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Department of Life Science, Gwangju Institute of Science and Technology, Gwangju, Korea
Correspondence: Address reprint requests to Chul-Seung Park, PhD, Dept. of Life Science, Gwangju Institute of Science and Technology, 1 Oryong-dong, Buk-gu, Gwangju, 500-712, Korea. Tel.: 82-62-970-2489; Fax: 82-62-970-2484; E-mail: cspark{at}gist.ac.kr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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indicating substantial permeability of small ions through the channel. Although a mutation near the selectivity filter mimicking other K+-selective channels influenced the size-selectivity for permeant ions, Na+ permeability of rSK2 channels was still retained. Since the reversal potential of endogenous SKCa current is determined by Na+ permeability in a physiological ionic environment, the ion selectivity of native SKCa channels should be reinvestigated and their in vivo roles may need to be restated. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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,2). These channels play important roles in a wide range of physiological processes, including neuronal excitability (3), rhythmic hormone release (4), smooth muscle tone (5,6), and gain control of the hearing organ (7,8). In many neurons, SKCa channels are thought to remain open after the action potential and to contribute to afterhyperpolarization (AHP), thereby modulating interspike interval and burst duration (9). Although SKCa channel currents were considered to underlie the medium and slow components of AHP (mAHP and sAHP, respectively) in various neurons, the functional role of these currents in different neurons has been controversial (10,11). Recent experimental evidence indicates that SKCa current is responsible for the intermediate, rather than the late, phase of AHP (12,13).
Three different but highly homologous membrane proteins, SK1–3, were identified as the main subunits of SKCa channels (14). These proteins can be classified as distinct members of the K+-channel superfamily with six transmembrane (TM) domains. However, it is the small region spanning the fifth and sixth TM domains and the pore-forming region (P-region) in between that shows high homology to other voltage-gated K+ channels. All three SKCa channel subunits contain the amino acid triplet, Gly-Tyr-Gly, essential for high potassium selectivity, in their P-regions. A series of functional and structural studies showed that the main subunits of SKCa channels bind an auxiliary protein, calmodulin, as the Ca2+-sensing gating machinery (15,16). Thus, functional SKCa channels are assembled as tetramers of identical or different subunits and four calmodulins constitutively bound at each carboxyl terminus. The opening of the channels is now understood as the result of the direct binding of cytosolic Ca2+ to calmodulin and the subsequent conformational change (16–18).
Although SKCa currents are known to play pivotal roles in the excitability of various neurons, it has been difficult to study the fundamental characteristics of the channels in neurons due to their heterogeneity in electrophysiology, pharmacology, and modulation (2). It is conceivable that individual neurons may express more than one type of SKCa channel subunit and that these subunits coassemble to form heteromultimers. In this study, we investigated one of the fundamental properties of an ion channel, the selectivity among permeant ions, for a cloned SKCa channel, SK2. SK2 channels are expressed most widely in the mammalian central nervous system, especially high in the hippocampal formations, the anterior olfactory nucleus, and the granular layer of the cerebellum (14). We expressed the rat SK2 channels in two different heterologous systems, mammalian cell lines and Xenopus oocytes. We initially noticed that the reversal potentials of SKCa channel currents deviated significantly from the Nernst relationship expected for channels of high K+ selectivity. To our surprise, the SK2 channel, known to be highly selective for K+, showed significant permeability for small ions such as Li+ and Na+. In fact, the permeability compared among monovalent cations showed the SK2 channel to be one of the least selective K+ channels known so far. These results are not only intriguing but alarming, since Na+ permeability of the SKCa channel means relatively depolarized reversal potentials for the SKCa currents.
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MATERIALS AND METHODS |
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). Complementary DNA for the rat SK2 channel (provided by Dr. Adelman, The Vollum Institute, Oregon Health Sciences University, Portland, OR), human IK (provided by Dr. Kaczmareck, Yale University, New Haven, CT), and rat Slo (the 
-subunit of the large-conductance Ca2+-activated K+ (BKCa) channel) (20) were subcloned into a modified pGH expression vector. The generation of a mutant rSK2 channel, S359A, was reported previously (21) and L358T mutant channel was constructed using PCR-based mutagenesis as described previously. Complementary RNAs for wild-type (rSK2, hIK, and rSlo) channels and two mutant rSK2 channels were synthesized in vitro from an NcoI-linearized plasmid using T7 polymerase (Promega, Madison, WI). Oocytes were injected with 
50 ng of RNA and incubated at 18°C for 3–7 days in ND96 solution containing 5 mM HEPES, 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 50 µg/ml gentamicin, pH 7.6 adjusted with NaOH.
Expression of rat SK2 channels in mammalian cells
The coding region of the rSK2 channel was subcloned into pcDNA3.1 (Invitrogen, Carlsbad, CA) and expressed transiently in Chinese hamster ovary (CHO) cells for electrophysiological recordings. Cells were maintained in F12K Nutrient Mixture medium (Invitrogen) supplemented with 10% fetal bovine serum at 37°C in the presence of 5% CO2. The day before transfection, 1 x 105 CHO cells were seeded on coverslips coated with 10 µg/ml poly-L-lysine and allowed to grow for 24 h. Transfections were carried out with Polyfect reagent (Qiagen, Valencia, CA) according to the instruction of the manufacturer. For each transfection reaction, 1.5 µg of pcDNA3.1 vector harboring the channel gene and 100 ng of pEGFP-N3 were used.
Whole-cell voltage clamp recording of SKCa channels expressed in CHO cells
Whole-cell currents of rSK2 channel were measured from transfected CHO cells using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). The recordings were made at room temperature (23°C) 1–2 days after transfection. Pipettes prepared from thin-walled borosilicate glass (World Precision Instruments, Sarasota, FL) had resistance of 3–4 M
. Signals were filtered at 1 kHz using a four-pole low-pass Bessel filter, digitized at the rate of 200 samples/ms using Digidata 1200 (Axon Instruments), and stored in a personal computer. pClamp8 software (Axon Instruments) was used to control the amplifier and to acquire the data. The cell membrane was held at 0 mV and ramped from –100 to 100 mV over 1 s. The pipette (or intracellular) solution contained 116 mM KOH, 4 mM KCl, 10 mM HEPES, 2 mM EGTA, and 2 µM CaCl2, and was adjusted to pH7.2 with MES. The bath (or extracellular) solution contained 116 mM NaOH, 4 mM KCl, 10 mM HEPES, and 2 mM EGTA (pH 7.2), and was perfused throughout the experiment. In a serial change of extracellular K+ concentrations, K+ was substituted by isotonic concentrations of other ions such as Na+ or NMDG+ (N-methyl-D-glucamine).
Inside-out patch clamp recording of SKCa, IKCa, and BKCa channels
Ionic currents carried by rSK2 (wild-type and mutants), hIK, and rSlo channels were recorded from patches of Xenopus oocyes or CHO cell membrane in the inside-out configuration. Patch recordings were made at room temperature (23°C) 3–7 days after injection or 1–3 days after transfection. Preparation of patch pipettes and electrophysiological instruments were the same as described above. To measure the reversal potentials under the bi-ionic Na+ and NMDG+ conditions, pipette (or extracellular) solutions were prepared to contain 120 mM NaOH, 10 mM HEPES, 2 mM EGTA, and 4 mM HCl, or 120 mM NMDG, 10 mM HEPES, 2 mM EGTA, and 4 mM HCl. The membrane was held at 0 mV and ramped from –130 to 50 mV for 0.8 s, and the intracellular solution contained 116 mM KOH, 10 mM HEPES, 2 mM EGTA, and 4 mM KCl. In macroscopic current recordings of rSK2 and hIK channels, the membrane was held at 0 mV and ramped from –100 to 100 mV over 1 s. For macroscopic current recordings of rSlo channels, the membrane was held at –140 mV and ramped from –50 to 150 mV over 1 s. Pipette extracellular solutions contained 10 mM HEPES, 2 mM EGTA, 116 mM KOH, and 4 mM KCl. Excised patches were perfused with an intracellular solution containing 10 mM HEPES, 2 mM EGTA, 4 mM HCl. For bi-ionic experiments using various monovalent cations, 120 mM K+ was substituted with the same concentrations of other monovalent cations, Na+, Li+, Rb+, 
or Cs+. Hydroxylated forms were used for all of the ions tested, which remain the concentration of both intracellular and extracellular solutions at 120 mM after adjusting the pH to 7.2 with MES and 4 mM HCl. To activate channel currents, the concentration of free Ca2+ in the intracellular solution was adjusted to 2 µM for SKCa, intermediate-conductance Ca2+-activated (IKCa), and BKCa K+ channels. Membrane patches bearing <30 pA of currents at –100 mV under symmetrical K+ in the absence of intracellular Ca2+ were used for bi-ionic experiments, and leaks were not subtracted.
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RESULTS |
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59 mV at room temperature (Fig. 1 A, inset, dotted line) for a perfectly selective K+ channel. The sub-Nernstian relationship between the reversal potential and the external K+ indicates that rSK channel has substantial permeability for Na+, the substituting ion.
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). When the intracellular solution was completely replaced by 120 mM NMDG+, the I/V relationship showed outward rectification and the currents reversed near –100 mV (NMDG:K) indicating that NMDG+ is virtually impermeable to rSK2 channel. However, subsequent replacement of extracellular solution to 120 mM Na+ gave a quite different outcome. We observed a significant amount of inward currents reversing around –60 mV (Na/K). Since the intracellular side of the membrane was continuously perfused with Ca2+-containing Na+ (or NMDG+) solutions, the internal accumulation of K+ from extracellular (pipette) solution should be negligible. In fact, the reversal potentials were independent of the direction of voltage ramps (data not shown). These results are not only surprising but also intriguing, since it has been generally assumed that native SKCa channels are highly selective for K+ and play a critical role in hyperpolarizing the membrane potential upon activation.
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and ionic currents were measured in the presence of 2 µM intracellular Ca2+. Fig. 2 A shows the typical current traces of ionic currents measured by a ramp pulse of –100 to 100 mV for each ion tested. Although most of the ions showed an inwardly rectifying relationship of I/V, internal 
exhibited outwardly rectifying currents at extreme positive voltages. We were able to estimate Erev accurately from expanded I/V curves such as those shown in Fig. 2 B.
From such traces, the relative permeability (PX/PK) of rSK2 was estimated for tested ions against K+ using Eq. 1,
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To confirm these results and rule out possible experimental artifacts, we measured the ion selectivity of BKCa channels, which belong to still another class of Ca2+-activated K+ channel with a quite different molecular structure and are proved to be highly selective for K+. The ion selectivity for BKCa channels was very different from that of either SKCa or IKCa channels. Under bi-ionic conditions using identical recording solutions, rat Slo channel, the -subunit of rat BKCa channel, gave the rank order of 
(Table 1). The permeability ratio of 0.01 estimated for Li+ and Na+ might be somewhat underestimated due to the large positive values of Erev >110 mV. Still, the permeability obtained using the rat Slo in this study is in a good agreement with a previous report for native BKCa channels (23).
In Fig. 3 A, the relative permeability of various monovalent cations was plotted against their ionic radii. Permeability of small ions is prominent for both SKCa and IKCa channels. Although K+ is >100-fold more permeable than Na+ for rSlo, the permeability of rSK2 channel for K+ is <10-fold better compared with that of Na+.
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Thus we asked whether we could increase the selectivity of the rSK2 channel for K+ over other monovalent cations by mutating the Leu residue to a Thr. L358T mutant channel was expressed well in Xenopus oocytes and activated by a submicromolar concentration of intracellular Ca2+ (Fig. 4 A). The Ca2+-activation curve of L358T mutant channel, with Hill coefficient n = 6.2 (half-activation constant K1/2 = 0.47), was similar to that of wild type (n = 5.1, K1/2 = 0.61) (Fig. 4 B). We then measured the ion selectivity of the mutant channel under bi-ionic conditions. The mutation did not improve the selectivity of SKCa channel for K+ over Na+ (Fig. 4, C and D) and the permeability ratios for Na+ and Li+ were almost identical to those of wild-type channels (Table 1). The most prominent effect of this Leu-to-Thr mutation was increased permeability for large cations, Rb+ and Cs+ (Fig. 4, C and D). In fact, Rb+ permeates much better than K+ in L358T mutant channel with PRb/PK of 1.45 (Table 1). It is also noteworthy that the current carried by shows a sharp outward rectification suggesting large conductance of through the mutant channel. Thus, the higher selectivity for K+ was not achieved by specific substitution of the hydrophobic residue near the cytosolic end of the K+-selectivity filter to a conserved hydroxyl-bearing residue alone.
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), the residue should also be involved in the permeation of K+ and other monovalent caions. In the case of the KcsA channel, the hydroxyl group of a Thr-residue (T74 in Fig. 3 C) counterpart of Ser-359 in rSK2 channel is involved in the direct coordination of K+ at the most cytosolic site ("Site 1") of the four K+-binding sites in the selectivity filter (24,25).
The current traces of two different mutants, S359A and S359T, are shown in Fig. 5, A and B, and C and D, respectively. The Ser-to-Ala mutation rendered significant changes in the selectivity for monovalent cations (Fig. 5 E). First of all, Rb+ was almost as permeable as K+ in S359A channels. Second, the permeability of smaller ions increased dramatically; the relative permeability of S359A for Na+ increases from 0.12 to 0.40 (Table 1). The apparent conductance of small ions, Li+ and Na+, also increases significantly. Third, the permeability of lager ions was increased slightly but significantly, from 0.19 to 0.26 for Cs+ (Table 1). When the Ser-359 was replaced with a conserved Thr residue, the permeation characteristics of wild-type channel were restored and only a minor change was observed in both selectivity and conductance among different monovalent cations (Fig. 5 E and Table 1). Thus, these results indicate that the hydroxyl group at Ser-359 is critical not only for high-affinity interaction of intracellular divalent cations as shown in a previous study (21) but also for size selectivity among permeant ions of monovalence.
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DISCUSSION |
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,27). A stretch of amino acids, the K+-channel signature sequence, determines the structure of the selectivity filter and provides the carbonyl oxygens that create the K+-binding sites (24,25,28). As members of the K+-channel family, the SKCa channels contain the amino acid sequence TFLSIGYG (14), highly homologous to the canonical "K+-channel signature", TXXTXGYG (29). The high selectivity for K+ is believed to be critical for the presumed physiological function of SKCa channels in inducing membrane hyperpolarization and modulating neuroexcitability. Although SKCa channels are known to underlie the prolonged afterhyperpolarization (or slow AHP) after trains of action potentials, AHP differs among tissues in time course (30,31) and pharmacology (32,33).
Based on the measurements of reversal potentials under bi-ionic conditions, we determined the selectivity of rSK2 channel for monovalent cations. Calculated permeability ratios (PX/PK) for the wild-type rSK2 yielded the order of 
(Table 1). The channel exhibits surprisingly high permeability for Li+ and Na+. Moreover, the IKCa channel, which is highly homologous to the SKCa channel, also shows a similar Na+ permeability. The permeability (PX/PK) of native SKCa currents was determined in chromaffin cells (34) and reported as 
markedly in contradiction to the results reported here. At this point the cause for this discrepancy is unclear. However, we wonder whether the endogenous SKCa currents in the previous study were properly measured for bi-ionic Na+ versus K+ and Li+ versus K+ conditions, since rapid run-down occurred after replacement of external K+ with Na+ or Li+ and the resulting currents were extremely small (<40 pA). We experienced no such run-down of rSK2 currents after serial replacement with various extracellular monovalent cations.
In an attempt to rationalize the high Na+ permeability in terms of amino acid sequence near the K+-selectivity filter, we compared the amino acid sequence of the P-regions in SKCa channels to that in other K+ channels (Fig. 3 B). Although both SKCa and IKCa channels contain a virtually perfect K+-channel signature sequence in their pore, the selectivity filter, or K+-binding sequence SIGYG, is preceded by a hydrophobic residue, Leu, instead of Thr or Ser in other K+ channels. Whereas three different residues, Thr, Ala, and Ser, at this position produced K+-selective channels, the mutation to Gly or Asn failed to express functional Shaker K+ channels (35). Intriguingly, the hyperpolarization-activated channel, HCN4, which is much less selective for K+, also has Leu at the corresponding position (36). However, the simple replacement of Leu with Thr in rSK2 failed to restore the high K+ selectivity seen in other K+ channels. Rather, L358T increased the permeability of larger ions, Rb+ and Cs+. It is also worth pointing out that the permeability of Rb+ becomes higher than K+ in the mutant channel. Although it is impossible to interpret these results without structural information at a high resolution, we can appreciate the fact that the identity of neighboring residues near the selectivity filter and the subtle changes in protein conformation in this area can influence permeation properties of ion channels, as observed in many previous studies.
The most important aspect of the results we report here is the physiological significance of the Na+ permeability of SKCa channels. Under the physiological ionic condition, i.e., 145 mM Na+ and 5 mM K+ in the extracellular milieu versus 12 mM Na+ and 140 mM K+ in cytoplasm, the relative permeability of Na+ (PNa/PK) measured for homomeric SK2 channel, 0.12, yields a membrane Erev of –46 mV. This value is significantly less negative than the reversal voltage that might be expected for afterhyperpolarization currents, IAHP. In recent studies using knock-out models and a dominant negative SKCa channel subunit, the SKCa channel was shown to underlie only the apamin-sensitive component of the medium-duration AHP and none of the three SKCa channel subunits contributes to the longer-lasting sAHP (12,13).
One intriguing possibility is the involvement of Na+-activated K+ channels in the generation of sAHP. Although the relative conductance of Na+ (GNa/GK) is small compared to that of K+, estimated as 0.11 based on the slope conductance at 0 mV under bionic condition (Fig. 2, A and B), we expect that the opening of SKCa channels allows significant Na+ permeation in the negative voltage range. In turn, the influx of Na+ might evoke Na+-activated K+ (KNa) channels that are known to express in many neuronal cells (37,38). These channels exhibit diverse characteristics depending on cell type and recording condition. Single-channel conductances of KNa channels are large (100–200 pS) and the channels are activated by tens of millimoles of intracellular Na+. Although KNa channels have been implicated in an apamin-insensitive and Na+-dependent slow afterhyperpolarization after a burst of action potentials (37), it remains questionable whether the concentration of intracellular Na+ can increase enough to activate the channel. Recently, the products of two different genes, slack and slick, were proposed as the subunits for Na+-activated K+ channels (39,40). Both Slack (Slo2.2) and Slick (Slo2.1) are highly expressed in the brain and generate Na+-activated K+ currents in heterologous systems. Thus, it will be critical to determine whether native SKCa channels also show significant permeability for Na+ under physiological condition and whether the local concentration of cytosolic Na+ can be high enough to activate Na+-activated K+ currents, especially in neuronal cells. It also remains to be shown whether the heteromerization of different subunits affects the selectivity of SKCa channel.
In summary, we determined the relative permeability of a cloned SKCa channel, rSK2, and recognized the significant permeability of Na+ through this channel. Although the mutations at the pore-forming region mimicking other K+-selective pores altered the size selectivity for permeant ions, they failed to restore a high K+ selectivity for SKCa channel. Since the permeability for Na+ determines the reversal potential of endogenous SKCa current in a physiological ionic environment, the ion selectivity of native SKCa channels should be determined and the role of Na+ permeability in neuronal excitability needs to be investigated.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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This work was supported by research grant's from the Ministry of Science and Technology of Korea, 21C Frontier (03K2201-00320) to C.S.P. and Systems Biology (M1050 301001) to D.H.K.
Submitted on July 1, 2005; accepted for publication August 16, 2005.
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  L. Grant and P. Fuchs Calcium- and Calmodulin-Dependent Inactivation of Calcium Channels in Inner Hair Cells of the Rat Cochlea J Neurophysiol, May 1, 2008; 99(5): 2183 - 2193. [Abstract] [Full Text] [PDF]
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 D. Bichet, M. Grabe, Y. N. Jan, and L. Y. Jan Electrostatic interactions in the channel cavity as an important determinant of potassium channel selectivity PNAS, September 26, 2006; 103(39): 14355 - 14360. [Abstract] [Full Text] [PDF]
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) and L358T mutant channel (•). Each data point was plotted against [Ca2+]i and fitted to a Hill equation. Half-activation constants (K1/2) and Hill coefficients (n), respectively, were estimated as 0.61 µM and 5.18 for wild-type rSK2, and 0.47 µM and 6.23 for L359T. (C) Representative traces of macroscopic currents measured from excised inside-out patches containing L359T mutant channel in K+ versus various monovalent cations. The currents were evoked by a ramp pulse from –100 to 100 mV in 1s. All recording solutions contained 120 mM of testing ions and the intracellular solutions included 2 µM Ca2+. Each current trace of different extracellular monovalent cation was indicated by an arrow. The current traces in expended scales were also shown (D). (E) Effect of mutation on size selectivity of rSK2 channel. Permeability ratios of wild-type rSK2 and L358T mutant were coplotted against the radius of each monovalent cation.