Shaker Potassium Channel Research Articles

Voltage-Clamp Fluorimetry Of Kv1.2 Channels Show Two Unique Phases Of Quenching Associated With Channel Activation

Voltage-clamp fluorimetry can be used to visualize real-time changes in protein structure relative to a locally introduced fluorophore, and thus enhance our understanding of ion channel gating. Since the crystal structure is available for Kv1.2, it seems important to compare the voltage-dependent fluorescence reports from this channel with those already recorded from Shaker potassium channels. In the absence of introduced cysteine residues, we were unable to record voltage-dependent fluorescence signals from wild-type channels incubated with tetramethylrhodamine maleimide (TMRM). However, signals were obtained from an introduced cysteine at A291 in the Kv1.2 S3-S4 linker. Depolarization resulted in two separate components of quenching, which may underlie two different conformational changes in the protein. A slow quenching phase was observed upon depolarization from a holding potential of −120 mV and was essentially complete by −50 mV. The rate of this quenching was not significantly voltage-dependent, with time constants between 35 ms at −70 mV and 29 ms at +80 mV. The voltage-dependence of the slow component suggests that it reports on conformational changes preceding opening. A more rapid quenching component was observed upon depolarizations positive to −40 mV, with time constants from 2-10 ms. It had a similar voltage-dependence to the conductance-voltage relationship for potassium currents through Kv1.2, suggesting that it may report on channel rearrangements associated with opening. Dissociation of channel gating charge movement from pore opening by the incorporation of the ILT triple mutation in the S4 domain abolished the fast phase of fluorescence quenching at potentials up to +80 mV. This suggests that the fast quenching reports on conformational changes associated with channel opening, and that slow fluorescence quenching reflects protein rearrangements occurring earlier in the gating process.

Regulated RNA Editing and Functional Epistasis inShakerPotassium Channels

Regulated point modification by an RNA editing enzyme occurs at four conserved sites in the Drosophila Shaker potassium channel. Single mRNA molecules can potentially represent any of 24 = 16 permutations (isoforms) of these natural variants. We generated isoform expression profiles to assess sexually dimorphic, spatial, and temporal differences. Striking tissue-specific expression was seen for particular isoforms. Moreover, isoform distributions showed evidence for coupling (linkage) of editing sites. Genetic manipulations of editing enzyme activity demonstrated that a chief determinant of Shaker editing site choice resides not in the editing enzyme, but rather, in unknown factors intrinsic to cells. Characterizing the biophysical properties of currents in nine isoforms revealed an unprecedented feature, functional epistasis; biophysical phenotypes of isoforms cannot be explained simply by the consequences of individual editing effects at the four sites. Our results unmask allosteric communication across disparate regions of the channel protein and between evolved and regulated amino acid changes introduced by RNA editing.

Light-Dependent Modulation of Shab Channels via Phosphoinositide Depletion in Drosophila Photoreceptors

The Drosophila phototransduction cascade transforms light into depolarizations that are further shaped by activation of voltage-dependent K+ (Kv) channels. In whole-cell recordings of isolated photoreceptors, we show that light selectively modulated the delayed rectifier (Shab) current. Shab currents were increased by light with similar kinetics to the light-induced current itself (latency approximately 20 ms), recovering to control values with a t(1/2) of approximately 60 s in darkness. Genetic disruption of PLCbeta4, responsible for light-induced PIP(2) hydrolysis, abolished this light-dependent modulation. In mutants of CDP-diaclyglycerol synthase (cds(1)), required for PIP(2) resynthesis, the modulation became irreversible, but exogenously applied PIP(2) restored reversibility. The modulation was accurately and reversibly mimicked by application of PIP(2) to heterologously expressed Shab channels in excised inside-out patches. The results indicate a functionally implemented mechanism of Kv channel modulation by PIP(2) in photoreceptors, which enables light-dependent regulation of signal processing by direct coupling to the phototransduction cascade.

More Gating Charges are Needed to Open a Shaker K + Channel than are Needed to Open an rBIIA Na + Channel

This study presents what is, to our knowledge, a novel technique by means of which the ratio of the single gating charges of voltage-gated rat brain IIA (rBIIA) sodium and Shaker potassium ion channels was estimated. In the experiment, multiple tandems of enhanced green fluorescent protein were constructed and inserted into the C-terminals of Na + and K + ion channels. cRNA of Na + and K + ion channels was injected and expressed in Xenopus laevis oocytes. The two electrode voltage-clamp technique allowed us to determine the total gating charge of sodium and potassium ion channels, while a relative measure of the amount of expressed channels could be established on the basis of the quantification of the fluorescence intensity of membrane-bound channels marked by enhanced green fluorescent proteins. As a result, gating charge and fluorescence intensity were found to be positively correlated. A relative comparison of the single gating charges of voltage-gated sodium and potassium ion channels could thus be established: the ratio of the single gating charges of the Shaker potassium channel and the rBIIA sodium channel was found to be 2.5 ± 0.4. Assuming the single channel gating charge of the Shaker K + channel to be ∼13 elementary charges (well supported by other studies), this leads to approximately six elementary charges for the rBIIA sodium channel, which includes a fraction of gating charge that is missed during inactivation.

An Extracellular Cu2+ Binding Site in the Voltage Sensor of BK and Shaker Potassium Channels

Copper is an essential trace element that may serve as a signaling molecule in the nervous system. Here we show that extracellular Cu2+ is a potent inhibitor of BK and Shaker K+ channels. At low micromolar concentrations, Cu2+ rapidly and reversibly reduces macrosocopic K+ conductance (GK) evoked from mSlo1 BK channels by membrane depolarization. GK is reduced in a dose-dependent manner with an IC50 and Hill coefficient of ∼2 μM and 1.0, respectively. Saturating 100 μM Cu2+ shifts the GK-V relation by +74 mV and reduces GKmax by 27% without affecting single channel conductance. However, 100 μM Cu2+ fails to inhibit GK when applied during membrane depolarization, suggesting that Cu2+ interacts poorly with the activated channel. Of other transition metal ions tested, only Zn2+ and Cd2+ had significant effects at 100 μM with IC50s > 0.5 mM, suggesting the binding site is Cu2+ selective. Mutation of external Cys or His residues did not alter Cu2+ sensitivity. However, four putative Cu2+-coordinating residues were identified (D133, Q151, D153, and R207) in transmembrane segments S1, S2, and S4 of the mSlo1 voltage sensor, based on the ability of substitutions at these positions to alter Cu2+ and/or Cd2+ sensitivity. Consistent with the presence of acidic residues in the binding site, Cu2+ sensitivity was reduced at low extracellular pH. The three charged positions in S1, S2, and S4 are highly conserved among voltage-gated channels and could play a general role in metal sensitivity. We demonstrate that Shaker, like mSlo1, is much more sensitive to Cu2+ than Zn2+ and that sensitivity to these metals is altered by mutating the conserved positions in S1 or S4 or reducing pH. Our results suggest that the voltage sensor forms a state- and pH-dependent, metal-selective binding pocket that may be occupied by Cu2+ at physiologically relevant concentrations to inhibit activation of BK and other channels.

Seymour Benzer 1921–2007

Seymour Benzer 1921–2007

On the role of ball and chain interactions in recovery from the inactivation of the shaker potassium channel

We describe a new factor in the recovery from inactivation in the ball and chain model. We propose a model in which the tension from the chain may help pull the ball away from its binding site, reducing the duration of the inactivation period. A corresponding model was built and analysed.

Gating of the shaker potassium channel is modulated differentially by N-glycosylation and sialic acids

N-linked glycans, including sialic acids, are integral components of ion channel complexes. To determine if N-linked sugars can modulate a rapidly inactivating K+ channel, the glycosylated Drosophila melanogaster Shaker K+ channel (ShB) and the N-glycosylation-deficient mutant (ShNQ), were studied under conditions of full and reduced sialylation. Through an apparent electrostatic mechanism, full sialylation induced uniform and significant hyperpolarizing shifts in all measured voltage-dependent ShB gating parameters compared to those measured under conditions of reduced sialylation. Steady-state gating of ShNQ was unaffected by changes in sialylation and was nearly identical to that observed for ShB under conditions of reduced sialylation, indicating that N-linked sialic acids were wholly responsible for the observed effects of sialic acid on ShB gating. Interestingly, the rates of transition among channel states and the voltage-independent rates of activation and inactivation were significantly slower for ShNQ compared to ShB. Both effects were independent of sialylation, indicating that N-linked sugars other than sialic acids alter ShB gating kinetics but have little to no effect on the steady-state distribution of channels among states. The effect of sialic acids on channel gating, particularly inactivation gating, and the impact of other N-linked sugars on channel gating kinetics are unique to the ShB isoform. Thus, ShB gating is modulated by two complementary but distinct sugar-dependent mechanisms, (1) an N-linked sialic acid-dependent surface charge effect and (2) a sialic acid-independent effect that is consistent with N-linked sugars affecting the stability of ShB among its functional states.

Hydropathic Analysis and Comparison of KcsA and Shaker Potassium Channels

The similarity in structure of potassium (K(+)) channels from different families has been revealed by only recently available crystallographic 3D structural data. The hydropathic analysis presented in this work illuminates whether homologous residues perform the same functions in channels that use different gating mechanisms. We calculated and compared the hydropathic profiles of two K(+) channels, KcsA and Kv1.2 (the latter a member of the Shaker family), at their pore-forming domain. Quantitative information describing important interactions stabilizing the protein beyond obvious secondary-structure elements was extracted from the analysis and applied as a template for subsequent molecular-dynamics (MD) analyses. For example, two key groups of interactions, defining the turns that connect the transmembrane helices and responsible for the orientation of the pore helix, were identified. Our results also indicate that Asp(80) and Asp(379) play a similar role in stabilizing the P-loop of KcsA and Kv1.2, respectively, but to significantly different extents.

In Vivo Measurement of Intramolecular Distances Using Genetically Encoded Reporters

The function of membrane proteins occurs in the context of the cell membrane in living cells acting in concert with various cell components such as other proteins, cofactors, etc. The understanding of the function at the molecular level requires structural techniques, but high resolution structural studies are normally obtained in vitro and in artificial membranes or detergent. Ideally the correlation of structure and function should be carried out in the native environment but most of the techniques applicable in vivo lack the high resolution necessary to track conformational changes on a molecular level. Here we report on the successful application of an improved variant of lanthanide-based resonance energy transfer a fluorescent based technique, to Shaker potassium channels expressed in live Xenopus oocytes. Lanthanide-based resonance energy transfer is particularly suitable to measure intramolecular distances with high resolution. The improvements reported in this work are mainly based on the use of two different small genetically encoded tags (the Lanthanide Binding Tag and the hexa-histidine tag), which due to their small size can be encoded at will in many positions of interest without distorting the protein's function. The technique reported here has the additional improvement that the two tags can be placed independently in contrast to previously described techniques that rely on chemical labeling procedures of thiols.

KDC1, a carrot Shaker-like potassium channel, reveals its role as a silent regulatory subunit when expressed in plant cells

The Shaker potassium channels are tetrameric proteins formed by the assembly of four alpha-subunits. The oligomerization can occur among both homo- and hetero-alpha-subunits. KDC1 is a carrot Shaker-like potassium channel expressed in the epidermis of plantlet roots and the protoderm of somatic embryos. KDC1 was previously characterised electrophysiologically in CHO and Xenopus oocytes cells, but the experiments performed in these systems did not provide conclusive evidence that KDC1 forms a functional homomeric channel in plant cells. In this report, we show that KDC1 localizes to the plasma membrane of root cells in transgenic tobacco plants transformed with a KDC1::GFP fusion construct. In tobacco mesophyll protoplasts, transiently transformed with KDC1::GFP, KDC1 was present on the endomembrane and the protoplasts did not show any inward potassium current, as demonstrated by patch-clamp experiments. The co-expression of KDC1::GFP with the Arabidopsis thaliana potassium channel AKT1 in tobacco mesophyll protoplasts has the effect of shifting KDC1 localization from endomembranes to the plasma membrane. Patch-clamp experiments performed on tobacco mesophyll protoplasts expressing AKT1 alone or in combination with KDC1::GFP showed voltage-activated inward potassium currents with different properties. In particular, the addition of Zn2+ to the bath solution induced a clear decrease of the potassium currents in protoplasts transformed with AKT1 alone, whereas a current potentiation (indicative of KDC1 presence) was observed in protoplasts co-transformed with AKT1 + KDC1::GFP. Split-Ubiquitin assay experiments performed in yeast cells confirmed the interaction between AKT1 and KDC1.

Post‐synaptic density perturbs insulin‐induced Kv1.3 channel modulation via a clustering mechanism involving the SH3 domain

The olfactory bulb (OB) contains the highest concentration of the insulin receptor (IR) kinase in the central nervous system; however, its functional role and modulation in this region remains poorly understood. IR kinase contains a number of proline-rich motifs, making it an excellent candidate for modulation by SH(3) domain-containing adaptor proteins. Kv1.3, a voltage-gated Shaker potassium channel and tyrosine phosphorylation substrate of IR kinase, contains several proline-rich sequences and a canonical post-synaptic density 95 (PSD-95)/discs large/zO-1 domain (PDZ) recognition motif common to most Shaker family members. We sought to determine if a functional relationship existed between Kv1.3, IR kinase, and the SH(3)/PDZ adaptor protein PSD-95. Through patch-clamp electrophysiology, immunochemistry, and co-immunoprecipitation, we found that while Kv1.3 and PSD-95 alone interact via the canonical C-terminal PDZ recognition motif of the channel, this molecular site of interaction acts to cluster the channels but the PSD-95 SH(3)-guanylate kinase domain functionally modulates Kv1.3 activity via two proline-rich domains in its N- and C-terminal. Therefore, these data suggest that adaptor domains responsible for ion-channel clustering and functional modulation are not necessarily coupled. Moreover, IR kinase and Kv1.3 can only be co-immunoprecipitated in the presence of PSD-95 as the adapting linker. Functionally, insulin-dependent Kv1.3 phosphorylation that causes channel current suppression is blocked via interaction with the PSD-95 SH(3)-guanylate kinase domain. Because all the three proteins co-localize in multiple lamina of the OB that are known to be rich in synaptic connections, membrane excitability and synaptic transmission at critical locations in the OB have the capacity to be finely regulated.

Rice Shaker Potassium Channel OsKAT1 Confers Tolerance to Salinity Stress on Yeast and Rice Cells

We screened a rice (Oryza sativa L. 'Nipponbare') full-length cDNA expression library through functional complementation in yeast (Saccharomyces cerevisiae) to find novel cation transporters involved in salt tolerance. We found that expression of a cDNA clone, encoding the rice homolog of Shaker family K(+) channel KAT1 (OsKAT1), suppressed the salt-sensitive phenotype of yeast strain G19 (Deltaena1-4), which lacks a major component of Na(+) efflux. It also suppressed a K(+)-transport-defective phenotype of yeast strain CY162 (Deltatrk1Deltatrk2), suggesting the enhancement of K(+) uptake by OsKAT1. By the expression of OsKAT1, the K(+) contents of salt-stressed G19 cells increased during the exponential growth phase. At the linear phase, however, OsKAT1-expressing G19 cells accumulated less Na(+) than nonexpressing cells, but almost the same K(+). The cellular Na(+) to K(+) ratio of OsKAT1-expressing G19 cells remained lower than nonexpressing cells under saline conditions. Rice cells overexpressing OsKAT1 also showed enhanced salt tolerance and increased cellular K(+) content. These functions of OsKAT1 are likely to be common among Shaker K(+) channels because OsAKT1 and Arabidopsis (Arabidopsis thaliana) KAT1 were able to complement the salt-sensitive phenotype of G19 as well as OsKAT1. The expression of OsKAT1 was restricted to internodes and rachides of wild-type rice, whereas other Shaker family genes were expressed in various organs. These results suggest that OsKAT1 is involved in salt tolerance of rice in cooperation with other K(+) channels by participating in maintenance of cytosolic cation homeostasis during salt stress and thus protects cells from Na(+).

Isolation of the first toxin from the scorpion Buthus occitanus israelis showing preference for Shaker potassium channels

We have purified BoiTx1, the first toxin from the venom of the Israeli scorpion, Buthus occitanus israelis, and studied its activity and genomic organization. BoiTx1 is a 37 amino acid-long peptide contained six conserved cysteines, and is classified as an α-KTx3.10 toxin. The pharmacological effects of BoiTx1 were studied on various cloned K + channels expressed in Xenopus laevis oocytes. BoiTx1 inhibited currents through Drosophila Shaker channels with an IC 50 value of 3.5 ± 0.5 nM, yet had much lesser effect on its mammalian orthologs. Thus, BoiTx1 is the first member of the α-KTx3 family that preferentially affects insect potassium channels.

The Src Homology 3 Domain of the β-Subunit of Voltage-gated Calcium Channels Promotes Endocytosis via Dynamin Interaction

High voltage-gated calcium channels enable calcium entry into cells in response to membrane depolarization. Association of the auxiliary beta-subunit to the alpha-interaction-domain in the pore-forming alpha1-subunit is required to form functional channels. The beta-subunit belongs to the membrane-associated guanylate kinase class of scaffolding proteins containing a Src homology 3 and a guanylate kinase domain. Although the latter is responsible for the high affinity binding to the alpha-interaction domain, the functional significance of the Src homology 3 domain remains elusive. Here, we show that injection of isolated beta-subunit Src homology 3 domain into Xenopus laevis oocytes expressing the alpha1-subunit reduces the number of channels in the plasma membrane. This effect is reverted by coexpressing alpha1 with a dominant-negative mutant of dynamin, a GTPase involved in receptor-mediated endocytosis. Full-length beta-subunit also down-regulates voltage-gated calcium channels but only when lacking the alpha-interaction domain. Moreover, isolated Src homology 3 domain and the full-length beta-subunit were found to interact in vitro with dynamin and to internalize the distantly related Shaker potassium channel. These results demonstrate that the beta-subunit regulates the turnover of voltage-gated calcium channels and other proteins in the cell membrane. This effect is mediated by dynamin and depends on the association state of the beta-subunit to the alpha1-pore-forming subunit. Our findings define a novel function for the beta-subunit through its Src homology 3 domain and establish a link between voltage-gated calcium channel activity and the cell endocytic machinery.

The twisted ion-permeation pathway of a resting voltage-sensing domain

Proteins containing voltage-sensing domains (VSDs) translate changes in membrane potential into changes in ion permeability or enzymatic activity. In channels, voltage change triggers a switch in conformation of the VSD, which drives gating in a separate pore domain, or, in channels lacking a pore domain, directly gates an ion pathway within the VSD. Neither mechanism is well understood. In the Shaker potassium channel, mutation of the first arginine residue of the S4 helix to a smaller uncharged residue makes the VSD permeable to ions ('omega current') in the resting conformation ('S4 down'). Here we perform a structure-guided perturbation analysis of the omega conductance to map its VSD permeation pathway. We find that there are four omega pores per channel, which is consistent with one conduction path per VSD. Permeating ions from the extracellular medium enter the VSD at its peripheral junction with the pore domain, and then plunge into the core of the VSD in a curved conduction pathway. Our results provide a model of the resting conformation of the VSD.

A Cation–π Interaction between Extracellular TEA and an Aromatic Residue in Potassium Channels

Open-channel blockers such as tetraethylammonium (TEA) have a long history as probes of the permeation pathway of ion channels. High affinity blockade by extracellular TEA requires the presence of an aromatic amino acid at a position that sits at the external entrance of the permeation pathway (residue 449 in the eukaryotic voltage-gated potassium channel Shaker). We investigated whether a cation–π interaction between TEA and such an aromatic residue contributes to TEA block using the in vivo nonsense suppression method to incorporate a series of increasingly fluorinated Phe side chains at position 449. Fluorination, which is known to decrease the cation–π binding ability of an aromatic ring, progressively increased the inhibitory constant K i for the TEA block of Shaker. A larger increase in K i was observed when the benzene ring of Phe449 was substituted by nonaromatic cyclohexane. These results support a strong cation–π component to the TEA block. The data provide an empirical basis for choosing between Shaker models that are based on two classes of reported crystal structures for the bacterial channel KcsA, showing residue Tyr82 in orientations either compatible or incompatible with a cation–π mechanism. We propose that the aromatic residue at this position in Shaker is favorably oriented for a cation–π interaction with the permeation pathway. This choice is supported by high level ab initio calculations of the predicted effects of Phe modifications on TEA binding energy.

Functional Interactions at the Interface between Voltage-Sensing and Pore Domains in the Shaker Kv Channel

Voltage-activated potassium (K(v)) channels contain a central pore domain that is partially surrounded by four voltage-sensing domains. Recent X-ray structures suggest that the two domains lack extensive protein-protein contacts within presumed transmembrane regions, but whether this is the case for functional channels embedded in lipid membranes remains to be tested. We investigated domain interactions in the Shaker K(v) channel by systematically mutating the pore domain and assessing tolerance by examining channel maturation, S4 gating charge movement, and channel opening. When mapped onto the X-ray structure of the K(v)1.2 channel the large number of permissive mutations support the notion of relatively independent domains, consistent with crystallographic studies. Inspection of the maps also identifies portions of the interface where residues are sensitive to mutation, an external cluster where mutations hinder voltage sensor activation, and an internal cluster where domain interactions between S4 and S5 helices from adjacent subunits appear crucial for the concerted opening transition.

Stoichiometry Studies Reveal Functional Properties of KDC1 in Plant Shaker Potassium Channels

Functional heteromeric plant Shaker potassium channels can be formed by the assembly of subunits from different tissues, as well as from diverse plant species. KDC1 (K + Daucus carota 1) produces inward-rectifying currents in Xenopus oocytes when coexpressed with KAT1 and other subunits appertaining to different plant Shaker subfamilies. Owing to the presence of KDC1, resulting heteromeric channels display slower activation kinetics, a shift of the activation threshold toward more negative membrane potentials and current potentiation upon the addition of external zinc. Despite available information on heteromerization of plant Shaker channels, very little is known to date on the properties of the various stoichiometric configurations formed by different subunits. To investigate the functional properties of heteromeric nKDC1/mKAT1 configurations, we realized a series of dimeric constructs combining KDC1 and KAT1 α-subunits. We found that homomeric channels, formed by monomeric or dimeric α-subunit constructs, show identical biophysical characteristics. Coinjections of diverse tandem constructs, instead, displayed significantly different currents proving that KDC1 has high affinity for KAT1 and participates in the formation of functional channels with at most two KDC1 subunits, whereas three KDC1 subunits prevented the formation of functional channels. This article brings a contribution to the understanding of the molecular mechanisms regulating plant Shaker channel functionality by association of modulatory subunits.

Fluorometric Measurements of Intermolecular Distances between the α- and β-Subunits of the Na+/K+-ATPase

The Na+/K+-ATPase maintains the physiological Na+ and K+ gradients across the plasma membrane in most animal cells. The functional unit of the ion pump is comprised of two mandatory subunits including the alpha-subunit, which mediates ATP hydrolysis and ion translocation, as well as the beta-subunit, which acts as a chaperone to promote proper membrane insertion and trafficking in the plasma membrane. To examine the conformational dynamics between the alpha- and beta-subunits of the Na+/K+-ATPase during ion transport, we have used fluorescence resonance energy transfer, under voltage clamp conditions on Xenopus laevis oocytes, to differentiate between two models that have been proposed for the relative orientation of the alpha- and beta-subunits. These experiments were performed by measuring the time constant of irreversible donor fluorophore destruction with fluorescein-5-maleimide as the donor fluorophore and in the presence or absence of tetramethylrhodamine-6-maleimide as the acceptor fluorophore following labeling on the M3-M4 or M5-M6 loop of the alpha-subunit and the beta-subunit. We have also used fluorescence resonance energy transfer to investigate the relative movement between the two subunits as the ion pump shuttles between the two main conformational states (E1 and E2) as described by the Albers-Post scheme. The results from this study have identified a model for the orientation of the beta-subunit in relation to the alpha-subunit and suggest that the alpha- and beta-subunits move toward each other during the E2 to E1 conformational transition.