A single charged voltage sensor is capable of gating the Shaker K+ channel

We sought to determine the contribution of an individual voltage sensor to Shaker''s function. Concatenated heterotetramers of Shaker zH4 Δ(6–46) wild type (wt) in combination with a neutral S4 segment Shaker mutant (mut) with stoichiometries 2wt/2mut and 1wt/3mut were studied and compared with the 4wt concatenated homotetramer. A single charged voltage sensor is sufficient to open Shaker conductance with reduced delay (<1 ms) and at more hyperpolarized voltages compared with 4wt. In addition, the wt-like slow inactivation of 1wt/3mut was almost completely eliminated by mutations T449V-I470C in its single wt subunit, indicating that the subunits bearing a neutral S4 were unable to trigger slow inactivation. Our results strongly suggest that a neutral S4 segment of Shaker''s subunit is voltage insensitive and its voltage sensor is in the activated position (i.e., ready for pore opening), and provide experimental support to the proposed model of independent voltage sensors with a final, almost voltage-independent concerted step.     

Extensive editing of mRNAs for the squid delayed rectifier K+ channel regulates subunit tetramerization

We report the extensive editing of mRNAs that encode the classical delayed rectifier K+ channel (SqK(v)1.1A) in the squid giant axon. Using a quantitative RNA editing assay, 14 adenosine to guanine transitions were identified, and editing efficiency varied tremendously between positions. Interestingly, half of the sites are targeted to the T1 domain, important for subunit assembly. Other sites occur in the channel's transmembrane spans. The effects of editing on K+ channel function are elaborate. Edited codons affect channel gating, and several T1 sites regulate functional expression as well. In particular, the edit R87G, a phylogenetically conserved position, reduces expression close to 50-fold by regulating the channel's ability to form tetramers. These data suggest that RNA editing plays a dynamic role in regulating action potential repolarization in the giant axon.     

Permeation of Na+ through a delayed rectifier K+ channel in chick dorsal root ganglion neurons

In whole-cell patch clamp recordings from chick dorsal root ganglion neurons, removal of intracellular K+ resulted in the appearance of a large, voltage-dependent inward tail current (Icat). Icat was not Ca2+ dependent and was not blocked by Cd2+, but was blocked by Ba2+. The reversal potential for Icat shifted with the Nernst potential for [Na+]. The channel responsible for Icat had a cation permeability sequence of Na+ >> Li+ >> TMA+ > NMG+ (PX/PNa = 1:0.33:0.1:0) and was impermeable to Cl-. Addition of high intracellular concentrations of K+, Cs+, or Rb+ prevented the occurrence of Icat. Inhibition of Icat by intracellular K+ was voltage dependent, with an IC50 that ranged from 3.0-8.9 mM at membrane potentials between -50 and -110 mV. This voltage- dependent shift in IC50 (e-fold per 52 mV) is consistent with a single cation binding site approximately 50% of the distance into the membrane field. Icat displayed anomolous mole fraction behavior with respect to Na+ and K+; Icat was inhibited by 5 mM extracellular K+ in the presence of 160 mM Na+ and potentiated by equimolar substitution of 80 mM K+ for Na+. The percent inhibition produced by both extracellular and intracellular K+ at 5 mM was identical. Reversal potential measurements revealed that K+ was 65-105 times more permeant than Na+ through the Icat channel. Icat exhibited the same voltage and time dependence of inactivation, the same voltage dependence of activation, and the same macroscopic conductance as the delayed rectifier K+ current in these neurons. We conclude that Icat is a Na+ current that passes through a delayed rectifier K+ channel when intracellular K+ is reduced to below 30 mM. At intracellular K+ concentrations between 1 and 30 mM, PK/PNa remained constant while the conductance at -50 mV varied from 80 to 0% of maximum. These data suggest that the high selectivity of these channels for K+ over Na+ is due to the inability of Na+ to compete with K+ for an intracellular binding site, rather than a barrier that excludes Na+ from entry into the channel or a barrier such as a selectivity filter that prevents Na+ ions from passing through the channel.     

Clustering of the K+ channel GORK of Arabidopsis parallels its gating by extracellular K+

GORK is the only outward‐rectifying Kv‐like K⁺ channel expressed in guard cells. Its activity is tightly regulated to facilitate K⁺ efflux for stomatal closure and is elevated in ABA in parallel with suppression of the activity of the inward‐rectifying K⁺ channel KAT1. Whereas the population of KAT1 is subject to regulated traffic to and from the plasma membrane, nothing is known about GORK, its distribution and traffic in vivo. We have used transformations with fluorescently‐tagged GORK to explore its characteristics in tobacco epidermis and Arabidopsis guard cells. These studies showed that GORK assembles in puncta that reversibly dissociated as a function of the external K⁺ concentration. Puncta dissociation parallelled the gating dependence of GORK, the speed of response consistent with the rapidity of channel gating response to changes in the external ionic conditions. Dissociation was also suppressed by the K⁺ channel blocker Ba²⁺. By contrast, confocal and protein biochemical analysis failed to uncover substantial exo‐ and endocytotic traffic of the channel. Gating of GORK is displaced to more positive voltages with external K⁺, a characteristic that ensures the channel facilitates only K⁺ efflux regardless of the external cation concentration. GORK conductance is also enhanced by external K⁺ above 1 mm . We suggest that GORK clustering in puncta is related to its gating and conductance, and reflects associated conformational changes and (de)stabilisation of the channel protein, possibly as a platform for transmission and coordination of channel gating in response to external K⁺.     

Voltage-dependent blockade of delayed rectifier K+ current by nitroprusside and ferricyanide

NO donor nitroprusside (NP) is a biologically active drug with hypotensive and neurotropic properties. Its effects are due to NO as well as to other derivates, specifically ferricyanide (FC) and ferrocyanide (F(2+)C) ions. In the present work we studied effects of NP, FC, and F(2+)C on delayed rectifier K⁺ current. The experiments were conducted on isolated neurons of land snail Helix using two-microelectrode voltageclamp technique. Delayed rectifier K⁺ current often displayed abnormal rectification. NP (1 mM) caused voltage-dependent reduction of K⁺ current, which looked like enhancement of the abnormal rectification. FC (1 mM)—but not F(2+)C—acted likewise; the effects of NP and FC were not additive. Dibutiryl cGMP (dbcGMP) had an opposite effect, weakening abnormal rectification of the K⁺ current. Protein kinase inhibitor H-8 (10 μM) caused voltage-dependent reduction of the K⁺ current, as NP and FC did. The effects of H-8 and NP and effects of H-8 and FC were not additive. The results suggest that: (i) NP effects on delayed rectifier K⁺ current are mediated by FC and (ii) the effects of these drugs involve phosphorylation processes.     

Intrinsic aqueduct orifices facilitate K+ channel gating

The ion-conducting pore of potassium channels, which can open and close to regulate ion passage, was at long thought to be a one-dimensional pore structure with a water-filled central cavity. Here, we find four orifices in the KcsA potassium channel, which are perpendicular to the pore and stretch out from the cavity. Equilibrium molecular dynamics simulations show that water molecules can flow between the cavity and orifices. Targeted molecular dynamics simulations show that during the opening process, water molecules can move into the cavity through the orifices to facilitate channel gating, whereas blocking the aqueduct orifices makes the channel difficult to open.     

Induction of a fast inactivation gating on delayed rectifier Shab K(+) channels by the anti-inflammatory drug celecoxib

Celecoxib is a drug designed to selectively inhibit COX-2, an inflammation-inducible cyclooxygenase isoform, over the constitutively expressed COX-1 isoform. In addition to this selective inhibition it is now known that celecoxib exerts a variety of effects on several types of ion channels, thus producing secondary physiological effects. In this work we demonstrate that at therapeutically relevant concentrations celecoxib interacts with Shab K(+) channels specifically promoting a fast inactivation gating (without blocking the pore or significantly affecting other gating processes). At least two celecoxib molecules bind to each channel promoting a fast inactivation that develops from both open and closed states. Channel inactivation in turn causes a reduction of the size of I(K). Taken together, our observations show that in addition to its intended therapeutic target celecoxib is a useful tool to further study the mechanism of Shab channel inactivation.     

Extracellular intersubunit interactions modulate epithelial Na+ channel gating

Epithelial Na⁺ channels (ENaCs) and related channels have large extracellular domains where specific factors interact and induce conformational changes, leading to altered channel activity. However, extracellular structural transitions associated with changes in ENaC activity are not well defined. Using crosslinking and two-electrode voltage clamp in Xenopus oocytes, we identified several pairs of functional intersubunit contacts where mouse ENaC activity was modulated by inducing or breaking a disulfide bond between introduced Cys residues. Specifically, crosslinking E499C in the β-subunit palm domain and N510C in the α-subunit palm domain activated ENaC, whereas crosslinking βE499C with αQ441C in the α-subunit thumb domain inhibited ENaC. We determined that bridging βE499C to αN510C or αQ441C altered the Na⁺ self-inhibition response via distinct mechanisms. Similar to bridging βE499C and αQ441C, we found that crosslinking palm domain αE557C with thumb domain γQ398C strongly inhibited ENaC activity. In conclusion, we propose that certain residues at specific subunit interfaces form microswitches that convey a conformational wave during ENaC gating and its regulation.     

Physical and molecular basis of ion channel gating: Can electrostatic interactions close the ion channel?

We analyzed voltage-dependent ion channel structure and conformational changes corresponding to channel gating. During the gating, S4 segments, as well as other parts of the channel, undergo a set of conformational modifications. These changes are accompanied by complicated movements of positive charges that are mostly located in the S4 segments. These charges electrostatically interact with the ions passing through the channel. The interaction energy depends on the conformational state of the channel, i.e., on the mutual positions of the permeant ions and these charges. Analyzing and making energetical estimations, we propose a hypothesis: the closed state of the ion channel corresponds to the S4 position when electrostatic interaction between positively charged groups of the S4 segments and permeant ions is strong enough to close the pathway for these ions.     

Molecular coupling between voltage sensor and pore opening in the Arabidopsis inward rectifier K+ channel KAT1

Animal and plant voltage-gated ion channels share a common architecture. They are made up of four subunits and the positive charges on helical S4 segments of the protein in animal K+ channels are the main voltage-sensing elements. The KAT1 channel cloned from Arabidopsis thaliana, despite its structural similarity to animal outward rectifier K+ channels is, however, an inward rectifier. Here we detected KAT1-gating currents due to the existence of an intrinsic voltage sensor in this channel. The measured gating currents evoked in response to hyperpolarizing voltage steps consist of a very fast (tau = 318 +/- 34 micros at -180 mV) and a slower component (4.5 +/- 0.5 ms at -180 mV) representing charge moved when most channels are closed. The observed gating currents precede in time the ionic currents and they are measurable at voltages (less than or equal to -60) at which the channel open probability is negligible ( approximately 10-4). These two observations, together with the fact that there is a delay in the onset of the ionic currents, indicate that gating charge transits between several closed states before the KAT1 channel opens. To gain insight into the molecular mechanisms that give rise to the gating currents and lead to channel opening, we probed external accessibility of S4 domain residues to methanethiosulfonate-ethyltrimethylammonium (MTSET) in both closed and open cysteine-substituted KAT1 channels. The results demonstrate that the putative voltage-sensing charges of S4 move inward when the KAT1 channels open.     

K+ binding sites and interactions between permeating K+ ions at the external pore mouth of an inward rectifier K+ channel (Kir2.1).

The arginine at position 148 is highly conserved in the inward rectifier K+ channel family. Increases of external pH decrease the single-channel conductance in mutant R148H of the Kir2.1 channel (arginine is mutated into histidine) but not in the wild type channel. Moreover, in 100 mM external K+, varying external pH induced biphasic changes of open channel noise, which peaks at around pH 7.4 in the R148H mutant but not in the wild type channel. The maximum single-channel conductances are higher in the wild type channel and R148H mutant at pH 6.0 than those in the R148H mutant at pH 7.4. However, the maximal conductance is achieved with much lower external [K+] for the latter. Interestingly, the single-channel conductances and open channel noise of the wild type channel at pH 6. 0 and the R148H mutant at pH 6.0 and 7.4 become the same in [K+] = 10 mM. These results indicate that the residue at position 148 is accessible to the external H+ and probably is involved in the formation of two K+ binding sites in the external pore mouth. Effective repulsion between permeating K+ ions in this area requires a positive charge at position 148, and such K+-K+ interaction is the essential mechanism underlying high K+ conduction rate through the Kir2.1 channel pore.     

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.     

Revisiting the role of Ca2+ in Shaker K+ channel gating

Shaker K+ channels were expressed in outside-out macropatches excised from Xenopus oocytes, and the effects on gating of removal of extracellular Ca2+ were examined in the complete absence of intracellular divalent cations. Removal of extracellular Ca2+ by perfusion with EDTA-containing solution caused a small negative shift in the channel's voltage-activation curve and led to an increased nonselective leak, but did not otherwise alter or disrupt the channels. The results contradict the proposal that Ca2+ is an essential component required for maintenance of ion selectivity and proper gating of Kv-type K+ channels. The large nonselective leak in Ca2+-free conditions was found to be a patch-seal phenomenon related to F- ion in the recording pipette.     

Molecular determinants of interactions between the N-terminal domain and the transmembrane core that modulate hERG K+ channel gating

A conserved eag domain in the cytoplasmic amino terminus of the human ether-a-go-go-related gene (hERG) potassium channel is critical for its slow deactivation gating. Introduction of gene fragments encoding the eag domain are able to restore normal deactivation properties of channels from which most of the amino terminus has been deleted, and also those lacking exclusively the eag domain or carrying a single point mutation in the initial residues of the N-terminus. Deactivation slowing in the presence of the recombinant domain is not observed with channels carrying a specific Y542C point mutation in the S4-S5 linker. On the other hand, mutations in some initial positions of the recombinant fragment also impair its ability to restore normal deactivation. Fluorescence resonance energy transfer (FRET) analysis of fluorophore-tagged proteins under total internal reflection fluorescence (TIRF) conditions revealed a substantial level of FRET between the introduced N-terminal eag fragments and the eag domain-deleted channels expressed at the membrane, but not between the recombinant eag domain and full-length channels with an intact amino terminus. The FRET signals were also minimized when the recombinant eag fragments carried single point mutations in the initial portion of their amino end, and when Y542C mutated channels were used. These data suggest that the restoration of normal deactivation gating by the N-terminal recombinant eag fragment is an intrinsic effect of this domain directed by the interaction of its N-terminal segment with the gating machinery, likely at the level of the S4-S5 linker.     

Modulation of 4-AP block of a mammalian A-type K channel clone by channel gating and membrane voltage.

We examined the state-, voltage-, and time dependences of interaction between 4-AP and a mammalian A-type K channel clone (rKv1.4) expressed in Xenopus oocytes using whole-cell and single-channel recordings. 4-AP blocked rKv1.4 from the cytoplasmic side of the membrane. The development of block required channel opening. Block was potentiated by removing the fast inactivation gate of the channel (deletion mutant termed "Del A"). A short-pulse train that activated rKv1.4 without inactivation induced more block by 4-AP than a long pulse that activated and then inactivated the channel. These observations suggest that both activation and inactivation gates limit the binding of 4-AP to the channel. Unblock of 4-AP also occurred during channel opening, because unblock required depolarization and was accelerated by more frequent or longer depolarization pulses (use-dependent unblock). Analysis of the concentration dependence of rate of block development indicated that 4-AP blocked rKv1.4 with slow kinetics (at -20 mV, binding and unbinding rate constants were 3.2 mM-1 s-1 and 4.3 s-1). This was consistent with single-channel recordings: 4-AP induced little or no changes in the fast kinetics of opening and closing within bursts, but shortened the mean burst duration and, more importantly, reduced the probability of channel opening by depolarization. Depolarization might decrease the affinity of 4-AP binding site in the open channel, because stronger depolarization reduced the degree of steady-state block by 4-AP. Furthermore, after 4-AP block had been established at a depolarized holding voltage, further depolarization induced a time-dependent unblock. Our data suggest that 4-AP binds to and unbinds from open rKv1.4 channels with slow kinetics, with the binding site accessibility controlled by the channel gating apparatus and binding site affinity modulated by membrane voltage.     

大鼠大脑皮层神经元上的延迟整流型钾离子单通道

延迟整流型钾通道在动作电位的复极化和时程控制以及绝对不应期的形成中充当重要角色.本文用细胞贴附式和内面向外式膜片箝技术研究了急性分离的SD大鼠大脑皮层神经元上延迟整流型钾通道的特性和阻断剂对其的作用,对推动钾通道的研究,了解皮层神经元电活动的规律有重要意义.     

Tectonics of a K+ channel: The importance of the N-terminus for channel gating

The small K⁺ channel Kcv represents the pore module of complex potassium channels. It was found that its gating can be modified by sensor domains, which are N-terminally coupled to the pore. This implies that the short N-terminus of the channel can transmit conformational changes from upstream sensors to the channel gates. To understand the functional role of the N-terminus in the context of the entire channel protein, we apply combinatorial screening of the mechanical coupling and long-range interactions in the Kcv potassium channel by reduced molecular models. The dynamics and mechanical connections in the channel complex show that the N-terminus is indeed mechanically connected to the pore domain. This includes a long rang coupling to the pore and the inner and outer transmembrane domains. Since the latter domains host the two gates of the channel, the data support the hypothesis that mechanical perturbation of the N-terminus can be transmitted to the channel gates. This effect is solely determined by the topology of the channel; sequence details only have an implicit effect on the coarse-grained dynamics via the fold and not through biochemical details at a smaller scale. This observation has important implications for engineering of synthetic channels on the basis of a K⁺ channel pore.