Ca2+ permeation in all principal subunits of vertebrates and for a principal subunit from Drosophila melanogaster. We measured the fractional Ca2+ current over the physiological range of Ca2+ concentrations and found that Ca2+ permeation is determined by subunit composition and modulated by membrane voltage and extracellular pH. Ca2+ permeation is controlled by the Ca2+-binding affinity of the intrapore cation-binding site, which varies profoundly between members of the CNG channel family, and gives rise to a surprising diversity in the ability to generate Ca2+ signals.     

Contributions of a negatively charged residue in the hydrophobic domain of the IRK1 inwardly rectifying K+ channel to K(+)-selective permeation.

Inwardly rectifying K+ channels are highly selective for K+ ions and show strong interaction with ions in the pore. Both features are important for the physiological functions of these channels and pose intriguing mechanistic questions of ion permeation. The aspartate residue in the second putative transmembrane segment of the IRK1 inwardly rectifying K+ channel, previously implicated in inward rectification gating due to cytoplasmic Mg2+ and polyamine block, is found in this study to be crucial for the channel's ability to distinguish between K+ and Rb+ ions. Mutation of this residue also perturbs the interaction between the channel pore and the Sr2+ blocking ion. Our studies suggest that this aspartate residue contributes to a selectivity filter near the cytoplasmic end of the pore.     

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.     

Plants do it differently. A new basis for potassium/sodium selectivity in the pore of an ion channel

Understanding of the molecular architecture necessary for selective K(+) permeation through the pore of ion channels is based primarily on analysis of the crystal structure of the bacterial K(+) channel KcsA, and structure:function studies of cloned animal K(+) channels. Little is known about the conduction properties of a large family of plant proteins with structural similarities to cloned animal cyclic nucleotide-gated channels (CNGCs). Animal CNGCs are nonselective cation channels that do not discriminate between Na(+) and K(+) permeation. These channels all have the same triplet of amino acids in the channel pore ion selectivity filter, and this sequence is different from that of the selectivity filter found in K(+)-selective channels. Plant CNGCs have unique pore selectivity filters; unlike those found in any other family of channels. At present, the significance of the unique pore selectivity filters of plant CNGCs, with regard to discrimination between Na(+) and K(+) permeation is unresolved. Here, we present an electrophysiological analysis of several members of this protein family; identifying the first cloned plant channel (AtCNGC1) that conducts Na(+). Another member of this ion channel family (AtCNGC2) is shown to have a selectivity filter that provides a heretofore unknown molecular basis for discrimination between K(+) and Na(+) permeation. Specific amino acids within the AtCNGC2 pore selectivity filter (Asn-416, Asp-417) are demonstrated to facilitate K(+) over Na(+) conductance. The selectivity filter of AtCNGC2 represents an alternative mechanism to the well-known GYG amino acid triplet of K(+) channels that has been identified as the critical basis for K(+) over Na(+) permeation through the pore of ion channels.     

The multi-ion nature of the pore in Shaker K+ channels.

We have investigated some of the permeation properties of the pore in Shaker K channels. We determined the apparent permeability ratio of K+, Rb+, and NH4+ ions and block of the pore by external Cs+ ions. Shaker channels were expressed with the baculovirus/Sf9 expression system and the channel currents measured with the whole-cell variant of the patch clamp technique. The apparent permeability ratio, PRb/PK, determined in biionic conditions with internal K+, was a function of external Rb+ concentration. A large change in PRb/PK occurred with reversed ionic conditions (internal Rb+ and external K+). These changes in apparent permeability were not due to differences in membrane potential. With internal K+, PNH4/PK was not a function of external NH4+ concentration (at least over the range 50-120 mM). We also investigated block of the pore by external Cs+ ions. At a concentration of 20 mM, Cs+ block had a voltage dependence equivalent to that of an ion with a valence of 0.91; this increased to 1.3 at 40 mM Cs+. We show that a 4-barrier, 3-site permeation model can simulate these and many of the other known properties of ion permeation in Shaker channels.     

Effect of changing temperature on the ionic permeation through the cyclic GMP-gated channel from vertebrate photoreceptors.

Native cGMP-gated channels were studied in rod outer segments of the larval tiger salamander Ambystoma tigrinum. The alpha subunit of the cGMP-gated channel from bovine rods, here referred to as the wild type (w.t.), and mutant channels were heterologously expressed in Xenopus laevis oocytes. These channels were studied in excised membrane patches in the inside-out configuration and were activated by the addition of 100 or 500 microM cGMP. The effect of temperature on the ionic permeation was studied. The macroscopic current flowing through the native channel at +100 mV had an activation energy of 35.8, 30, 31.8, 34.5, 41.3, and 22.4 kJ mol-1 in the presence of Li+, Na+, K+, Rb+, Cs+, and NH4+, respectively. The macroscopic current flowing through the w.t. channel at +100 mV had an activation energy of 45.2, 38.2, 37.5, 47.3, 49.4, and 38.9 kJ mol-1 in the presence of Li+, Na+, K+, Rb+, Cs+, and NH4+, respectively. The activation energy of the macroscopic current flowing through the native and w.t. channels did not vary significantly when the ionic concentration of the permeant ion was changed between 2.5 and 110 mM. The activation energy of the single-channel current of the w.t. channel at +100 mV was 40.4 and 33 kJ mol-1 for Na+ and NH4+, respectively. The reversal potential of biionic solutions changed significantly with temperature. These results can be used to obtain an estimate of the enthalpic and entropic contributions to the barrier of the Gibbs free energy experienced by an ion during its permeation through the open channel. These estimates indicate that the ionic permeation and selectivity of the cGMP-gated channel are controlled both by enthalpic and entropic factors and that the selectivity of the native channel for Li+ over Na+ is primarily caused by entropic effects.     

Bead-like passage of chloride ions through ClC chloride channels

The ClC chloride channels control the ionic composition of the cytoplasm and the volume of cells, and regulate electrical excitability. Recently, it has been proposed that prokaryotic ClC channels are H+-Cl- exchange transporter. Although X-ray and molecular dynamics (MD) studies of bacterial ClC channels have investigated the filter open-close and ion permeation mechanism of channels, details have remained unclear. We performed MD simulations of ClC channels involving H+, Na+, K+, or H3O+ in the intracellular region to elucidate the open-close mechanism, and to clarify the role of H+ ion an H+-Cl- exchange transporter. Our simulations revealed that H+ and Na+ caused channel opening and the passage of Cl- ions. Na+ induced a bead-like string of Cl- -Na+-Cl--Na+-Cl- ions to form and permeate through ClC channels to the intracellular side with the widening of the channel pathway.     

Symmetry and asymmetry of permeation through toxin-modified Na+ channels.

Single Na+ channels from rat skeletal muscle were inserted into planar lipid bilayers in the presence of either 200 nM batrachotoxin (BTX) or 50 microM veratridine (VT). These toxins, in addition to their ability to shift inactivation of voltage-gated Na+ channels, may be used as probes of ion conduction in these channels. Channels modified by either of the toxins have qualitatively similar selectivity for the alkali cations (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). Biionic reversal potentials, for example, were concentration independent for all ions studied. Na+/K+ and Na+/Rb+ reversal potentials, however, were dependent on the orientation of the ionic species with respect to the intra- or extracellular face of the channel, whereas Na+/Li+ biionic reversal potentials were not orientation dependent. A simple, four-barrier, three-well, single-ion occupancy model was used to generate current-voltage relationships similar to those observed in symmetrical solutions of Na, K, or Li ions. The barrier profiles for Na and Li ions were symmetric, whereas that for K ions was asymmetric. This suggests the barrier to ion permeation for K ions may be different than that for Na and Li ions. With this model, these hypothetical energy barrier profiles could predict the orientation-dependent reversal potentials observed for Na+/K+ and Na+/Rb+. The energy barrier profiles, however, were not capable of describing biionic Na/Li ion permeation. Together these results support the hypothesis that Na ions have a different rate determining step for ion permeation than that of K and Rb ions.     

The cytosolic inactivation domains of BKi channels in rat chromaffin cells do not behave like simple, open-channel blockers.

Most BK-type voltage- and Ca(2+)-dependent K+ channels in rat chromaffin cells exhibit rapid inactivation. This inactivation is abolished by brief trypsin application to the cytosolic face of membrane patches. Here we examine the effects of cytosolic channel blockade and pore occupancy on this inactivation process, using inside-out patches and whole-cell recordings. Occupancy of a superficial pore-blocking site by cytosolic quaternary blockers does not slow inactivation. Occupancy of a deeper pore-blocking site by cytosolic application of Cs+ is also without effect on the onset of inactivation. Although the rate of inactivation is relatively unaffected by changes in extracellular K+, the rate of recovery from inactivation (at -80 and -140 mV with 10 microM Ca2+) is faster with increases in extracellular K+ but is unaffected by the impermeant ion, Na+. When tail currents are compared after repolarization, either while channels are open or after inactivation, no channel reopening is detectable during recovery from inactivation. BK inactivation appears to be mechanistically distinct from that of other inactivating voltage-dependent channels. Although involving a trypsin-sensitive cytosolic structure, the block to permeation does not appear to occur directly at the cytosolic mouth or inner half of the ion permeation pathway.     

Role of surface electrostatics in the operation of a high-conductance Ca2+-activated K+ channel

This paper demonstrates that local electric fields originating from negatively charged groups on a K+-specific ion channel modify its behavior. Single high-conductance, Ca2+-activated K+ channels were studied in neutral phospholipid bilayers. The channel protein surface charges were manipulated experimentally by carboxyl group esterification using trimethyloxonium (TMO) or by electrolyte screening. Three channel properties--ion conduction, ion blockade, and voltage-dependent gating--are affected by surface electrostatics. Negative charges increase the affinity of cationic pore blockers by establishing a local negative potential at the pore entrance; these charges modify channel gating by establishing a potential gradient across the ion channel; finally, both effects influence ion permeation through the pore.     

Redox-sensitive extracellular gates formed by auxiliary beta subunits of calcium-activated potassium channels

An important step to understanding ion channels is identifying the structural components that act as the gates to ion movement. Here we describe a new channel gating mechanism, produced by the beta3 auxiliary subunits of Ca2+-activated, large-conductance BK-type K+ channels when expressed with their pore-forming alpha subunits. BK beta subunits have a cysteine-rich extracellular segment connecting two transmembrane segments, with small cytosolic N and C termini. The extracellular segments of the beta3 subunits form gates to block ion permeation, providing a mechanism by which current can be rapidly diminished upon cellular repolarization. Furthermore, this gating mechanism is abolished by reduction of extracellular disulfide linkages, suggesting that endogenous mechanisms may regulate this gating behavior. The results indicate that auxiliary beta subunits of BK channels reside sufficiently close to the ion permeation pathway defined by the alpha subunits to influence or block access of small molecules to the permeation pathway.     

Voltage-dependent Ba2+ permeation through store-operated CRAC channels: implications for channel selectivity

Store-operated Ca2+ entry through CRAC channels is a major route for Ca2+ influx in non-excitable cells. Studies on store-operated channel selectivity using fluorescent dyes have found that the channels are impermeable to Ba2+. Furthermore, in such studies, agonists have been reported to increase Ba2+ influx, leading to the conclusion that additional Ca2+ entry pathways (permeable to Ba2+) co-exist with the Ba2+-impermeable store-operated channels. However, patch clamp experiments demonstrate that CRAC channels are permeable to Ba2+. We have addressed this paradox using fluorescence measurements and whole cell patch clamp recordings of ICRAC. In store-depleted cells loaded with fura 2, Ba2+ application results in a slower and smaller rise in fluorescence than is the case with Ca2+. Ba2+, unlike Ca2+, depolarises the membrane potential by approximately 40 mV, due to rapid block of an inwardly rectifying K+ current. Although Ba2+ permeates CRAC channels at very negative potentials in patch clamp recordings, Ba2+ permeation is steeply voltage-dependent. This combination of Ba2+-dependent depolarisation and voltage-dependent Ba2+ permeation accounts for the apparent lack of Ba2+ permeation through store-operated channels seen in fluorescence experiments. Our findings identify major limitations with the use of Ba2+ as a surrogate for Ca2+ in probing Ca2+ entry pathways and raise the possibility that some of the previous reports proposing multiple Ca2+ entry pathways based on Ba2+ entry into fura 2-loaded cells could be explained by voltage-dependent Ba2+ permeation through CRAC channels.     

On the structural basis for ionic selectivity among Na+, K+, and Ca2+ in the voltage-gated sodium channel.

Voltage-sensitive sodium channels and calcium channels are homologous proteins with distinctly different selectivity for permeation of inorganic cations. This difference in function is specified by amino acid residues located within P-region segments that link presumed transmembrane elements S5 and S6 in each of four repetitive Domains I, II, III, and IV. By analyzing the selective permeability of Na+, K+, and Ca2+ in various mutants of the mu 1 rat muscle sodium channel, the results in this paper support the concept that a conserved motif of four residues contributed by each of the Domains I-IV, termed the DEKA locus in sodium channels and the EEEE locus in calcium channels, determines the ionic selectivity of these channels. Furthermore, the results indicate that the Lys residue in Domain III of the sodium channel is the critical determinant that specifies both the impermeability of Ca2+ and the selective permeability of Na+ over K+. We propose that the alkylammonium ion of the Lys(III) residue acts as an endogenous cation within the ion binding site/selectivity filter of the sodium channel to tune the kinetics and affinity of inorganic cation binding within the pore in a manner analogous to ion-ion interactions that occur in the process of multi-ion channel conduction.     

Defining the conductance of the closed state in a voltage-gated K+ channel

The opening and closing of the ion conduction pathway in ion channels underlies the generation and propagation of electrical signals in biological systems. Although electrophysiological approaches to measuring the flow of ions in the open state have contributed profoundly to our understanding of ion permeation and gating, it remains unclear how much the ion-throughput rate decreases upon closure of the ion conduction pore. To address this fundamental question, we expressed the Shaker Kv channel at high levels and then measured macroscopic K+ currents at negative membrane voltages and counted the number of channels by quantifying the translocation of gating charge. Our results show that the conductance of the closed state is between 0 and 0.16 fS, or at least 100,000 times lower than for the open state of the channel, indicating that the flow of ions is very tightly regulated in this class of K+ channels.     

Molecular basis of ion selectivity, block, and rectification of the inward rectifier Kir3.1/Kir3.4 K(+) channel

The glycine-tyrosine-glycine (GYG) sequence in the p-loop of K+ channel subunits lines a narrow pore through which K+ ions pass in single file intercalated by water molecules. Mutation of the motif can give rise to non-selective channels, but it is clear that other structural features are also required for selectivity because, for instance, a recently identified class of cyclic nucleotide-gated pacemaker channels has the GYG motif but are poorly K+ selective. We show that mutation of charged glutamate and arginine residues behind the selectivity filter in the Kir3.1/Kir3.4 K+ channel reduces or abolishes K+ selectivity, comparable with previously reported effects in the Kir2.1 K+ channel. It has been suggested that a salt bridge exists between the glutamate-arginine residue pair. Molecular modeling indicates that the salt bridge does exist, and that it acts as a "bowstring" to maintain the rigid bow-like structure of the selectivity filter and restrict selectivity to K+. The modeling shows that relaxation of the bowstring by mutation of the residue pair leads to enhanced flexibility of the p-loop, allowing permeation of other cations, including polyamines. In experiments, mutation of the residue pair can also abolish polyamine-induced inward rectification. The latter effect occurs because polyamines now permeate rather than block the channel, to the remarkable extent that large polyamine currents can be measured.     

Quaternary ammonium compounds as structural probes of single batrachotoxin-activated Na+ channels

Quaternary ammonium (QA) blockers are well-known structural probes for studying the permeation pathway of voltage-gated K+ channels. In this study we have examined the effects of a series of n-alkyl-trimethylammonium compounds (Cn-QA) on batrachotoxin (BTX)-activated Na+ channels from skeletal muscle incorporated into planar lipid bilayers. We found that these amphipathic QA compounds (Cn-QA where n = 10-18) block single Na+ channels preferentially from the internal side with equilibrium dissociation constants (KD) in the submicromolar to micromolar range. External application of amphipathic QA compounds is far less effective, by a factor of greater than 200. The block can be described by a QA molecule binding to a single site in the Na+ channel permeation pathway. QA binding affinity is dependent on transmembrane voltage with an effective valence (delta) of approximately 0.5. QA dwell times (given as mean closed times, tau c) increase as a function of n-alkyl chain length, ranging from approximately 13 ms for C10-QA to 500 ms for C18-QA at +50 mV. The results imply that there is a large hydrophobic region within the Na+ channel pore which accepts up to 18 methylene groups of the Cn-QA cation. This hydrophobic domain may be of clinical significance since it also interacts with local anesthetics such as cocaine and mepivacaine. Finally, like BTX-activated Na+ channels in bilayers, unmodified Na+ channels in GH3 cells are also susceptible to QA block. Amphipathic QA cations elicit both tonic and use-dependent inhibitions of normal Na+ currents in a manner similar to that of local anesthetic cocaine. We conclude that amphipathic QA compounds are valuable structural probes to study the permeation pathway of both normal and BTX-activated Na+ channels.     

Ionic permeation and blockade in Ca2+-activated K+ channels of bovine chromaffin cells

Single channel currents through Ca2+-activated K+ channels of bovine chromaffin cells were measured to determine the effects of small ions on permeation through the channel. The channel selects strongly for K+ over Na+ and Cs+, and Rb+ carries a smaller current through the channel than K+. Tetraethylammonium ion (TEA+) blocks channel currents when applied to either side of the membrane; it is effective at lower concentrations when applied externally. Millimolar concentrations of internal Na+ reduce the average current through the channel and produce large fluctuations (flicker) in the open channel currents. This flickery block is analyzed by a new method, amplitude distribution analysis, which can measure block and unblock rates in the microsecond time range even though individual blocking events are not time-resolved by the recording system. The analysis shows that the rate of block by Na+ is very voltage dependent, but the unblock rate is voltage independent. These results can be explained easily by supposing that current flow through the channel is diffusion limited, a hypothesis consistent with the large magnitude of the single channel current.     

Na+ permeation and block of hERG potassium channels

The inactivation gating of hERG channels is important for the channel function and drug-channel interaction. Whereas hERG channels are highly selective for K+, we have found that inactivated hERG channels allow Na+ to permeate in the absence of K+. This provides a new way to directly monitor and investigate hERG inactivation. By using whole cell patch clamp method with an internal solution containing 135 mM Na+ and an external solution containing 135 mM NMG+, we recorded a robust Na+ current through hERG channels expressed in HEK 293 cells. Kinetic analyses of the hERG Na+ and K+ currents indicate that the channel experiences at least two states during the inactivation process, an initial fast, less stable state followed by a slow, more stable state. The Na+ current reflects Na+ ions permeating through the fast inactivated state but not through the slow inactivated state or open state. Thus the hERG Na+ current displayed a slow inactivation as the channels travel from the less stable, fast inactivated state into the more stable, slow inactivated state. Removal of fast inactivation by the S631A mutation abolished the Na+ current. Moreover, acceleration of fast inactivation by mutations T623A, F627Y, and S641A did not affect the hERG Na+ current, but greatly diminished the hERG K+ current. We also found that external Na+ potently blocked the hERG outward Na+ current with an IC50 of 3.5 mM. Mutations in the channel pore and S6 regions, such as S624A, F627Y, and S641A, abolished the inhibitory effects of external Na+ on the hERG Na+ current. Na+ permeation and blockade of hERG channels provide novel ways to extend our understanding of the hERG gating mechanisms.