Cloning and functional expression of a TEA-sensitive A-type potassium channel from rat brain.

A rat brain cDNA (Raw3) related to the Drosophila Shaw K+ channel family has been characterized. Raw3 cRNA leads to the formation of TEA-insensitive, fast inactivating (A-type) K+ channels when injected into Xenopus laevis oocytes. Raw3 channels have markedly different properties from the previously cloned rat A-type K+ channel RCK4, Raw3 channels operate in the positive voltage range.     

Cloning and expression of a human voltage-gated potassium channel. A novel member of the RCK potassium channel family.

We have isolated and characterized a human cDNA (HBK2) that is homologous to novel member (RCK2) of the K+ channel RCK gene family expressed in rat brain. RCK2 mRNA was detected predominantly in midbrain areas and brainstem. The primary sequences of the HBK2/RCK2 K+ channel proteins exhibit major differences to other members of the RCK gene family. The bend region between segments S1 and S2 is unusually long and does not contain the N-glycosylation site commonly found in this region. They might be O-glycosylated instead. Functional characterization of the HBK2/RCK2 K+ channels in Xenopus laevis oocytes following micro-injection in in vitro transcribed HBK2 or RCK2 cRNA showed that the HBK2/RCK2 proteins form voltage-gated K+ channels with novel functional and pharmacological properties. These channels are different to RCK1, RCK3, RCK4 and RCK5 K+ channels.     

Gating mechanism of a cloned potassium channel expressed in frog oocytes and mammalian cells

We have cloned a cDNA coding for a delayed rectifier K+ channel from rat brain (RCK1) and rat muscle (RMK1) and expressed it in Xenopus oocytes and in a myoblast cell line (Sol-8). Stably transfected Sol-8 cells exhibited large outward K+ currents, which were indistinguishable from the K+ currents induced in Xenopus oocytes by injection of mRNA transcribed in vitro. RCK1 encodes a K+ channel with a unitary conductance of approximately 14 pS. The steep voltage dependence of channel opening resides in transitions between closed states, whereas the direct transitions into and out of the open state are very rapid and not markedly voltage-dependent. Channel inactivation is very slow, voltage-independent, and occurs from the open state only. We present a simple model that incorporates our findings and is consistent with the presumed structural symmetry of a functional K+ channel.     

Electrophysiological characterization of a new member of the RCK family of rat brain K+ channels

A novel member of the RCK family of rat brain K+ channels, called RCK2, has been sequenced and expressed in Xenopus oocytes. The K+ currents were voltage-dependent, activated within 20 ms (at 0 mV), did not inactivate in 5 s, and had a single channel conductance in frog Ringers of 8.2 pS. Compared to other members of the RCK family the pharmacological profile of RCK2 was unique in that the channel was resistant to block (IC50 = 3.3 microM) by charybdotoxin [(1988) Proc. Natl. Acad. Sci. USA 85, 3329-3333] but relatively sensitive to 4-aminopyridine (0.3 mM), tetraethylammonium (1.7 mM), alpha-dendrotoxin (25 nM), noxiustoxin (200 nM), and mast cell degranulating peptide (200 nM). Thus, RCK2 is a non-inactivating delayed rectifier K+ channel with interesting pharmacological properties.     

Molecular basis of functional diversity of voltage-gated potassium channels in mammalian brain.

Cloning and sequencing of cDNAs isolated from a rat cortex cDNA library reveals that a gene family encodes several highly homologous K+ channel forming (RCK) proteins. Functional characterization of the channels expressed in Xenopus laevis oocytes following microinjection of in vitro transcribed RCK-specific RNAs shows that each of the RCK proteins forms K+ channels that differ greatly in both their functional and pharmacological properties. This suggests that the molecular basis for the diversity of voltage-gated K+ channels in mammalian brain is based, at least partly, on the expression of several RCK proteins by a family of genes and their assembly to homooligomeric K+ channels with different functional properties.     

Members of the RCK potassium channel family are differentially expressed in the rat nervous system.

mRNAs encoding four members of the RCK potassium channel family, named RCK1, RCK3, RCK4 and RCK5 have been analyzed by RNA blot hybridization experiments using specific RNA probes. Each probe recognizes a single mRNA species, their sizes ranging from approximately 4600 nucleotides up to approximately 11,000 nucleotides. The expression of RCK mRNAs as well as their developmental appearance in different regions of the central and peripheral rat nervous system has been investigated. The two most abundant RCK potassium channel mRNAs (RCK1 and RCK5) are predominantly expressed in the adult nervous system. RCK3 and RCK4 mRNAs are present throughout all developmental stages studied. The temporal and regional patterns observed are specific for each RCK potassium channel mRNA indicating that specific regulation of expression occurs. Differential mRNA expression might provide one mechanism for the generation of potassium channel diversity in vivo.     

Potassium channels expressed from rat brain cDNA have delayed rectifier properties

Injection into Xenopus oocytes of RNA synthesized in vitro using the rat brain cDNA RCK1 as a template or nuclear injection of the cDNA results in the expression of functional potassium channels. These channels exhibit properties similar to those of the non-inactivating delayed rectifier channel found in mammalian neurons and other excitable cells.     

Ile-177 and Ser-180 in the S1 segment are critically important in Kv1.1 channel function

Ile-177 and Ser-180 are conserved residues in the first transmembrane segment (S1) of the Shaker, Shab, Shaw, and Shal subfamilies of voltage-gated K+ channels. Here we report that the mutation of these residues in Kv1.1 to leucine, proline, or arginine abolished the expression of outward potassium currents in Xenopus oocytes. Co-injection of these mutant cRNAs and wild type Kv1.1 cRNA into Xenopus oocytes exerted a potent dominant negative effect resulting in the suppression of Kv1.1-encoded currents. Transient transfection experiments of COS-7 cells revealed that the S1 mutants directed the synthesis of Kv1.1 polypeptides. Quantitative co-immunoprecipitation assays revealed that most of the S1 mutants co-assembled and formed both homo- and heteromultimeric complexes. Furthermore, the mutated polypeptides could reach the plasma membranes of transfected Sol8 cells. We conclude that mutations of Ile-177 and Ser-180 do not interfere with either the assembly of multimeric channel complexes or the targeting of these complexes to the plasma membrane. It is likely that these residues are involved in helix-helix interactions that are critical to the proper functioning of voltage-gated potassium channels.     

AtKC1 is a general modulator of Arabidopsis inward Shaker channel activity

A functional Shaker potassium channel requires assembly of four α-subunits encoded by a single gene or various genes from the Shaker family. In Arabidopsis thaliana, AtKC1, a Shaker α-subunit that is silent when expressed alone, has been shown to regulate the activity of AKT1 by forming heteromeric AtKC1-AKT1 channels. Here, we investigated whether AtKC1 is a general regulator of channel activity. Co-expression in Xenopus oocytes of a dominant negative (pore-mutated) AtKC1 subunit with the inward Shaker channel subunits KAT1, KAT2 or AKT2, or the outward subunits SKOR or GORK, revealed that the three inward subunits functionally interact with AtKC1 while the outward ones cannot. Localization experiments in plant protoplasts showed that KAT2 was able to re-locate AtKC1 fused to GFP from endomembranes to the plasma membrane, indicating that heteromeric AtKC1-KAT2 channels are efficiently targeted to the plasma membrane. Functional properties of heteromeric channels involving AtKC1 and KAT1, KAT2 or AKT2 were analysed by voltage clamp after co-expression of the respective subunits in Xenopus oocytes. AtKC1 behaved as a regulatory subunit within the heterotetrameric channel, reducing the macroscopic conductance and negatively shifting the channel activation potential. Expression studies showed that AtKC1 and its identified Shaker partners have overlapping expression patterns, supporting the hypothesis of a general regulation of inward channel activity by AtKC1 in planta. Lastly, AtKC1 disruption appeared to reduce plant biomass production, showing that AtKC1-mediated channel activity regulation is required for normal plant growth.     

Distribution and functional properties of human KCNH8 (Elk1) potassium channels

The Elk subfamily of the Eag K⁺ channel gene family is represented in mammals by three genes that are highly conserved between humans and rodents. Here we report the distribution and functional properties of a member of the human Elk K⁺ channel gene family, KCNH8. Quantitative RT-PCR analysis of mRNA expression patterns showed that KCNH8, along with the other Elk family genes, KCNH3 and KCNH4, are primarily expressed in the human nervous system. KCNH8 was expressed at high levels, and the distribution showed substantial overlap with KCNH3. In Xenopus oocytes, KCNH8 gives rise to slowly activating, voltage-dependent K⁺ currents that open at hyperpolarized potentials (half-maximal activation at -62 mV). Coexpression of KCNH8 with dominant-negative KCNH8, KCNH3, and KCNH4 subunits led to suppression of the KCNH8 currents, suggesting that Elk channels can form heteromultimers. Similar experiments imply that KCNH8 subunits are not able to form heteromultimers with Eag, Erg, or Kv family K⁺ channels. electrophysiology; human nervous system; potassium current     

Multiple subunits of a voltage-dependent potassium channel contribute to the binding site for tetraethylammonium.

RNAs encoding a wild-type (RBK1) and a mutant (RBK1(Y379V,V381T); RBK1*) subunit of voltage-dependent potassium channels were injected into Xenopus oocytes. When expressed separately, they made homotetrameric channels that differed about 100-fold in sensitivity to tetraethylammonium (TEA). Mixtures of channels having one, two, or three low affinity subunits were expressed by injecting various proportions of RBK1 and RBK1* RNAs. The affinity for TEA of these three channel species was deduced by fitting concentration-response curves for the inhibition of potassium currents. DNAs were also concatenated to construct a sequence that encoded two connected subunits, and channels that contained four, two, or no TEA-sensitive subunits were expressed. The results suggest that bound TEA interacts simultaneously with all four subunits.     

Distinct behavior of connexin56 and connexin46 gap junctional channels can be predicted from the behavior of their hemi-gap-junctional channels.

The gap-junctional protein rat connexin46 (Cx46) has the unusual ability to form voltage-gated channels in the nonjunctional plasma membrane of Xenopus oocytes (Paul et al., 1991; Ebihara and Steiner, 1993). These have been suggested to be gap-junctional hemichannels or connexons. The Xenopus oocyte system was used to characterize the functional properties of a closely related lens gap-junctional protein, chicken connexin56 (Cx56) (Rup et al., 1993) and to contrast them to those of rat Cx46. Single oocytes injected with either Cx56 or Cx46 cRNA developed time-dependent, outward currents that activated on depolarization. The currents induced by Cx56 and Cx46 showed differences in steady-state voltage dependence and in their degree of rectification. Furthermore, the voltage-dependent properties of the nonjunctional channels induced by the connexin cRNAs in external solutions containing low concentrations of calcium ions could account remarkably well for the behavior of the intercellular channels formed by Cx56 and Cx46 in paired oocytes. These results suggest that many of the voltage-dependent properties of the hemi-gap-junctional channels are retained by the intercellular channels.     

Endogenous KCNE subunits govern Kv2.1 K+ channel activation kinetics in Xenopus oocyte studies

Kv2.1 is a voltage-gated potassium (Kv) channel that generates delayed rectifier currents in mammalian heart and brain. The biophysical properties of Kv2.1 and other ion channels have been characterized by functional expression in heterologous systems, and most commonly in Xenopus laevis oocytes. A number of previous oocyte-based studies of mammalian potassium channels have revealed expression-level-dependent changes in channel properties, leading to the suggestion that endogenous oocyte factors regulate channel gating. Here, we show that endogenous oocyte potassium channel KCNE ancillary subunits xMinK and xMiRP2 slow the activation of oocyte-expressed mammalian Kv2.1 channels two-to-fourfold. This produces a sigmoidal relationship between Kv2.1 current density and activation rate in oocyte-based two-electrode voltage clamp studies. The effect of endogenous xMiRP2 and xMinK on Kv2.1 activation is diluted at high Kv2.1 expression levels, or by RNAi knockdown of either endogenous subunit. RNAi knockdown of both xMiRP2 and xMinK eliminates the correlation between Kv2.1 expression level and activation kinetics. The data demonstrate a molecular basis for expression-level-dependent changes in Kv channel gating observed in heterologous expression studies.     

Single-Channel Properties of IKs Potassium Channels

Expressed in Xenopus oocytes, KvLQT1 channel subunits yield a small, rapidly activating, voltage- dependent potassium conductance. When coexpressed with the minK gene product, a slowly activating and much larger potassium current results. Using fluctuation analysis and single-channel recordings, we have studied the currents formed by human KvLQT1 subunits alone and in conjunction with human or rat minK subunits. With low external K⁺, the single-channel conductances of these three channel types are estimated to be 0.7, 4.5, and 6.5 pS, respectively, based on noise analysis at 20 kHz bandwidth of currents at +50 mV. Power spectra computed over the range 0.1 Hz–20 kHz show a weak frequency dependence, consistent with current interruptions occurring on a broad range of time scales. The broad spectrum causes the apparent single-channel current value to depend on the bandwidth of the recording, and is mirrored in very “flickery” single-channel events of the channels from coexpressed KvLQT1 and human minK subunits. The increase in macroscopic current due to the presence of the minK subunit is accounted for by the increased apparent single-channel conductance it confers on the expressed channels. The rat minK subunit also confers the property that the outward single-channel current is increased by external potassium ions.     

Molecular cloning of a novel form (two-repeat) protein related to voltage-gated sodium and calcium channels

Voltage-gated Ca and Na channels share similar structure: four homologous domains (I-IV), each with six transmembrane segments (S1-S6). They may be formed by two rounds of duplication of a single channel domain similar to voltage-activated potassium channels. However, the channels with the intermediate structure, namely, two-domain channels have not yet been identified. We report here the cloning of a novel protein from rat kidney that contains two domains (I-II), each with S1-S6 segments that are found in voltage-gated Ca and Na channels. Because of unusual structure, the protein was named two-pore channel 1 (TPC1). TPC1 encodes 819 amino acids with two conserved positively charged voltage sensor segments (S4) but the pore segments are not conserved. Northern blot analysis showed that TPC1 mRNA (5 kb) was expressed widely. It was expressed at relatively high level in kidney, liver, and lung. Immunohistochemistry of kidney revealed that TPC1 was expressed at inner medullary collecting ducts. In expression studies, no functional currents could be detected in CHO cells and Xenopus oocytes. Based on its primary structure, we propose that TPC1 might be a predecessor of the conventional four repeat voltage-gated Ca and Na channels and will give insights into the evolution of ion channels.     

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.     

The Human Red Cell Voltage-regulated Cation Channel. The Interplay with the Chloride Conductance,the Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> Channel and the Ca<Superscript>2+</Superscript> Pump

Electrocytes from the electric organ of Electrophorus electricus exhibited sodium action potentials that have been proposed to be repolarized by leak currents and not by outward voltage-gated potassium currents. However, patch-clamp recordings have suggested that electrocytes may contain a very low density of voltage-gated K⁺ channels. We report here the cloning of a K⁺ channel from an eel electric organ cDNA library, which, when expressed in mammalian tissue culture cells, displayed delayed-rectifier K⁺ channel characteristics. The amino-acid sequence of the eel K⁺ channel had the highest identity to Kv1.1 potassium channels. However, different important functional regions of eel Kv1.1 had higher amino-acid identity to other Kv1 members, for example, the eel Kv1.1 S4-S5 region was identical to Kv1.5 and Kv1.6. Northern blot analysis indicated that eel Kv1.1 mRNA was expressed at appreciable levels in the electric organ but it was not detected in eel brain, muscle, or cardiac tissue. Because electrocytes do not express robust outward voltage-gated potassium currents we speculate that eel Kv1.1 channels are chronically inhibited in the electric organ and may be functionally recruited by an unknown mechanism.     

Expression of an outward-rectifying potassium channel from maize mRNA and complementary RNA in Xenopus oocytes.

Injection of Xenopus oocytes with poly(A)+ mRNA isolated from different plants (maize, cucumber, and squash) results in the appearance of a voltage- and time-dependent, potassium-selective, outward current that is similar to the outward-rectifying potassium current recorded in many higher plant cells. Maize shoots were found to be especially enriched in mRNA encoding such activity. A cDNA library of maize shoot mRNA was constructed in the vector lambda ZAPII and was used to synthesize RNA complementary to the cDNA (cRNA). Injection of the cRNA gave rise to an outward-rectifying potassium current with properties similar to the currents obtained by poly(A)+ mRNA injection. These results demonstrate that higher plant mRNA can be properly translated into a product that produces a voltage-regulated potassium channel in the plasma membrane of Xenopus oocytes. Thus, Xenopus oocytes can be used as a heterologous expression system for the functional identification and isolation of plant ion channel genes as well as for the study of structure-function relationship of plant ion channels.     

Bufo marinus oocytes as a model for ion channel protein expression and functional characterization for electrophysiological studies.

Oocytes from Xenopus laevis are commonly used as an expression system for ion channel proteins. The aim of this study was to determine whether oocytes from the Colombian native toad, Bufo marinus, could be used as an alternative expression system for ion channel protein expression and functional characterization using the two-microelectrode voltage clamp method. B. marinus oocytes and X. laevis were isolated and cultured in similar conditions. The mean resting membrane potential of B. marinus oocytes was similar to that of X. laevis oocytes as well as the whole-cell basal currents. The potassium ion channel Kv1.1 was successfully expressed in B. marinus oocytes and showed a typical outward rectifying current. Potassium channel blockers reduced these currents. The similarities on electrical properties and expression of ion channel proteins show that B. marinus oocytes can be used effectively to express these proteins, making these cells a viable heterologous system for the expression of ion channel proteins and their electrophysiological characterization.