©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Amino Terminus and the First Four Membrane-spanning Segments of the Arabidopsis K Channel KAT1 Confer Inward-rectification Property of Plant-Animal Chimeric Channels (*)

(Received for publication, February 15, 1995; and in revised form, May 17, 1995)

Yongwei Cao (§) Nigel M. Crawford Julian I. Schroeder (¶)

From the Department of Biology and the Center for Molecular Genetics, University of California at San Diego, La Jolla, California 92093-0116

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Arabidopsis hyperpolarization-activated (inward-rectifying) K channel KAT1 is structurally more similar to animal depolarization-activated (outward-rectifying) K channels than to animal hyperpolarization-activated K channels. To gain insight into the structural basis for the opposite voltage dependences of plant inward-rectifying and animal outward-rectifying K channels, we constructed recombinant chimeric channels between the hyperpolarization-activated K channel KAT1 and a Xenopus depolarization-activated K channel. We report here that two of the chimeric constructs, which contain the first third of the KAT1 sequence, including the first four membrane-spanning segments (S1-S4) and the linker sequence between the fourth and fifth membrane-spanning segments, express functional channels that retain activation by hyperpolarization, but not depolarization. These two chimeric channels are no longer selective for K. The chimeras are selective for cations over anions and are permeable to Ca. Therefore, unlike animal hyperpolarizationactivated K channels, in which the carboxyl terminus is important for inward rectification induced by Mg and polyamine block, the plant KAT1 channel has its major determinants for inward rectification in the amino-terminal region, which ends at the end of the S4-S5 linker.


INTRODUCTION

Voltage-dependent K channels can be divided into at least two major classes with respect to the membrane potential ranges that produce activation. One class belongs to the outward-rectifying K (K)()channels, which are activated by membrane depolarization. This class comprises the most diverse group of K channels, showing many distinct biophysical and pharmacological properties(1, 2) . Potassium channels of this class belong to a superfamily of genes that display common structural motifs characterized by six membrane-spanning segments (S1-S6), a putative voltage sensor (S4 segment), and an S5-S6 linker (pore or H5 region) involved in K permeability and ion selectivity (reviewed in (1) and (2) ).

K channels that belong to a different class are the inward-rectifying K (K) channels, which are activated by membrane hyperpolarization. Genes coding for this class of K channels have recently been cloned from both plants (3, 4) and animals(5, 6, 7) . However, the general structures of these K channels differ significantly between animals and plants. Animal inward-rectifying K channels possess only two predicted membrane-spanning segments (M1 and M2), which surround a consensus region corresponding to the H5 or pore region of the K channels. Activation of animal inward-rectifying K channels is mainly mediated by the alleviation of cytosolic Mg and polyamine block at hyperpolarized membrane potentials(8, 9, 10, 11) . Guard cells and other plant cells express time- and voltage-dependent inward-rectifying K channels ((12) ; for review, see (13) ). The Arabidopsis KAT1 cDNA (3) has been shown to confer the voltage and time dependence, the cation selectivity, and TEA and Ba block properties of typical plant inward-rectifying K channels (14) . However, plant K channels are structurally more similar to K channels in animals(3, 4) . Plant K channels have at least six putative membrane-spanning segments, a typical voltage sensor segment (S4), and a K-selective pore (H5 or P) domain. Furthermore, activation of inward-rectifying K channels in guard cells is independent of cytosolic Mg(15) . It is possible that activation of plant K channels is mediated by intrinsic voltage-dependent gating, similar to that of animal K channels.

To investigate the molecular structures that determine the direction of rectification, we engineered a series of chimeric constructs between a plant inward-rectifying K channel (KAT1) (3) and an animal outward-rectifying K channel (Xsha2) (16) and expressed corresponding mRNAs in Xenopus oocytes. Our results demonstrate that the region between the N terminus and the end of the S4-S5 linker of the KAT1 channel is sufficient for hyperpolarization-induced activation.


MATERIALS AND METHODS

The Xsha2 cDNA clone (16) was kindly provided by Dr. Nick Spitzer (University of California at San Diego). The KAT1 cDNA clone (3) was reisolated from a YES cDNA library of Arabidopsis(17) . Chimeric channels were produced by a recombinant polymerase chain reaction method described by Higuchi(18) . The chimeric constructs were subcloned into the pBluescript KS vector (Stratagene, La Jolla, CA) or into a modified pBluescript KS vector containing a poly(A) segment (50 A residues) downstream of the cloning sites (kindly provided by Dr. C. Labarca, California Institute of Technology) to increase the level of expression. All polymerase chain reaction-generated DNA fragments were verified by DNA sequencing. Capped mRNA was transcribed from linearized plasmids in vitro using a T7 RNA polymerase kit (Ambion Inc., Austin, TX). DNA sequence alignment was performed with PCGENE computer software (IntelliGenetics Inc.) initially and then slightly modified to maximize the alignment of identical amino acids.

Oocytes were isolated as described (19) and injected with 5-20 ng of mRNA in 50 nl of water. Ionic currents after leakage subtraction were recorded 1-5 days after injection using the two-electrode voltage-clamp technique as described previously(14) . Membrane currents were normally measured in a K solution containing 115 mM KCl, 1 mM CaCl, 2 mM MgCl, 10 mM Hepes (pH 7.2), or in a Na solution, which was the same as the K solution except that 115 mM KCl was replaced with 115 mM NaCl. For experiments with varying Cl concentrations (see Fig. 3F), KCl in the K solution was partially or completely replaced with K glutamate to obtain the desired Cl concentrations. For experiments with varying K or Ca concentrations (see Fig. 4), KCl in the 115 mM K solution was removed, and different amounts of K glutamate or Ca glutamate were added. The osmotic strength of these solutions was adjusted to 240-260 mosm/kg with mannitol. To determine the conductance ratios of the chimeric channels to monovalent cations, oocytes were bathed in solutions containing 1 mM MgCl, 90 mM mannitol, 10 mM Hepes, and a 15 mM concentration of one of the following salts: KCl, NaCl, CsCl, LiCl, and RbCl. pH was adjusted to 7.2 with 1 N Tris-Cl. Bath electrode potentials were kept stable by using 3 M KCl-agar electrode bridges or by maintaining the Cl concentration constant for individual oocytes. BAPTA was prepared as a 50 mM stock solution (pH 7.2 with KOH) and was injected into oocytes (50 nl/oocyte) 15-60 min before recording. The final concentration of BAPTA in oocytes was estimated to be 2.5 mM assuming a spherical oocyte volume of 1 µl.


Figure 3: Effect of BAPTA injection on reversal potentials and macroscopic currents recorded from KXH5 mRNA-injected oocytes. Currents were recorded from a KXH5-expressing oocyte before (A and B) and after (C and D) BAPTA injection and from a control oocyte after BAPTA injection (E). A and C, inward current and relaxation tail currents were elicited by a step to -120 mV (A) or -140 mV (C), followed by depolarizing pulses to values indicated to the left of tail current traces. B, D, and E, macroscopic currents were elicited by voltage pulse protocols described in the legend to Fig. 2. F, reversal potentials are plotted as a function of external Cl concentration. Reversal potentials were measured from tail current experiments as shown in A and C. Error bars indicate S.D. (n = 4 for all points). G, normalized macroscopic currents such as those shown in B and D were plotted as a function of the membrane potential. Current amplitudes were measured at the end of 1.5 s of stimulation after leakage subtraction from the same oocytes before and after BAPTA injection and normalized with respect to current levels recorded at -140 mV before BAPTA injection. Errorbars indicate S.D. (n = five KXH5-injected oocytes). All recordings were performed in 115 mM KCl solution with the exception of F (see ``Materials and Methods'').




Figure 4: Current-voltage relationship of chimeric channels. Macroscopic currents were recorded from KXH5-injected (A) and KXS5-injected (B) oocytes with extracellular K concentrations in the range of 0-115 mM K (as indicated by the symbols). Oocytes were injected with 2.5 mM BAPTA before recording. Currents were elicited by voltage protocols as described in the legend to Fig. 2and measured at the end of 1.5 s of stimulation after linear leakage subtraction.




Figure 2: Macroscopic currents recorded from uninjected or mRNA-injected oocytes. A, KAT1; B, Xsha2 (16) ; C, uninjected oocyte showing measurable endogenous currents(17, 22) ; D, KXH5; E, KXS5. The membrane potential of oocytes was held at -40 to -60 mV and stepped for 1.5 s to potentials ranging from -160 to +60 mV in 20-mV increments. The values of some of the voltage pulses are indicated to the right of the corresponding current traces. Currents were recorded in either a 115 mM KCl solution (A and C-E) or a 115 mM NaCl solution (B).




RESULTS

Sequence comparison between plant K channels and animal K channels has revealed that the two types of channels share some similarities in the hydrophobic core region(3, 4, 13, 17) . As illustrated in the alignment between KAT1 and Xsha2 (Fig. 1A), these similarities are not limited to the voltage sensor segment (S4), the K-selective pore region (H5), and the overall hydrophobicity profile, but are extended to some amino acids in other membrane-spanning segments as well. For instance, the two negatively charged residues in S2 (Asp, Asp), the negatively charged aspartic acid in S3 (Asp), the leucine residue in S5 (Leu), and the alanine residue in S6 (Ala), which are all highly conserved among animal K channels(20) , are also conserved in plant K channels (Fig. 1A). These amino acids have been hypothesized to be important in forming ionic interactions with the positively charged residues in S4 and to stabilize the channel conformation in the open or closed state(21) . The conservation of these residues in both animal K channels and plant K channels suggests that the overall membrane topology and the possible arrangements of the six membrane-spanning segments are similar between the two types of channels and that chimeric constructs made by joining segments of the two types of channels at conserved residues may retain the gross membrane topology.


Figure 1: Sequence alignment of the hydrophobic portions of the KAT1 and Xsha2 channels and the design of chimeric constructs. A, KAT1 and Xsha2 sequences were first aligned with two Arabidopsis K channels (AKT1 and AKT2) (4, 17) and members of four separate Drosophila K channel families (Shaker, Shal, Shab, and Shaw)(30, 31, 32) , respectively. The identical residues among all three Arabidopsis K channels or all five animal K channels are shown in boldface upper-case letters and are underlined. The conservatively substituted residues among all three Arabidopsis K channels or at least four animal K channels are shown in lightface upper-case letters. The nonconserved residues are shown in lower-case letters. Amino acids that are identical or conservatively substituted between KAT1 and Xsha2 are marked by verticallines and asterisks, respectively. Members of the following groups of amino acids were considered to be conserved: (Met, Ile, Leu, Val); (Ala, Gly); (Ser, Thr); (Gln, Asn); (Asp, Glu); and (Lys, Arg), (Phe, Tyr, Trp). Gaps were introduced for optimal alignment and are shown as dashes. The junction points for the construction of the recombinant chimeric channels are marked by arrows. B, schematic representation of KAT1, Xsha2, and eight of the chimeric cDNA constructs between KAT1 and Xsha2. The openbox in the C-terminal half of KAT1 represents a potential cyclic nucleotide-binding site(3) .



Four reciprocal pairs of chimeric constructs were made to fuse fragments of the KAT1 and Xsha2 channels at conserved residues at Asp (S2 segment), Ile (S4 segment), Leu (S5 segment), and Trp (H5 region) (Fig. 1, A and B). Messenger RNAs were synthesized in vitro from these chimeric constructs. 1-5 days after injection into Xenopus oocytes, currents elicited by both depolarizing and hyperpolarizing pulses were analyzed. Control KAT1 mRNA induced an inward-rectifying K current (Fig. 2A)(14) , and Xsha2 mRNA induced an outward-rectifying K current in Xenopus oocytes (Fig. 2B)(16, 19) . These currents were significantly larger than endogenous currents recorded in some batches of uninjected or water-injected oocytes (Fig. 2C) (see (19) and (22) ). However, not all chimeric constructs induced exogenous currents. Careful analysis of these nonfunctional chimeras (XKS2, KXS2, XKS4, KXS4, XKS5, and XKH5) showed that they did not give rise to voltage-dependent currents, and they also did not increase linear leakage currents in oocytes as judged by oocyte background resistance measurements between -120 and +20 mV (n > 100 oocytes). Five additional chimeras were constructed: two in which the KAT1 regions from Asp (S2) to Leu (S5) or to Trp (H5) were inserted into XSha2, two in which the equivalent regions from XSha2 were inserted into KAT1, and one in which only the XSha2 S4/55 linker was inserted into KAT1.()These chimeras were also nonfunctional. Only the chimeric KXH5 (n = 186 oocytes) and KXS5 (n = 69 oocytes) mRNAs (Fig. 1B) consistently induced large inward currents upon membrane hyperpolarization (Fig. 2, D and E). Upon membrane depolarization, no outward currents that were significantly larger than endogenous currents were detected (Fig. 2, D and E). Thus, both KXH5 and KXS5 produced inward-rectifying currents in oocytes. Similarly, the equivalent chimera to KXH5 between KAT1 and the Drosophila K channel EAG showed inward-rectifying currents (data not shown). Further analysis focused on KXS5 and KXH5 (Fig. 1B). The current induced by KXH5 was generally larger than that induced by KXS5, but otherwise similar to the current induced by KXS5. Both currents were activated at membrane potentials negative to approximately -70 mV, which is similar to the activation potential for KAT1(14, 17) . However, the activation and deactivation time courses for both chimeras were much slower than those for the KAT1 channel (Fig. 2).

To determine the ionic selectivity of the chimeric channels, the reversal potential was measured by relaxation (``tail'') current analysis. Surprisingly, both KXH5 and KXS5 tail currents reversed direction at -18.4 ± 2.1 mV (n = 9) in a 115 mM KCl solution (Fig. 3, A and F; data not shown). This reversal potential was closer to the Cl equilibrium potential than to the K equilibrium potential. Substituting K with Na had a negligible effect on the reversal potential (data not shown), whereas varying the external Cl concentration shifted the reversal potential for both KXH5 and KXS5 channels with a slope of 42 mV/10-fold change in external Cl concentration (Fig. 3F, opencircles), which is indicative of channels selective for Cl over K. In addition, tail currents of both chimeric channels showed an unusually large outward conductance and a very slow relaxation time course (Fig. 3A), which are characteristic of endogenous Cl channel currents observed infrequently in some uninjected control oocyte batches (see (22) for details). Thus, initial results indicated that the currents induced by KXH5 and KXS5 were mostly carried by Cl ions.

Since oocytes contain endogenous Ca-activated Cl channels(23) , it is likely that the observed Cl current may be activated by Ca if the chimeric channels are permeable to Ca such that activation of chimeric channels induces endogenous Ca-activated Cl currents. To test this possibility, we injected oocytes with BAPTA before recording to chelate intracellular Ca and thus to inhibit Ca-activated Cl channels in oocytes. As predicted, injection of BAPTA greatly reduced the current amplitude and changed the time dependence of KXH5-injected (n = 26) and KXS5-injected (n = 9) oocytes (Fig. 3, B, D, and G). BAPTA injections had no effect in control oocytes, which showed no endogenous currents (n = 5) (Fig. 3E). Furthermore, BAPTA injections completely abolished hyperpolarization-induced Cl currents in the subpopulation of oocyte batches in which uninjected oocytes showed large hyperpolarization-activated Cl currents (n = 4) (see (22) ). The KXH5 and KXS5 currents after BAPTA injection continued to show inward rectification, a slow activation time course, and no activation by depolarization (Fig. 3, D and G). In addition, the typical large and slowly relaxing Cl tail currents (Fig. 3A) were also completely abolished by BAPTA injection (Fig. 3C). The reversal potential after BAPTA injection was +2.9 ± 3.5 mV for KXH5 (n = 5) (Fig. 3, C and F) and +4.2 ± 2.8 mV for KXS5 (n = 4) in 115 mM KCl solution. These reversal potentials were not dependent on the external Cl concentration (Fig. 3F, closedcircles), indicating that the remaining currents were no longer carried by Cl ions.

To determine whether the remaining currents after BAPTA injection were carried by cations rather than anions, we measured inward-rectifying current-voltage relationships in bath solutions with varying cation concentrations. As illustrated in Fig. 4, removal of external K abolished the remaining inward currents, while readdition of K (as glutamate salt to keep the Cl concentration unchanged) recovered the inward current. In control uninjected oocytes, no K-dependent inward currents were observed (n = 3). Therefore, the chimeras KXH5 and KXS5 produced a hyperpolarization-activated conductance that was dependent on the external K concentration.

The chimeric channels were also permeable to other cations. As shown in Table 1, replacing K in the bath solution with equimolar concentrations of Rb, Na, Cs, or Li did not change the amplitude of inward currents significantly in both KXH5- and KXS5-injected oocytes. Furthermore, replacing K in the 115 mM K solution with Ca also induced inward currents, which depended on the extracellular Ca level (KXH5, n = 3; and KXS5, n = 3). Control oocytes did not show significant inward currents in a bath solution with 90 mM Ca after BAPTA injection (n = 3). These results showed that both chimeric channels encode nonselective cation channels, which are activated by hyperpolarization, but not depolarization.




DISCUSSION

These data show that functional chimeric channels can be formed between a plant inward-rectifying K channel and an animal outward-rectifying K channel. The two functional chimeric channels retain hyperpolarization-induced activation while losing depolarization-induced activation. Based on the sequence composition of the two functional chimeras, the region that is responsible for inward rectification is located in the first third of the plant KAT1 channel, which includes the first four putative membrane-spanning segments and the S4-S5 linker. This result is in marked contrast to results obtained by chimeric analysis and site-directed mutagenesis in animal inward-rectifying K channels, in which the C terminus and a single charged amino acid in the last putative membrane-spanning segment (M2) play a major role in determining Mg and polyamine block-dependent inward rectification(9, 10, 24, 25) . This regional difference indicates different mechanisms for the generation of inward rectification between plant and animal inward-rectifying K channels. Plant inward-rectifying K channels usually display a steep voltage-dependent activation (for review, see (13) ). This voltage dependence is not induced by Mg block(15) . The sequence and the predicted structural similarities between plant K channels and animal K channels and the presence of a typical S4 segment in plant K channels (3, 4) (Fig. 1A) further suggest that the activation of plant K channels may be induced by intrinsic voltagedependent gating, a process similar to the gating of animal K channels(26) . The S4 segment of plant K channels may respond to membrane hyperpolarization by ``moving'' inwardly across the membrane electrical field and in turn open the channel. This hypothesis would be consistent with our results.

Unlike the wild-type KAT1 inward-rectifying K channel, the two chimeric channels both display a much slower activation time course and lose K selectivity. These results are not surprising because several regions of voltage-dependent ion channels affect pore function and activation kinetics. Furthermore, removal of the N- and C-terminal regions of a delayed-rectifying K channel (drk) have been shown to produce a large increase in channel activation times and inactivation times, similar to those found here, while retaining depolarization-induced activation(27) . It is possible that functional regions may not be completely compatible with each other if they are derived from different sources as in our plant-animal chimeric channels, which likely leads to nonfunctional chimeras, indicating a limitation. Furthermore, the two functional chimeric channels may have an aberrant structure, which results in changes in activation kinetics and loss of K selectivity while retaining activation by hyperpolarization and cation over anion selectivity.

Both the KXH5 and KXS5 chimeric channels behave similarly, indicating that the S5 segment and part of the S5-H5 linker are not critical for determining the hyperpolarization-induced activation, in spite of a significantly longer S5-H5 linker in plant K channels when compared with animal K channels (Fig. 1A). In addition, the lack of outward rectification in both chimeric channels suggests that the structural component that links S4 movement to channel opening is located in the region from the N terminus to the beginning of the S5 domain of the KAT1 channel. Durell and Guy (21) have proposed, based on experimental and computational data, that the small S4-S5 linker region in animal K channels may function as a channel gate. Site-directed mutagenesis studies in animal K channels have also shown that the S4-S5 linker contributes to formation of the inner mouth of K channels(28, 29) . It is worth noting that the S4-S5 linker sequences of plant K channels are very different from those of animal K channels (Fig. 1A). Plant S4-S5 linkers are shorter and, most noticeably, have two conserved negatively charged residues at the beginning (Glu and Asp) and a positively charged residue (Arg) at the end, which are absent in animal S4-S5 linkers (Fig. 1A). These charge differences would imply structurally distinct gates that may be relevant to the opposite voltage-dependent activation of plant K channels and animal K channels. More specific domain-swap and site-directed mutagenesis analyses may allow further definition of regions or amino acids that are important for the difference between hyperpolarization- and depolarizationinduced activation.


FOOTNOTES

*
This work was supported by Department of Energy Grant De-FG03-94-ER20148 and a National Science Foundation grant (to J. I. S.) and in part by National Institutes of Health Grant GM40672 (to N. M. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a Ph.D. fellowship from the Rockefeller Foundation. Present address: Div. of Biology, 156-29, California Institute of Technology, Pasadena, CA 91125.

To whom correspondence should be addressed: Dept. of Biology, 0116, University of California at San Diego, La Jolla, CA 92093-0116. Tel.: 619-534-7759; Fax: 619-534-7108.

The abbreviations used are: K+, outward-rectifying; K, inward-rectifying; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N, N, N`, N`-tetraacetic acid.

Y. Cao, N. Uozumi and J. I. Schroeder, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Nick Spitzer for the Xsha2 cDNA clone, Dr. C. Labarca for the pBS-KSAMV-pA50 vector, and Audrey Ichida for comments on the manuscript.


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