Shaker and ether-à-go-go K+ channel subunits fail to coassemble in Xenopus oocytes.

Members of different voltage-gated K+ channel subfamilies usually do not form heteromultimers. However, coassembly between Shaker and ether-à-go-go (eag) subunits, members of two distinct K+ channel subfamilies, was suggested by genetic and functional studies (Zhong and Wu. 1991. Science. 252: 1562-1564; Chen, M.-L., T. Hoshi, and C.-F. Wu. 1996. Neuron. 17:535-542). We investigated whether Shaker and eag form heteromultimers in Xenopus laevis oocytes using electrophysiological and biochemical approaches. Coexpression of Shaker and eag subunits produced K+ currents that were virtually identical to the sum of separate Shaker and eag currents, with no change in the kinetics of Shaker inactivation. According to the results of dominant negative and reciprocal coimmunoprecipitation experiments, the Shaker and eag proteins do not interact. We conclude that Shaker and eag do not coassemble to form heteromultimers in Xenopus oocytes.     

Shaker K+ channel subunits from heteromultimeric channels with novel functional properties.

A large number of related genes (the Sh gene family) encode potassium channel subunits which form voltage-dependent K+ channels by aggregating into homomulitimers. One of these genes, the Shaker gene in Drosophila, generates several products by alternative splicing. These products encode proteins with a constant central region flanked by variable amino and carboxyl domains. Coinjection of two Shaker RNAs with different amino or different carboxyl ends into Xenopus oocytes produces K+ currents that display functional properties distinct from those observed when each RNA is injected separately, indicating the formation of heteromultimeric channels. The analysis of Shaker heteromultimers suggests certain rules regarding the roles of variable amino and carboxyl domains in determining kinetic properties of heteromultimeric channels. Heteromultimers with different amino ends produce currents in which the amino end that produces more inactivation dominates the kinetics. In contrast, heteromultimers with different carboxyl ends recover from inactivation at a rate closer to that observed in homomultimers of the subunit which results in faster recovery. While this and other recent reports demonstrate that closely related Sh family proteins form functional heteromultimers, we show here that two less closely related Sh proteins do not seem to form functional heteromultimeric channels. The data suggest that sites for subunit recognition may be found in sequences within a core region, starting about 130 residues before the first membrane spanning domain of Shaker and ending after the last membrane spanning domain, which are not conserved between Sh Class I and Class III genes.     

Gating currents in Shaker K+ channels. Implications for activation and inactivation models.

We have studied ionic and gating currents in mutant and wild-type Shaker K+ channels to investigate the mechanisms of channel activation and the relationship between the voltage sensor of the channel and its inactivation particle. The turn on of the gating current shows a rising phase, indicating that the hypothetical identical activation subunits are not independent. Hyperpolarizing prepulses indicate that most of the voltage-dependence occurs in the transitions between closed states. The open-to-closed transition is voltage independent, as suggested by the presence of a rising phase in the off gating currents. In Shaker channels showing fast inactivation, the off gating charge is partially immobilized as a result of depolarizing pulses that elicit inactivation. In mutant channels lacking inactivation, the charge is recovered quickly at the end of the pulse. Internal TEA mimics the inactivation particle in its behavior but the charge immobilization is established faster and is complete. We conclude that the activation mechanism cannot be due to the movement of identical independent gating subunits, each undergoing first order transitions, and that the inactivation particle is responsible for charge immobilization in this channel.     

Kvbeta1.2 subunit coexpression in HEK293 cells confers O2 sensitivity to kv4.2 but not to Shaker channels.

Voltage-gated K+ (KV) channels are protein complexes composed of ion-conducting integral membrane alpha subunits and cytoplasmic modulatory beta subunits. The differential expression and association of alpha and beta subunits seems to contribute significantly to the complexity and heterogeneity of KV channels in excitable cells, and their functional expression in heterologous systems provides a tool to study their regulation at a molecular level. Here, we have studied the effects of Kvbeta1.2 coexpression on the properties of Shaker and Kv4.2 KV channel alpha subunits, which encode rapidly inactivating A-type K+ currents, in transfected HEK293 cells. We found that Kvbeta1.2 functionally associates with these two alpha subunits, as well as with the endogenous KV channels of HEK293 cells, to modulate different properties of the heteromultimers. Kvbeta1.2 accelerates the rate of inactivation of the Shaker currents, as previously described, increases significantly the amplitude of the endogenous currents, and confers sensitivity to redox modulation and hypoxia to Kv4.2 channels. Upon association with Kvbeta1.2, Kv4.2 can be modified by DTT (1,4 dithiothreitol) and DTDP (2,2'-dithiodipyridine), which also modulate the low pO2 response of the Kv4.2+beta channels. However, the physiological reducing agent GSH (reduced glutathione) did not mimic the effects of DTT. Finally, hypoxic inhibition of Kv4.2+beta currents can be reverted by 70% in the presence of carbon monoxide and remains in cell-free patches, suggesting the presence of a hemoproteic O2 sensor in HEK293 cells and a membrane-delimited mechanism at the origin of hypoxic responses. We conclude that beta subunits can modulate different properties upon association with different KV channel subfamilies; of potential relevance to understanding the molecular basis of low pO2 sensitivity in native tissues is the here described acquisition of the ability of Kv4. 2+beta channels to respond to hypoxia.     

Level of expression controls modes of gating of a K+ channel.

Several distinct subfamilies of K+ channel genes have been discovered by molecular cloning, however, in some cases the structural differences among them do not account for the diversity of K+ current types, ranging from transient A-type to slowly inactivating delayed rectifier-type, as members within each subfamily have been shown to code for K+ channels of different inactivation kinetics and pharmacological properties. We show that a single K+ channel cDNA of the Shaker subfamily (ShH4) can express in Xenopus oocytes not only a transient A-type K+ current but also, upon increased level of expression, slowly inactivating K+ currents with markedly reduced sensitivity to tetraethylammonium. In correlation with the macroscopic currents there are single-channel gating modes ranging from the fast-inactivation mode which underlies the transient A-type current, to slow-inactivation modes characterized by bursts of longer openings, and corresponding to the slowly inactivating macroscopic currents.     

Regulation of K+ channel activities in plants: from physiological to molecular aspects

Plant voltage-gated channels belonging to the Shaker family participate in sustained K+ transport processes at the cell and whole plant levels, such as K+ uptake from the soil solution, long-distance K+ transport in the xylem and phloem, and K+ fluxes in guard cells during stomatal movements. The attention here is focused on the regulation of these transport systems by protein-protein interactions. Clues to the identity of the regulatory mechanisms have been provided by electrophysiological approaches in planta or in heterologous systems, and through analogies with their animal counterparts. It has been shown that, like their animal homologues, plant voltage-gated channels can assemble as homo- or heterotetramers associating polypeptides encoded by different Shaker genes, and that they can bind auxiliary subunits homologous to those identified in mammals. Furthermore, several regulatory processes (involving, for example, protein kinases and phosphatases, G proteins, 14-3-3s, or syntaxins) might be common to plant and animal Shakers. However, the molecular identification of plant channel partners is still at its beginning. This paper reviews current knowledge on plant K+ channel regulation at the physiological and molecular levels, in the light of the corresponding knowledge in animal cells, and discusses perspectives for the deciphering of regulatory networks in the future.     

The K+ channel SKT1 is co-expressed with KST1 in potato guard cells--both channels can co-assemble via their conserved KT domains

An appreciable number of potassium channels mediating K+ uptake have been identified in higher plants. Promoter-beta-glucuronidase reporter gene studies were used here to demonstrate that SKT1, encoding a potato K+ inwardly rectifying channel, is expressed in guard cells in addition to KST1 previously reported. However, whereas KST1 was found to be expressed in essentially all mature guard cells, SKT1 expression was almost exclusively restricted to guard cells of the abaxial leaf epidermis. This suggests that different types of K+ channel subunits contribute to channel formation in potato guard cells and therefore differential regulation of stomatal movements in the two leaf surfaces. The overlapping expression pattern of SKT1 and KST1 in abaxial guard cells indicates that K+in channels of different sub-families contribute to ionic currents in this cell type, thus explaining the different properties of channels expressed solely in heterologous systems and those endogenous to guard cells. Interaction studies had previously suggested that plant K+ inward rectifiers form clusters via their conserved C-terminal domain, KT/HA. K+ channels co-expressed in one cell type may therefore form heteromers, which increase functional variability of K+ currents, a phenomenon well described for animal voltage-gated K+ channels. Co-expression of KST1 and SKT1 in Xenopus oocytes resulted in currents with an intermediate sensitivity towards Cs+, suggesting the presence of heteromers, and a sensitivity towards external Ca2+, which reflected the property of the endogenous K+in current in guard cells. Modulation of KST1 currents in oocytes by co-expressing KST1 with a SKT1 pore-mutant, which by itself was not able to confer activating K+ currents, demonstrated the possibility that KST1 and SKT1 co-assemble to hetero-oligomers. Furthermore, various C-terminal deletions of the mutated SKT1 channel restored KST1 currents, showing that the C-terminal KT motif is essential for heteromeric channel formation.     

Conduction properties of the cloned Shaker K+ channel.

The conduction properties of the cloned Shaker K+ channel were studied using electrophysiological techniques. Single channel conductance increases in a sublinear manner with symmetric increases in K+ activity, reaching saturation by 0.6 M K+. The Shaker K+ channel is highly selective among monovalent cations; under bi-ionic conditions, its selectivity sequence is K+ > Rb+ > NH+4 > Cs+ > Na+, whereas, by relative conductance in symmetric solutions, it is K+ > NH+4 > Rb+ > Cs+. In Cs+ solutions, single channel currents were too small to be measured directly, so nonstationary fluctuation analysis was used to determine the unitary Cs+ conductance. The single channel conductance displays an anomalous molefraction effect in symmetric mixtures of K+ and NH+4, suggesting that the conducting pore is occupied by multiple ions simultaneously.     

In vivo functional role of the Drosophila hyperkinetic beta subunit in gating and inactivation of Shaker K+ channels.

The physiological roles of the beta, or auxiliary, subunits of voltage-gated ion channels, including Na+, Ca2+, and K+ channels, have not been demonstrated directly in vivo. Drosophila Hyperkinetic (Hk) mutations alter a gene encoding a homolog of the mammalian K+ channel beta subunit, providing a unique opportunity to delineate the in vivo function of auxiliary subunits in K+ channels. We found that the Hk beta subunit modulates a wide range of the Shaker (Sh) K+ current properties, including its amplitude, activation and inactivation, temperature dependence, and drug sensitivity. Characterizations of the existing mutants in identified muscle cells enabled an analysis of potential mechanisms of subunit interactions and their functional consequences. The results are consistent with the idea that via hydrophobic interaction, Hk beta subunits modulate Sh channel conformation in the cytoplasmic pore region. The modulatory effects of the Hk beta subunit appeared to be specific to the Sh alpha subunit because other voltage- and Ca(2+)-activated K+ currents were not affected by Hk mutations. The mutant effects were especially pronounced near the voltage threshold of IA activation, which can disrupt the maintenance of the quiescent state and lead to the striking neuromuscular and behavioral hyperexcitability previously reported.     

Substitution of a hydrophobic residue alters the conformational stability of Shaker K+ channels during gating and assembly.

A leucine residue at position 370 (L370) in 29-4 Shaker K+ channels resides within two overlapping sequence motifs conserved among most voltage-gated channels: the S4 segment and a leucine heptad repeat. Here we investigate the effects observed upon substitution of L370 with many other uncharged amino acid residues. We find that smaller or more hydrophilic residues produce greater alterations in both activation and inactivation gating than does substitution with other large hydrophobic residues. In addition, subunits containing less conservative substitutions at position 370 are restricted in their assembly with wild-type subunits and are unlikely to form homomultimeric channel complexes. Consistent with the idea that L370 influences the tertiary structure of these channels, the results indicate that L370 undergoes specific hydrophobic interactions during the conformational transitions of gating; similar interactions may take place during the folding, insertion, or assembly of Shaker K+ channel subunits.     

Transient outward K+ currents across the plasma membrane of laticifer from Hevea brasiliensis.

Non-inactivating outward rectifying K+ channel currents have been identified in a variety of plant cell types and species. The present study of laticifer protoplasts from Hevea brasiliensis, cells which are specialized for stress response, has revealed, through a switch-clamp method, an outward rectifying current displaying rapid inactivation. The inactivation depended on the external K+ concentration and on the voltage. This current inactivation appeared clearly different from all those previously described in plant cells and it shared homology with current kinetics of animal Shaker family channels. These results, given the recent cloning of plant K+ channel beta-subunits, shed new light on possible plant K+ channel regulation.     

Mapping the receptor site for charybdotoxin, a pore-blocking potassium channel inhibitor.

The Shaker K+ channel belongs to a family of structurally related voltage-activated cation channels that play a central role in cellular electrical signaling. By studying multiple site-directed mutants of the Shaker K+ channel, a region that forms the binding site for a pore-blocking scorpion toxin has been identified. The region contains a sequence that is highly conserved among cloned K+ channels and may contribute to the formation of the ion conduction pore.     

Tandem linkage of Shaker K+ channel subunits does not ensure the stoichiometry of expressed channels.

Shaker K+ channels are multimeric, probably tetrameric proteins. Substitution of a conserved leucine residue to valine (V2) at position 370 in the Drosophila Shaker 29-4 sequence results in large alterations in the voltage dependence of gating in the expressed channels. In order to determine the effects of this mutation in hybrid channels with a fixed stoichiometry of V2 and wild-type (WT) subunits we generated cDNA constructs of two linked-monomeric subunits similar to the tandem constructs previously reported by Isacoff, E. Y., Y. N. Jan, and L. Y. Jan. (1990. Nature (Lond.). 345:530-534). In addition, we constructed a tandem cDNA containing a wild-type subunit and a truncated nonfunctional subunit (Sh102) that suppresses channel expression. We report that the voltage-dependence of the channels produced with WT and V2 subunits varied significantly with the order of the subunits in the construct (WT-V2 or V2-WT), while the WT-Sh102 construct yielded currents that were much larger than expected. These results suggest that the tandem linkage of Shaker subunits does not guarantee the stoichiometry of the expressed channel proteins.     

Functional expression of Shaker K+ channels in a baculovirus-infected insect cell line

We constructed a recombinant baculovirus, A. californica nuclear polyhedrosis virus, containing the Drosophila Shaker H4 K+ channel cDNA under control of the polyhedrin promoter. When infected with this recombinant baculovirus, the cell line Sf9, derived from the army-worm caterpillar S. frugiperda, expresses fully functional Shaker transient K+ currents, as assayed by whole-cell recording. K+ currents begin to appear at about 15 hr after infection, and they continue to increase over the next 3 days. Over the same period of time, a 75 kd band appears on SDS gels stained with Coomassie blue. The identity of this band as a Shaker gene product is confirmed by Western blot analysis using an anti-Shaker antiserum. The 75 kd band accounts for a substantial fraction of the membrane protein in Shaker-infected Sf9 cells. These results give hope that the baculovirus system, which has been used successfully for high-level expression of soluble proteins from higher eukaryotes, may be appropriate for producing large amounts of cloned ion channel proteins as well.     

Binding of a gating modifier toxin induces intersubunit cooperativity early in the Shaker K channel's activation pathway

Potassium currents from voltage-gated Shaker K channels activate with a sigmoid rise. The degree of sigmoidicity in channel opening kinetics confirms that each subunit of the homotetrameric Shaker channel undergoes more than one conformational change before the channel opens. We have examined effects of two externally applied gating modifiers that reduce the sigmoidicity of channel opening. A toxin from gastropod mucus, 6-bromo-2-mercaptotryptamine (BrMT), and divalent zinc are both found to slow the same conformational changes early in Shaker's activation pathway. Sigmoidicity measurements suggest that zinc slows a conformational change independently in each channel subunit. Analysis of activation in BrMT reveals cooperativity among subunits during these same early steps. A lack of competition with either agitoxin or tetraethylammonium indicates that BrMT binds channel subunits outside of the external pore region in an allosterically cooperative fashion. Simulations including negatively cooperative BrMT binding account for its ability to induce gating cooperativity during activation. We conclude that cooperativity among K channel subunits can be greatly altered by experimental conditions.     

Expanded Functional Diversity of Shaker K+ Channels in Cnidarians Is Driven by Gene Expansion

The genome of the cnidarian Nematostella vectensis (starlet sea anemone) provides a molecular genetic view into the first nervous systems, which appeared in a late common ancestor of cnidarians and bilaterians. Nematostella has a surprisingly large and diverse set of neuronal signaling genes including paralogs of most neuronal signaling molecules found in higher metazoans. Several ion channel gene families are highly expanded in the sea anemone, including three subfamilies of the Shaker K⁺ channel gene family: Shaker (Kv1), Shaw (Kv3) and Shal (Kv4). In order to better understand the physiological significance of these voltage-gated K⁺ channel expansions, we analyzed the function of 18 members of the 20 gene Shaker subfamily in Nematostella. Six of the Nematostella Shaker genes express functional homotetrameric K⁺ channels in vitro. These include functional orthologs of bilaterian Shakers and channels with an unusually high threshold for voltage activation. We identified 11 Nematostella Shaker genes with a distinct “silent” or “regulatory” phenotype; these encode subunits that function only in heteromeric channels and serve to further diversify Nematostella Shaker channel gating properties. Subunits with the regulatory phenotype have not previously been found in the Shaker subfamily, but have evolved independently in the Shab (Kv2) family in vertebrates and the Shal family in a cnidarian. Phylogenetic analysis indicates that regulatory subunits were present in ancestral cnidarians, but have continued to diversity at a high rate after the split between anthozoans and hydrozoans. Comparison of Shaker family gene complements from diverse metazoan species reveals frequent, large scale duplication has produced highly unique sets of Shaker channels in the major metazoan lineages.     

Shaker potassium channel gating. III: Evaluation of kinetic models for activation

Predictions of different classes of gating models involving identical conformational changes in each of four subunits were compared to the gating behavior of Shaker potassium channels without N-type inactivation. Each model was tested to see if it could simulate the voltage dependence of the steady state open probability, and the kinetics of the single-channel currents, macroscopic ionic currents and macroscopic gating currents using a single set of parameters. Activation schemes based upon four identical single-step activation processes were found to be incompatible with the experimental results, as were those involving a concerted, opening transition. A model where the opening of the channel requires two conformational changes in each of the four subunits can adequately account for the steady state and kinetic behavior of the channel. In this model, the gating in each subunit is independent except for a stabilization of the open state when all four subunits are activated, and an unstable closed conformation that the channel enters after opening. A small amount of negative cooperativity between the subunits must be added to account quantitatively for the dependence of the activation time course on holding voltage.     

Dual-color quantum dot detection of a heterotetrameric potassium channel (hKCa3.1)

Potassium channels play a key role in establishing the cell membrane potential and are expressed ubiquitously. Today, more than 70 mammalian K(+) channel genes are known. The diversity of K(+) channels is further increased by the fact that different K(+) channel family members may assemble to form heterotetramers. We present a method based on fluorescence microscopy to determine the subunit composition of a tetrameric K(+) channel. We generated artificial "heteromers" of the K(+) channel hK(Ca)3.1 by coexpressing two differently tagged hK(Ca)3.1 constructs containing either an extracellular hemagglutinin (HA) or an intracellular V5 epitope. hK(Ca)3.1 channel subunits were detected in the plasma membrane of MDCK-F cells or HEK293 cells by labeling the extra- and intracellular epitopes with differently colored quantum dots (QDs). As previously shown for the extracellular part of hK(Ca)3.1 channels, its intracellular domain can also bind only one QD label at a time. When both channel subunits were coexpressed, 27.5 ± 1.8% and 24.9 ± 2.1% were homotetramers consisting of HA- and V5-tagged subunits, respectively. 47.6 ± 3.2% of the channels were heteromeric and composed of both subunits. The frequency distribution of HA- and V5-tagged homo- and heteromeric hK(Ca)3.1 channels is reminiscent of the binomial distribution (a + b)(2) = a(2) + 2ab + b(2). Along these lines, our findings are consistent with the notion that hK(Ca)3.1 channels are assembled from two homomeric dimers and not randomly from four independent subunits. We anticipate that our technique will be applicable to other heteromeric membrane proteins, too.