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.     

Stable cation coordination at a single outer pore residue defines permeation properties in Kir channels

In epithelial Kir7.1 channels a non-conserved methionine in the outer pore region adjacent to the G-Y-G selectivity filter (position +2) was found to determine unique properties for permeant and blocking ions characteristic of a K(+) channel in a single-occupancy state. The monovalent cation permeability sequence of Kir7.1 channels expressed in Xenopus oocytes was Tl(+)>K(+)>Rb(+)NH(4)(+)>Cs(+)>Na(+)>Li(+), but the macroscopic conductance for Rb(+) was approximately 8-fold larger than for the smaller K(+) ions, and decreased approximately 40-fold with the conserved arginine at the +2 position (Kir7.1M125R). Moreover, in Kir7.1 Rb(+) restored the typical permeation properties of other multi-ion channels indicating that a stable coordination of permeant ions at the +2 position defines the initial step in the conduction pathway of Kir channels.     

TEA(+)-sensitive KCNQ1 constructs reveal pore-independent access to KCNE1 in assembled I(Ks) channels

I(Ks), a slowly activating delayed rectifier K(+) current through channels formed by the assembly of two subunits KCNQ1 (KvLQT1) and KCNE1 (minK), contributes to the control of the cardiac action potential duration. Coassembly of the two subunits is essential in producing the characteristic and physiologically critical kinetics of assembled channels, but it is not yet clear where or how these subunits interact. Previous investigations of external access to the KCNE1 protein in assembled I(Ks) channels relied on occlusion of the pore by extracellular application of TEA(+), despite the very low TEA(+) sensitivity (estimated EC(50) > 100 mM) of channels encoded by coassembly of wild-type KCNQ1 with the wild type (WT) or a series of cysteine-mutated KCNE1 constructs. We have engineered a high affinity TEA(+) binding site into the h-KCNQ1 channel by either a single (V319Y) or double (K318I, V319Y) mutation, and retested it for pore-delimited access to specific sites on coassembled KCNE1 subunits. Coexpression of either KCNQ1 construct with WT KCNE1 in Chinese hamster ovary cells does not alter the TEA(+) sensitivity of the homomeric channels (IC(50) approximately 0.4 mM [TEA(+)](out)), providing evidence that KCNE1 coassembly does not markedly alter the structure of the outer pore of the KCNQ1 channel. Coexpression of a cysteine-substituted KCNE1 (F54C) with V319Y significantly increases the sensitivity of channels to external Cd(2+), but neither the extent of nor the kinetics of the onset of (or the recovery from) Cd(2+) block was affected by [TEA(+)](o) at 10x the IC(50) for channel block. These data strongly suggest that access of Cd(2+) to the cysteine-mutated site on KCNE1 is independent of pore occlusion caused by TEA(+) binding to the outer region of the KCNE1/V319Y pore, and that KCNE1 does not reside within the pore region of the assembled channels.     

KCNQ1 subdomains involved in KCNE modulation revealed by an invertebrate KCNQ1 orthologue

KCNQ1 channels are voltage-gated potassium channels that are widely expressed in various non-neuronal tissues, such as the heart, pancreas, and intestine. KCNE proteins are known as the auxiliary subunits for KCNQ1 channels. The effects and functions of the different KCNE proteins on KCNQ1 modulation are various; the KCNQ1-KCNE1 ion channel complex produces a slowly activating potassium channel that is crucial for heartbeat regulation, while the KCNE3 protein makes KCNQ1 channels constitutively active, which is important for K(+) and Cl(-) transport in the intestine. The mechanisms by which KCNE proteins modulate KCNQ1 channels have long been studied and discussed; however, it is not well understood how different KCNE proteins exert considerably different effects on KCNQ1 channels. Here, we approached this point by taking advantage of the recently isolated Ci-KCNQ1, a KCNQ1 homologue from marine invertebrate Ciona intestinalis. We found that Ci-KCNQ1 alone could be expressed in Xenopus laevis oocytes and produced a voltage-dependent potassium current, but that Ci-KCNQ1 was not properly modulated by KCNE1 and totally unaffected by coexpression of KCNE3. By making chimeras of Ci-KCNQ1 and human KCNQ1, we determined several amino acid residues located in the pore region of human KCNQ1 involved in KCNE1 modulation. Interestingly, though, these amino acid residues of the pore region are not important for KCNE3 modulation, and we subsequently found that the S1 segment plays an important role in making KCNQ1 channels constitutively active by KCNE3. Our findings indicate that different KCNE proteins use different domains of KCNQ1 channels, and that may explain why different KCNE proteins give quite different outcomes by forming a complex with KCNQ1 channels.     

Gating and flickery block differentially affected by rubidium in homomeric KCNQ1 and heteromeric KCNQ1/KCNE1 potassium channels

The voltage-gated potassium channel KCNQ1 associates with the small KCNE1 subunit to form the cardiac IKs delayed rectifier potassium current and mutations in both genes can lead to the long QT syndrome. KCNQ1 can form functional homotetrameric channels, however with drastically different biophysical properties compared to heteromeric KCNQ1/KCNE1 channels. We analyzed gating and conductance of these channels expressed in Xenopus oocytes using the two-electrode voltage-clamp and the patch-clamp technique and high extracellular potassium (K) and rubidium (Rb) solutions. Inward tail currents of homomeric KCNQ1 channels are increased about threefold upon substitution of 100 mM potassium with 100 mM rubidium despite a smaller rubidium permeability, suggesting an effect of rubidium on gating. However, the kinetics of tail currents and the steady-state activation curve are only slightly changed in rubidium. Single-channel amplitude at negative voltages was estimated by nonstationary noise analysis, and it was found that rubidium has only a small effect on homomeric channels (1.2-fold increase) when measured at a 5-kHz bandwidth. The apparent single-channel conductance was decreased after filtering the data at lower cutoff frequencies indicative of a relatively fast "flickery/block" process. The relative conductance in rubidium compared to potassium increased at lower cutoff frequencies (about twofold at 10 Hz), suggesting that the main effect of rubidium is to decrease the probability of channel blockage leading to an increase of inward currents without large changes in gating properties. Macroscopic inward tail currents of heteromeric KCNQ1/KCNE1 channels in rubidium are reduced by about twofold and show a pronounced sigmoidal time course that develops with a delay similar to the inactivation process of homomeric KCNQ1, and is indicative of the presence of several open states. The single channel amplitude of heteromers is about twofold smaller in rubidium than in potassium at a bandwidth of 5 kHz. Filtering at lower cutoff frequencies reduces the apparent single-channel conductance, the ratio of the conductance in rubidium versus potassium is, however, independent of the cutoff frequency. Our results suggest the presence of a relatively rapid process (flicker) that can occur almost independently of the gating state. Occupancy by rubidium at negative voltages favors the flicker-open state and slows the flickering rate in homomeric channels, whereas rubidium does not affect the flickering in heteromeric channels. The effects of KCNE1 on the conduction properties are consistent with an interaction of KCNE1 in the outer vestibule of the channel.     

Block of tetrodotoxin-resistant Na+ channel pore by multivalent cations: gating modification and Na+ flow dependence

Tetrodotoxin-resistant (TTX-R) Na(+) channels are much less susceptible to external TTX but more susceptible to external Cd(2+) block than tetrodotoxin-sensitive (TTX-S) Na(+) channels. Both TTX and Cd(2+) seem to block the channel near the "DEKA" ring, which is probably part of a multi-ion single-file region adjacent to the external pore mouth and is involved in the selectivity filter of the channel. In this study we demonstrate that other multivalent transitional metal ions such as La(3+), Zn(2+), Ni(2+), Co(2+), and Mn(2+) also block the TTX-R channels in dorsal root ganglion neurons. Just like Cd(2+), the blocking effect has little intrinsic voltage dependence, but is profoundly influenced by Na(+) flow. The apparent dissociation constants of the blocking ions are always significantly smaller in inward Na(+) currents than those in outward Na(+) current, signaling exit of the blocker along with the Na(+) flow and a high internal energy barrier for "permeation" of these multivalent blocking ions through the pore. Most interestingly, the activation and especially the inactivation kinetics are slowed by the blocking ions. Moreover, the gating changes induced by the same concentration of a blocking ion are evidently different in different directions of Na(+) current flow, but can always be correlated with the extent of pore block. Further quantitative analyses indicate that the apparent slowing of channel activation is chiefly ascribable to Na(+) flow-dependent unblocking of the bound La(3+) from the open Na(+) channel, whereas channel inactivation cannot happen with any discernible speed in the La(3+)-blocked channel. Thus, the selectivity filter of Na(+) channel is probably contiguous to a single-file multi-ion region at the external pore mouth, a region itself being nonselective in terms of significant binding of different multivalent cations. This region is "open" to the external solution even if the channel is "closed" ("deactivated"), but undergoes imperative conformational changes during the gating (especially the inactivation) process of the channel.     

Conduction properties of the M-channel in rat sympathetic neurons.

We have investigated the conduction properties of the M-channel in rat superior cervical ganglion neurons. Reversal potentials measured under bi-ionic conditions yielded a permeation sequence of Tl > K > Rb > Cs > NH4 > Na. Slope conductances gave a conductance sequence of K > Tl > NH4 > Rb > Cs. M-current was shown to exhibit a number of features atypical of potassium channels. First, the conduction of monovalent cations relative to K was very low. Second, the nature of the permeant ion did not affect the deactivation kinetics. Third, M-current did not exhibit anomalous mole-fraction behavior, a property suggestive of a multi-ion pore. Finally, external Ba, which is a blocker of M-current, showed a preferential block of outward current and had much less effect on inward current. The permeability sequence of the M-channel is very similar to other K-selective channels, implying a high degree of conservation in the selectivity filter. However, other conduction properties suggest that the pore structure outside of the selectivity filter is very different from previously cloned potassium channels.     

Made for "anchorin": Kv7.2/7.3 (KCNQ2/KCNQ3) channels and the modulation of neuronal excitability in vertebrate axons

Kv7.2 and Kv7.3 (encoded by KCNQ2 and KCNQ3) are homologous subunits forming a widely expressed neuronal voltage-gated K(+) (Kv) channel. Hypomorphic mutations in either KCNQ2 or KCNQ3 cause a highly penetrant, though transient, human phenotype-epilepsy during the first months of life. Some KCNQ2 mutations also cause involuntary muscle rippling, or myokymia, which is indicative of motoneuron axon hyperexcitability. Kv7.2 and Kv7.3 are concentrated at axonal initial segments (AISs), and at nodes of Ranvier in the central and peripheral nervous system. Kv7.2 and Kv7.3 share a novel ～80 residue C-terminal domain bearing an "anchor" motif, which interacts with ankyrin-G and is required for channel AIS (and likely, nodal) localization. This domain includes the sequence IAEGES/TDTD, which is analogous (not homologous) to the ankyrin-G interaction motif of voltage-gated Na(+) (Na(V)) channels. The KCNQ subfamily is evolutionarily ancient, with two genes (KCNQ1 and KCNQ5) persisting as orthologues in extant bilaterian animals from worm to man. However, KCNQ2 and KCNQ3 arose much more recently, in the interval between the divergence of extant jawless and jawed vertebrates. This is precisely the interval during which myelin and saltatory conduction evolved. The natural selection for KCNQ2 and KCNQ3 appears to hinge on these subunits' unique ability to be coordinately localized with Na(V) channels by ankyrin-G, and the resulting enhancement in the reliability of neuronal excitability.     

Pore- and state-dependent cadmium block of I(Ks) channels formed with MinK-55C and wild-type KCNQ1 subunits

Human MinK and KCNQ1 subunits assemble to form I(Ks) channels. When MinK position 55 is mutated to cysteine (MinK-55C), I(Ks) channels can be blocked by external cadmium (Cd(2+)). We have supported a pore-associated location for MinK-55C because Cd(2+) block is sensitive to voltage, permeant ions on the opposite side of the membrane (trans-ions), and external tetraethylammonium (TEA), an I(Ks) pore-blocker. Two recent reports argue that MinK-55C is distant from the pore: one finds TEA does not affect Cd(2+) block if channels are formed with a KCNQ1 mutant (K318I, V319Y) that increases TEA affinity; the second proposes that Cd(2+) binds between MinK-55C and a cysteine in KCNQ1 that is posited to lie toward the channel periphery. Here, these discrepancies are considered. First, Cd(2+) block of MinK-55C channels formed with wild-type KCNQ1 is shown to depend not only on voltage and trans-ions but state (showing decreased on-rate with increased open time and blocker trapping on channel closure). Conversely, MinK-55C channels with K318I, V319Y KCNQ1 are found to demonstrate Cd(2+) block that is independent of voltage, trans-ions and state (and to have a lower unitary conductance): thus, the KCNQ1 mutations alter the process under study, yielding Cd(2+) inhibition that is pore-independent and, perforce, TEA-insensitive. Second, MinK-55C channels are found to remain sensitive to Cd(2+) despite mutation of any single native cysteine in KCNQ1 or all nine simultaneously; this suggests no KCNQ1 cysteine binds Cd(2+) and can serve to localize MinK-55C. Despite many concerns that are enumerated, we remain obliged to conclude that Cd(2+) enters and leaves the pore to reach MinK-55C, placing that residue in or near the pore.     

The role of S4 charges in voltage-dependent and voltage-independent KCNQ1 potassium channel complexes

Voltage-gated potassium (Kv) channels extend their functional repertoire by coassembling with MinK-related peptides (MiRPs). MinK slows the activation of channels formed with KCNQ1 alpha subunits to generate the voltage-dependent I(Ks) channel in human heart; MiRP1 and MiRP2 remove the voltage dependence of KCNQ1 to generate potassium "leak" currents in gastrointestinal epithelia. Other Kv alpha subunits interact with MiRP1 and MiRP2 but without loss of voltage dependence; the mechanism for this disparity is unknown. Here, sequence alignments revealed that the voltage-sensing S4 domain of KCNQ1 bears lower net charge (+3) than that of any other eukaryotic voltage-gated ion channel. We therefore examined the role of KCNQ1 S4 charges in channel activation using alanine-scanning mutagenesis and two-electrode voltage clamp. Alanine replacement of R231, at the N-terminal side of S4, produced constitutive activation in homomeric KCNQ1 channels, a phenomenon not observed with previous single amino acid substitutions in S4 of other channels. Homomeric KCNQ4 channels were also made constitutively active by mutagenesis to mimic the S4 charge balance of R231A-KCNQ1. Loss of single S4 charges at positions R231 or R237 produced constitutively active MinK-KCNQ1 channels and increased the constitutively active component of MiRP2-KCNQ1 currents. Charge addition to the CO2H-terminal half of S4 eliminated constitutive activation in MiRP2-KCNQ1 channels, whereas removal of homologous charges from KCNQ4 S4 produced constitutively active MiRP2-KCNQ4 channels. The results demonstrate that the unique S4 charge paucity of KCNQ1 facilitates its unique conversion to a leak channel by ancillary subunits such as MiRP2.     

Two different conformational states of the KirBac3.1 potassium channel revealed by electron crystallography

Potassium channels allow the selective flow of K(+) ions across membranes. In response to external gating signals, the potassium channel can move reversibly through a series of structural conformations from a closed to an open state. 2D crystals of the inwardly rectifying K(+) channel KirBac3.1 from Magnetospirillum magnetotacticum have been captured in two distinct conformations, providing "snap shots" of the gating process. Analysis by electron cryomicroscopy of these KirBac3.1 crystals has resulted in reconstructed images in projection at 9 A resolution. Kir channels are tetramers of four subunits arranged as dimers of dimers. Each subunit has two transmembrane helices (inner and outer). In one crystal form, the pore is blocked; in the other crystal form, the pore appears open. Modeling based on the KirBac1.1 (closed) crystal structure shows that opening of the ion conduction pathway could be achieved by bending of the inner helices and significant movements of the outer helices.     

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.     

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.     

KCNQ4, a novel potassium channel expressed in sensory outer hair cells, is mutated in dominant deafness

Potassium channels regulate electrical signaling and the ionic composition of biological fluids. Mutations in the three known genes of the KCNQ branch of the K+ channel gene family underlie inherited cardiac arrhythmias (in some cases associated with deafness) and neonatal epilepsy. We have now cloned KCNQ4, a novel member of this branch. It maps to the DFNA2 locus for a form of nonsyndromic dominant deafness. In the cochlea, it is expressed in sensory outer hair cells. A mutation in this gene in a DFNA2 pedigree changes a residue in the KCNQ4 pore region. It abolishes the potassium currents of wild-type KCNQ4 on which it exerts a strong dominant-negative effect. Whereas mutations in KCNQ1 cause deafness by affecting endolymph secretion, the mechanism leading to KCNQ4-related hearing loss is intrinsic to outer hair cells.     

Novel role of KCNQ2/3 channels in regulating neuronal cell viability

Overactivation of certain K(+) channels can mediate excessive K(+) efflux and intracellular K(+) depletion, which are early ionic events in apoptotic cascade. The present investigation examined a possible role of the KCNQ2/3 channel or M-channel (also named Kv7.2/7.3 channels) in the pro-apoptotic process. Whole-cell recordings detected much larger M-currents (212 ± 31 pA or 10.5 ± 1.5 pA/pF) in cultured hippocampal neurons than that in cultured cortical neurons (47 ± 21 pA or 2.4 ± 0.8 pA/pF). KCNQ2/3 channel openers N-ethylmaleimide (NEM) and flupirtine caused dose-dependent K(+) efflux, intracellular K(+) depletion, and cell death in hippocampal cultures, whereas little cell death was induced by NEM in cortical cultures. The NEM-induced cell death was antagonized by co-applied KCNQ channel inhibitor XE991 (10 μM), or by elevated extracellular K(+) concentration. Supporting a mediating role of KCNQ2/3 channels in apoptosis, expression of KCNQ2 or KCNQ2/3 channels in Chinese hamster ovary (CHO) cells initiated caspase-3 activation. Consistently, application of NEM (20 μM, 8 h) in hippocampal cultures similarly caused caspase-3 activation assessed by immunocytochemical staining and western blotting. NEM increased the expression of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), induced mitochondria membrane depolarization, cytochrome c release, formation of apoptosome complex, and apoptosis-inducing factor (AIF) translocation into nuclear. All these events were attenuated by blocking KCNQ2/3 channels. These findings provide novel evidence that KCNQ2/3 channels could be an important regulator in neuronal apoptosis.     

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.     

Regulation of the voltage-gated K(+) channels KCNQ2/3 and KCNQ3/5 by serum- and glucocorticoid-regulated kinase-1

The voltage-gated KCNQ2/3 and KCNQ3/5 K(+) channels regulate neuronal excitability. We recently showed that KCNQ2/3 and KCNQ3/5 channels are regulated by the ubiquitin ligase Nedd4-2. Serum- and glucocorticoid-regulated kinase-1 (SGK-1) plays an important role in regulation of epithelial ion transport. SGK-1 phosphorylation of Nedd4-2 decreases the ability of Nedd4-2 to ubiquitinate the epithelial Na(+) channel, which increases the abundance of channel protein in the cell membrane. In this study, we investigated the mechanism(s) of SGK-1 regulation of M-type KCNQ channels expressed in Xenopus oocytes. SGK-1 significantly upregulated the K(+) current amplitudes of KCNQ2/3 and KCNQ3/5 channels approximately 1.4- and approximately 1.7-fold, respectively, whereas the kinase-inactive SGK-1 mutant had no effect. The cell surface levels of KCNQ2-hemagglutinin/3 were also increased by SGK-1. Deletion of the KCNQ3 channel COOH terminus in the presence of SGK-1 did not affect the K(+) current amplitude of KCNQ2/3/5-mediated currents. Coexpression of Nedd4-2 and SGK-1 with KCNQ2/3 or KCNQ3/5 channels did not significantly alter K(+) current amplitudes. Only the Nedd4-2 mutant (S448A)Nedd4-2 exhibited a significant downregulation of the KCNQ2/3/5 K(+) current amplitudes. Taken together, these results demonstrate a potential mechanism for regulation of KCNQ2/3 and KCNQ3/5 channels by SGK-1 regulation of the activity of the ubiquitin ligase Nedd4-2.     

Altered KCNQ3 Potassium Channel Function Caused by the W309R Pore-Helix Mutation Found in Human Epilepsy

The second tryptophan (W) residue of the conserved WW motif in the pore helix of many K⁺ channel subunit is thought to interact with the tyrosine (Y) residues of the selectivity filter. A missense mutation causing the replacement of the corresponding residues with an arginine (W309R) occurs in KCNQ3 subunits forming part of M-channels. In this study, we examined the functional consequences of the W309R mutation in heterogously expressed KCNQ channels. Homomeric KCNQ3^W309R channels lacked KCNQ currents. Heteromeric KCNQ2/KCNQ3^W309R channels displayed a dominant-negative suppression of current and a significant modification in gating properties when compared with heteromeric KCNQ3/KCNQ2 channels mimicking the M-channels. A three-dimensional homology model in the W309R mutant indicated that the R side chain of pore helices is too far from the Y side chain of the selectivity filter to interact via hydrogen bonds with each other and stabilize the pore structure. Collectively, the present results suggest that the second W residues of pore helices and their chemical interaction with the Y residues of the selectivity filter are essential for normal K⁺ channel function. This pore-helix mutation, if occurs in the brain M channels, could thus lead to a channel dysfunction sufficient to trigger epileptic hyperexcitability.     

Influence of permeating ions on Kv1.5 channel block by nifedipine

Nifedipine can block K(+) currents through Kv1.5 channels in an open-channel manner (32). Replacement of internal and external K(+) with equimolar Rb(+) or Cs(+) reduced the potency of nifedipine block of Kv1.5 from an IC(50) of 7.3 microM (K(+)) to 16.0 microM (Rb(+)) and 26.9 microM (Cs(+)). The voltage dependence of block was unaffected, and a single binding site block model was used to describe block for all three ions. By varying ion species at the intra- and extracellular mouth of the channel and by using a nonconducting W472F-Kv1.5 mutant, we demonstrated that block was conditioned by the ion permeating the pore and, to a lesser extent, by the extracellular ion species alone. In Kv1.5, the outer pore mutations R487V and R487Y reduced nifedipine potency close to that of Kv4.2 and other Kv channels with an equivalent valine. Although changing this residue can affect C-type inactivation of Kv channels, the normalized reduction and time course of currents blocked by nifedipine in 5, 135, and 300 mM extracellular K(+) concentration was the same. Similarly, a mean recovery time constant from nifedipine block of 316 ms was unchanged (332 ms) after 5-s prepulses to allow C-type inactivation. This is consistent with the conclusion that nifedipine block and C-type inactivation in the Kv1.5 channel can coexist but are mediated by distinct mechanisms coordinated by outer pore conformation.     

KCNE beta subunits determine pH sensitivity of KCNQ1 potassium channels.

BACKGROUND/AIMS: Heteromeric KCNEx/KCNQ1 (=KvLQT1, Kv7.1) K(+) channels are important for repolarization of cardiac myocytes, endolymph secretion in the inner ear, gastric acid secretion, and transport across epithelia. They are modulated by pH in a complex way: homomeric KCNQ1 is inhibited by external acidification (low pH(e)); KCNE2/KCNQ1 is activated; and for KCNE1/KCNQ1, variable effects have been reported. Methods: The role of KCNE subunits for the effect of pH(e) on KCNQ1 was analyzed in transfected COS cells and cardiac myocytes by the patch-clamp technique. RESULTS: In outside-out patches of transfected cells, hKCNE2/hKCNQ1 current was increased by acidification down to pH 4.5. Chimeras with the acid-insensitive hKCNE3 revealed that the extracellular N-terminus and at least part of the transmembrane domain of hKCNE2 are needed for activation by low pH(e). hKCNE1/hKCNQ1 heteromeric channels exhibited marked changes of biophysical properties at low pH(e): The slowly activating hKCNE1/hKCNQ1 channels were converted into constitutively open, non-deactivating channels. Experiments on guinea pig and mouse cardiac myocytes pointed to an important role of KCNQ1 during acidosis implicating a significant contribution to cardiac repolarization under acidic conditions. CONCLUSION: External pH can modify current amplitude and biophysical properties of KCNQ1. KCNE subunits work as molecular switches by modulating the pH sensitivity of human KCNQ1.