Na -induced inward rectification in the two-pore domain K channel, TASK-2

轻羽

School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom% e7 b- x7 y7 m/ J3 v4 J1 f

+ w6 D/ ^6 I9 l; Q; f% t0 h' AABSTRACT
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  R6 `% @# Z! t( E2 A$ e% w' KTASK-2 is a member of the two-pore domain K  (K2P) channel family that is expressed at high levels in several epithelia, including the proximal tubule. In common with the other TASK channels, TASK-2 is sensitive to changes in extracellular pH. We have expressed human TASK-2 in Chinese hamster ovary cells and studied whole cell and single-channel activity by patch clamp. The open probability of K2P channels is generally independent of voltage, yielding linear current-voltage (I-V) curves. Despite these properties, we found that these channels showed distinct inward rectification immediately on the establishment of whole cell clamp, which became progressively less pronounced with time. This rectification was due to intracellular Na  but was unaffected by polyamines or Mg2  (agents that cause rectification in Kir channels). Rectification was concentration- and voltage-dependent and could be reversibly induced by switching between Na -rich and Na -free bath solutions. In excised inside-out patches, Na  reduced the amplitude of single-channel currents, indicative of rapid block and unblock of the pore. Mutations in the selectivity filter abolished Na -induced rectification, suggesting that Na  binds within the selectivity filter in wild-type channels. This sensitivity to intracellular Na  may be an additional potential regulatory mechanism of TASK-2 channels.
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intracellular sodium; voltage dependent; pore block
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- g  O  w) }/ j* BTASK-2 IS A MEMBER OF THE tandem-pore domain K  channel (K2P) family and is located in various epithelial tissues, including the pancreas, placenta, lung, small intestine, colon, and especially the kidney but is not found in the nervous system, skeletal muscle, or heart (23, 25). It displays 90% of the maximum current at pH 8.8, and only 10% at pH 6.5 (25). In addition to gating by pH, TASK-2 currents are inhibited by lidocaine and bupivacaine and potentiated by halothane and chloroform (6). TASK-2 is also sensitive to changes in the osmotic potential of the external medium and participates in cell volume regulation (1, 21). Recently, the importance of TASK-2 in NaHCO3 absorption in the proximal tubule has been established, and TASK-2-deficient animals have metabolic acidosis and hypotension (30).( |, d* c2 \* a3 `% [, |
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K2P channels are generally classified as background or "leak" K channels and exist as dimers, each subunit comprising four transmembrane domains (TMDs) and two pore-forming domains (16). Current flow through K2P channels is determined by the prevailing electrochemical gradient for K ; current-voltage (I-V) curves can be described by the GHK equation and, in the absence of a concentration gradient, they pass current equally well in both inward and outward directions. In vivo, where the intracellular concentration of K  is many-fold higher than extracellular, these channels would be predicted to show mild "outward rectification" (4). The open probability is independent of voltage and currents, with the exception of TWIK-2 (26), which are noninactivating; thus K2P channels are open at the resting membrane potential (RMP) and are involved in the determination of the RMP.8 l3 t+ V8 K. p! {7 \
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Despite the above published properties, in an unrelated series of experiments, we noticed that currents in Chinese hamster ovary (CHO) cells heterologously expressing TASK-2 showed weak inward rectification immediately on the establishment of whole cell clamp. However, this rectification became progressively less pronounced with time. Inward rectification is a functional characteristic of Kir channels, which preferentially conduct K  ions into the cell at membrane potentials negative to the K  equilibrium potential (EK), whereas currents at voltages positive to EK are substantially reduced due to blockage of the pore by intracellular Mg2 , or polyamines such as spermine and spermidine (20). Thus we supposed that the transient rectification observed in our experiments was due to the initial presence, and subsequent loss, of one of these cations. The following experiments were carried out to determine the basis of this rectification.5 H0 [" K  e7 d" c/ X- x

  b8 F7 R( @/ {) c* r6 WMATERIALS AND METHODS# n: s0 K1 U2 q" W' _% y! I
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Expression of mouse TASK-2 and cell culture. TASK-2 (GenBank accession no. AF319542) was cloned from mouse kidney into the bicistronic mammalian expression vector pIRES-GFP (Clontech). The GFP coding sequence was replaced with the coding sequence for hCD8 antigen, which is expressed on the cell surface of transfectants. When TASK-2 is expressed from this vector, CD8 is expressed too, and transfected cells could be identified by their ability to bind anti-CD8 immunomagnetic beads (Dynal). CHO-K1 cells were routinely cultured in F-12 medium supplemented with 10% fetal calf serum, 200 U/ml penicillin, and 200 μg/ml streptomycin. Cells were transiently transfected using Fugene 6 transfection reagent according to the manufacturer's instructions (Roche). Cells were dissociated by 5-min incubation in Ca2 -free PBS 24–48 h posttransfection and incubated for a further 15 min with anti-CD8 beads. Beaded cells were isolated by magnetic separation and plated onto coverslips. Cells were subject to whole cell patch-clamp analysis within 72 h of transfection (8). CD8 expression in individual cells was confirmed visually at this stage by the adherence of three or more beads; the beads did not appear to interfere with seal formation., j& q' \1 e! e: U' p  s
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Generation of mutants. A G102A/G207A double mutant of mouse TASK-2 was generated in two steps by the QuikChange site-directed mutagenesis technique (Stratagene) and confirmed by automated fluorescence DNA sequencing (Lark).; I! h# p5 N- E/ G8 K

% I$ v+ A0 }& i9 x. yElectrophysiology and solutions. Whole cell currents were recorded in either Na -rich or Na -free (NMDG) mammalian Ringer with a K -rich pipette solution. Bath solutions consisted of the following (in mM): 1) Na -containing Ringer (145 NaCl, 4.5 KCl, 1 MgCl2, 2 CaCl2, 5 HEPES, and 5 PIPES, pH 7.8); and 2) Na -free Ringer, as above with NaCl substituted by 145 NMDG-Cl or KCl. Pipette solutions consisted of 135 K-gluconate, 1 MgCl2, 1 K2ATP, 0.98 CaCl2, 5 EGTA, and 5 HEPES, pH 7.2. The calcium activity of this solution was 10 nM as determined by the React program (Godfrey Smith, Glasgow University). The polyamines spermine (100 μM and 1 mM), spermidine (1 mM), and putrescine (100 μM) and Na  (5–50 mM) were added to pipette solutions without osmotic compensation. Polyamines bind to ATP, potentially reducing their free concentration; however, at least for spermine and spermidine, where the binding constants have been determined (31), we calculated (using React) that the amount of polyamine bound to ATP was only 4 and 2%, respectively. For single-channel experiments, the pipette contained Ringer solution, and the bath was either nominally Na  free (in mM: 115 K-gluconate, 20 KF, 10 KCl, 5 MgCl2, 5 K2ATP, 0.61 CaCl2, 5 EGTA, and 10 HEPES, pH 7.2, 10 nM free Ca2 ) or 50 mM Na  (as above, with 50 mM K  replaced with Na ). Recordings were low-pass filtered at 2 kHz and sampled at 5 kHz. All chemicals were from Sigma. Currents were recorded over the range –100 to  100 mV using either a voltage pulse or voltage ramp protocol.  |, w6 y& o( B% ^( o% j& m

6 a6 k! L" d6 r6 u0 R' OAnalysis and statistics. Results are given as means ± SE, and significance was assumed at the 5% level (P 5 c* u0 a8 [, F  \$ w
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Expression of wild-type TASK-2. As expected, CHO cells transiently transfected with mouse TASK-2 expressed an outwardly rectifying current sensitive to changes in extracellular pH, as shown in Fig. 1. In general, immediately after whole cell patch clamp was achieved, initial traces showed a rectification, consistent with voltage-dependent block of outward currents at positive potentials (Fig. 2). With time this rectification diminished and the I-V curves became much more linear, as expected for TASK-2 (Fig. 2) (25). We supposed the loss of rectification was due to loss of an intracellular inhibitor such as a polyamine (17) as the cell became dialyzed with the pipette solution.+ L# ~" j2 b  E
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Effect of polyamines and Mg2  on TASK-2. To identify the intracellular mediator of this weak inward rectification, we first examined whole cell currents in the presence of different polyamines and Mg2 , known mediators of inward rectification in Kir channels (17). Neither polyamines nor Mg2  caused significant channel block at any of the concentrations tested (Fig. 3). The percent inhibition values at 100 mV were –1.51 ± 3.0 for 100 μM putrescine, 1.51 ± 7.0 for 1 mM spermine, 3.53 ± 7.0 for 1 mM spermidine, and 2.12 ± 1.0 for 20 mM Mg2 .
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3 n+ h: d" \9 n3 QEffect of Na  on TASK-2. Another cation previously shown to cause inward rectification in BK (calcium-activated) and delayed rectifier K  channels is Na  (13, 18, 33). We therefore examined whether Na  contributes to the weak inward rectification in TASK-2. When Na  was added to the pipette solution, a concentration- and voltage-dependent block of whole cell currents was observed (Fig. 4). In each case, the observed block was statistically significant. The block was most pronounced at positive potentials.
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' m" z, ]. `3 uTo better understand the disappearance of rectification with time (Fig. 2), we supposed that this initial inhibition was due to the existence of a relatively high resting intracellular Na  concentration and that this was washed out after the establishment of whole cell clamp with a Na -free pipette solution. To reduce intracellular Na , TASK-2-transfected CHO cells were incubated for 1 h in Na -free (NMDG) Ringer solution and subjected to whole cell patch clamp with 50 mM Na  in the pipette. Initially, I-V plots were linear. However, rectification developed with time, consistent with diffusion, and a gradual increase in the cytosolic concentration, of Na  from the pipette into the cell (Fig. 5). We have attempted to simulate this result with a simple model that incorporates the time-dependent increase in Na  as it diffuses into the cell from the pipette, combined with a voltage-dependent block. The increase in Na  was calculated according to Pusch and Neher (Eq. 17 in Ref. 24), assuming a Na  diffusion coefficient of 131 x 10–11 m2/s, a cell capacitance of 10 pF, and an access resistance of 15 M. The open probability was calculated using the Woodhull equation (32) assuming a Kd at 0 mV of 250 mM and a binding site that senses 75% of the membrane potential (an electrical distance of 0.75). The unblocked currents were calculated from the GHK equation (11), the permeability chosen to give current amplitudes similar to those shown by the cells, and a leak conductance was included to enable the reversal potential to differ from EK. The inset to Fig. 5 shows the result of these calculations for successive ramps at the same time interval, 5 s, as was used in experiments. Although the results are not described perfectly well by this model, the general form, and the time course in particular, is similar.
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Following on from the finding that cells displayed spontaneous rectification, we hypothesized that this Na  must be derived by influx across the cell membrane; i.e., the steady-state intracellular Na  concentration would reflect the relative magnitudes of Na  influx and efflux pathways. Surprisingly, we found this also to be the case with cells during conventional whole cell clamp. Figure 6 shows the reversible development of rectification as the bath solution was alternated between Na -free (NMDG) and Na -containing Ringer. Thus intracellular Na  varies with the extracellular concentration, and the inference is that even in the nominal absence of Na  in the pipette, Na  influx elevates the cytosolic or subplasmalemmal Na  concentration sufficiently to produce rectification.$ g6 s5 ^% J2 d
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Single-channel studies. To seek further evidence that intracellular Na  was responsible for the rectification seen in the whole cell experiments, we made single-channel recordings from excised, inside-out patches, in which the "cytosolic" or intracellular face of the channel is exposed to the bath solution. With an outwardly directed K  gradient, single-channel events were evident in the absence of an applied voltage (Fig. 7), as expected for a K -selective channel. These currents were rapidly and reversibly inhibited by 50 mM Na . Single-channel I-V relationships show that with a Na -free bath solution, currents increased monotonically as the potential was made more positive. However, with 50 mM Na  in the bath, the single-channel currents increased from –40 to  20 mV but became progressively smaller at more positive potentials, indicating a voltage-dependent block, as seen in the whole cell studies.
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What is the mechanism of rectification The voltage dependence of Na  block suggests that it is acting as a pore blocker, its binding residing within the membrane voltage field and probably within the pore. In the strongly rectifying K  channel, Kir 2.1, several residues, particularly an aspartate residue (D172) present in the pore-lining second TMD, have been shown to be critical for the binding of polyamines and Mg2  and in the development of rectification (5, 28). The signature sequence (P-loop) and subsequent TMDs of mouse TASK-2 and Kir2.1 (a strong inward rectifier) were aligned using the CLUSTAL algorithm. There was very little amino acid sequence identity (12%), and there was no charged residue equivalent to D172 of Kir2.1; the only negatively charged residues close to that of aspartate (D) 172 of Kir 2.1 are glutamate (E) at positions 228 and 251 in mouse TASK-2 TMD 2 and COOH terminus, respectively; these are located 4 residues upstream and 20 residues downstream of the tyrosine residue, Y232, equivalent to D172 in Kir2.1. Of these two, residue 228 was potentially interesting because homology modeling suggests that it should face the pore cavity and is only one helical turn above the residue equivalent to D172 of Kir2.1 (22), whereas residue 251 is predicted to lie outside the conduction pathway (14). Nevertheless, because of their proximity, both of these residues were mutated to the neutral amino acid glutamine (Q). Whole cell currents for E228Q and E251Q recorded in the presence of 50 mM Na  in the pipette showed a weak inward rectification indistinguishable from that of wild-type TASK-2 (data not shown).
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Perhaps Na  is blocking within the selectivity filter The selectivity filter comprises a TXGXG motif and is critical to potassium channel function. Previously, in Shaker voltage-gated channels, mutation of either glycine to alanine resulted in the loss of selectivity between K  and Na  (9). We have introduced mutations of the second glycine in both pore regions of TASK-2 (the double mutant G102A/G207A). As expected, the channel displays little K  selectivity with respect to Na  but retains some cation selectivity (Fig. 8); the calculated PK/PNa ratio was 1.4:1, and the cation/anion selectivity was 5.2:1 . Of more importance to this study, however, is that the channels were no longer blocked by Na ; 50 mM Na  was present in the pipette solution in all traces in Fig. 8, but there was no rectification.' H, G& g, ^+ K! ?, `% ~
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Finally, we determined whether Na -induced rectification was affected by pH. To test this, we collected ramp-generated whole cell I-V curves, with 50 mM Na  in the pipette, as the bath solution was switched between pH 7.8 and 5.8 (Fig. 9). In this experiment, we deliberately slowed the bath flow rate to extend the bath exchange time to 1 min. Figure 9A shows the currents initially at pH 7.8, and the subsequent inhibition as the bath became more acid; although the bath pH was not determined during this procedure, it must lie between pH 7.8 and 5.8. Figure 9B shows the same data after leak subtraction, normalized to the currents at pH 7.8; in this case, we have assumed that at pH 5.8 most of the remaining current was leak. The juxtaposition of the traces in Fig. 9B indicates that the degree of block is independent of pH over this range.1 F- a- ]; U. ?9 ^
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DISCUSSION, i8 }1 w& ~- k' ?7 k! \3 u5 X
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TASK-2 is a member of the recently discovered K2P channel family and is highly expressed in epithelia. Typically, the TASK subfamily show mild outward rectification of K  currents that approximate those predicted by the GHK equation. These channels are open at the RMP of the cell and therefore act as molecular sensors, translating changes in the composition of the extracellular fluid to a change in membrane potential. K2P channels are gated by a variety of external factors. In the case of TASK-2, these are the pH and osmotic potential of the external milieu.1 {& i; q) b  ], ]4 l+ f* _. L- c. e3 m

3 R# z/ Z4 w0 W. N( GIn general, immediately after achieving whole cell clamp, we would observe a weak inward rectification at depolarizing potentials in excess of 40 mV or so, where outward currents would decrease in a voltage-dependent manner (Fig. 2). This weak inward rectification would disappear within 1 min, leaving an almost linear I-V plot consistent with GHK kinetics. We supposed that this rectification was due to the initial presence, and subsequent disappearance, of an intracellular factor. Given that cations such as Mg2  or polyamines cause rectification in Kir channels, we began by investigating the effect of these cations on wild-type TASK-2 currents. As can be seen in Fig. 3, despite the inclusion of polyamines or Mg2  at concentrations that would cause significant rectification in Kir channels (17), we observed no rectification of TASK-2 currents.
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* N' l" N3 n" |: |! Q/ X6 B+ O- z9 ~A less well-known endogenous blocker of K  channels is Na ; block by intracellular Na  has been observed in Ca2 -activated and delayed rectifier K channels (13, 18, 33). In the current study, it can be seen clearly that there was a concentration- and voltage-dependent block of the channel (Fig. 4)., M) }3 b5 D2 a% z" r+ a- g: d

. p) K0 p( e# J3 `, s# iWas the block seen immediately on establishment of whole cell clamp due to intracellular Na  We investigated this question by incubating cells in Na -free (NMDG) Ringer for 1 h before beginning patch experiments; after this time, we assumed the cells were nominally Na  free. Cells were then subject to patch clamp with a pipette solution containing 50 mM Na  (Fig. 5). Immediately after whole cell clamp was achieved, TASK-2 currents were essentially linear. However, rectification developed over 60 s or so, presumably as Na  diffused from the pipette into the cell. Another interesting feature of Na  block, apparent from inspection of Figs. 5 and 2, is that block is apparent over a wider voltage range than is immediately apparent from the composite Fig. 4, where we normalized the data to the slope conductance between –60 and –40 mV. However, we can see from Figs. 2 and 5 that block is also apparent in the negative-voltage range. This is confirmed in the single-channel recordings (Fig. 8).
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" T; Q' M, Y8 v1 d9 e+ TThe effect of Na  was reversible in both whole cell and inside-out patch configurations. In the whole cell configuration, cells were studied with a Na -free pipette solution, and the bath solution alternated between Na -containing Ringer and Na -free (NMDG) Ringer. In the absence of Na , the currents showed little or no rectification. When Na  was introduced to the bath, it presumably entered the cells by native Na -entry pathways and hence caused a weak inward rectification (Fig. 6). This effect was fully reversible when the bath solution was switched back to Na  free. This reversibility was also evident in inside-out patches (Fig. 7).: P7 b: d6 y& l# e* P% `9 i- X
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What is the mechanism of Na  block We considered, first, the rectification mechanism in Kir channels, in which polyamines and Mg2  apparently bind within the inner vestibule to negatively charged residues in TMD 2 (D172) and the COOH terminus (E224 and E299) (5, 28, 29). Neutralization of any of these residues has been shown previously to reduce significantly the rectification caused by polyamines or Mg2 . Alignment of TMD2 of Kir2.1 and its equivalent TMDs in TASK-2 showed very little sequence identity. Importantly, there was no residue in TASK-2 equivalent to D172 of Kir2.1. The closest negatively charged residues in the sequence were glutamate residues at position 228, within TMD2, and 251, in the COOH terminus, predicted to be intracellular. Both residues were mutated to the neutral amino acid glutamine. Cells transfected with each mutant were subject to patch clamp with pipette solutions containing 50 mM Na . In each case, mutant channels still showed weak inward rectification indistinguishable from wild type. These findings, together with the lack of effect of polyamines or Mg2 , suggest that the weak inward rectification in TASK-2 occurs by a mechanism different to that seen in Kir channels.
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A possible model for the mechanism by which Na  causes inward rectification is by pore block. According to this model, Na  may be able to bind to ion binding sites normally occupied by K . Although a dehydrated Na  ion is smaller than an equivalent K  ion, in the archetypal K  channel KcsA, Na  cannot coordinate with the backbone carbonyl groups within the selectivity filter, and Na  does not enter the selectivity filter, even when in huge excess (35). However, the asymmetry of pore residues in K2P channels allows for rather more flexibility with respect to cation permeability, and Na  entry to the selectivity filter may occur (3). In this case, differences in the binding energies of Na  and K  would disrupt the normal balance between electrostatic repulsion and ion binding; the poorly bound Na  ion would be unable to displace the more tightly bound K  ion, or vice versa, such that the selectivity filter would effectively become blocked (10). To investigate this hypothesis, we made mutations in the selectivity filter in an attempt to relieve pore block by Na . Previously, it has been shown that mutation of either glycine residue in the pore of the Shaker K channel gives a nonselective cation channel that does not discriminate between Na  and K. We mutated the second (outermost) glycine residue of both TASK-2 pore domains (G102A/G207A double mutant). The resulting I-V plots were approximately linear in both Na  and K  Ringer and had reversal potentials close to 0 mV, demonstrating the nonselective nature of the mutated channel (Fig. 8). In this case, the pore is now capable of conducting Na  and, even with 50 mM Na  in the pipette, there was no rectification observed. These experiments suggest that in wild-type TASK-2 channels, Na  is able to enter the selectivity filter from the cytoplasm but is either unable to pass through the filter or is conducted at a much lower rate than K , which thereby reduces current flow.
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Further information concerning the site of the block may be inferred from the voltage dependence of the block. Fitting the whole cell and single-channel data at 50 mM Na  with the Woodhull equation gives best fit values for electrical distance () of 0.74 ± 0.06 and 0.79 ± 0.04, respectively. Thus the site at which Na  blocks is estimated to be 75–80% of the way across the membrane relative to the inside (32). This is similar to the electrical distance of 0.7 for Ba2  in Ca2 -activated K  channels (19), and later crystallographic studies in KcsA showed this binding site to lie at the innermost part of the selectivity filter, close to the pore cavity (12). Because the estimated electrical distance for Na  in the current study is slightly greater than 0.7, this would tend to indicate that Na  also binds within the selectivity filter but slightly externally to Ba2 .
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Na  is a fast blocker of TASK-2. Intracellular Na  apparently acts as a fast blocker of TASK-2 channels (11). This is indicated by the apparent reduction in single-channel current amplitudes at positive potentials, where Na  presumably binds and unbinds from the channel at rates greater than the resolution of the patch-clamp amplifier. In agreement with the whole cell data, the progressive increase in the degree of block with depolarization indicates that Na  is binding within the pore (11).9 h( T1 E  T( r
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Is Na  block a physiological event The normal resting membrane potential of cells generally lies toward EK, i.e., between –70 and –80 mV, and the resting intracellular Na  concentration of 10–15 mM. Thus there would be little interference by intracellular Na  (/ U. B( D/ h/ k- z$ U3 q$ A
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In conclusion, intracellular Na  causes rectification of TASK-2 currents in a voltage- and concentration-dependent manner via pore block, probably within the selectivity filter. This sensitivity to intracellular Na  may be considered a potential regulatory mechanism of TASK-2 channels.- C0 O8 v" n0 w! ^3 }( t
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ACKNOWLEDGMENTS
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( k0 f- t0 t. l( j+ LWe thank the Wellcome Trust for generous financial support.
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; \! T  e- \! s! ~$ [  UThe costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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