(ROMK) to intracellular pH - NCBI

The EMBO Journal vol.15 no.16 pp.4093-4099, 1996

Identification of a titratable lysine residue that determines sensitivity of kidney potassium channels (ROMK) to intracellular pH B.Faklerl, J.H.Schultz, J.Yang2, U.Schulte, U.Brandle, H.RZenner, L.Y.Jan2 and J.RRuppersberg' Department of Sensory Biophysics, ENT-Hospital of the University of Tubingen, Rontgenweg II and Department of Physiology, GmelinstraBe 5. 72076 Tilbingen, Germany and 'Howard Hughes Medical Institute, Department of Physiology. University of California. San Francisco, CA 94143. USA

'Corresponding authors Potassium (K+) homeostasis is controlled by the secretion of K+ ions across the apical membrane of renal collecting duct cells through a low-conductance inwardly rectifying K+ channel. The sensitivity of this channel to intracellular pH is particularly high and assumed to play a key role in K+ homeostasis. Recently, the apical K+ channel has been cloned (ROMK1, 2,3 = Kirl.la, Kirl.lb and Kirl.lc) and the pH dependence of ROMK1 was shown to resemble closely that of the native apical K+ channel. It is reported here that the steep pH dependence of ROMK channels is determined by a single amino acid residue located in the N-terminus close to the first hydrophobic segment MI. Changing lysine (K) at position 80 to methionine (M) removed the sensitivity of ROMK1 channels to intracellular pH. In pH-insensitive IRK1 channels, the reverse mutation (M84K) introduced dependence on intracellular pH similar to that of ROMK1 wild-type. A detailed mutation analysis suggests that a shift in the apparent pKa of K80 underlies the pH regulation of ROMK1 channels in the physiological pH range. Keytords: intracellular pH/inward-rectifier potassium channels/kidney/pK, shift/ROMK

Introduction Potassium (K+) homeostasis is controlled by the secretion of K+ ions across the apical membrane of cortical collecting duct cells in the kidney. Low-conductance (35 pS) inwardly rectifying K+ channels were identified as the channels primarily responsible for K+ secretion (Wang et al., 1990, 1992). These low-conductance K+ channels have been shown to be particularly sensitive to changes in intracellular pH (pH-) (Wang et al., 1990; Wang and Giebisch, 1991). Intracellular acidification in the physiological range reversibly reduced channel open probability and is thought to account for the subsequent decrease in K+ secretion (Wang et al., 1990). Thus, the sensitivity of the apical K+ channel to intracellular pH is assumed to play a key role in K+ homeostasis (Wang et al., 1992; Wright and Giebisch, 1992). Efforts to identify channel molecules responsible for renal K+ secretion initially resulted in the cloning of © Oxford University Press

ROMKl (Ho et al., 1993) and, subsequently, in cloning of ROMK2 and ROMK3 (Zhou et al., 1994; Boim et al., 1995) which are splice variants of ROMK1. Splicing results in N-terminal variations: ROMK2 lacks the first 19 amino acids (aa) of ROMK1, while ROMK3 contains a seven amino acid extension. All three clones [ROMK1, 2, 3 = Kirl.la, Kirl.lb and Kirl.Ic according to Doupnik et al. (1995)] encode weak inward-rectifier K+ channels. were shown to be expressed in renal tubular cells (Boim et al., 1995; Lee and Hebert, 1995) and are presently believed to represent the native apical K+ channels. ROMK channels are members of a superfamily of structurally and functionally related K' channel proteins (Kir channels) (Ho et al., 1993; Kubo et al., 1993a,b; Ashford et al., 1994; Bond et al., 1994; Lessage et al.. 1994). As deduced from the primary sequence, these K+ channel subunits are made up of hydrophilic amino and carboxyl termini which flank a well-conserved core region. The latter consists of two hydrophobic segments (M 1 and M2) flanking a putative P-region. which is highly homologous to the P-region found in KV-type and Ca2dependent K' channels (Stuihmer et a!., 1989; Butler et al., 1993). Similar to K,-type K' channels, Kir channels assemble as tetramers (Glowatzki et al., 1995; Yang et al., 1995) and may be either homo- or heteromeric channel proteins (Duprat et al., 1995; Glowatzki et al., 1995; Kofuji et al., 1995; Krapivinsky et al., 1995). The common functional property shared by the Kir channels is their inwardly rectifying current-voltage relationship (I-V). Rectification may be weak or strong and is due to a voltagedependent block of the channel pore by intracellular magnesium (Matsuda, 1991) and polyamines (Fakler et al., 1994b, 1995; Ficker et al., 1994; Lopatin et al., 1994). Among the members of the Kir family cloned so far. regulation by pH, has only been described for ROMK1 channels (Tsai et al., 1995). Dependence of the ROMK1 slope conductance on pHi was found to be positively cooperative and intracellular histidine residues were assumed to be involved in high pH sensitivity (Tsai et al., 1995). This paper describes the structural determinant and molecular mechanism underlying the steep dependence of Kirl. 1 channels on intracellular pH.

Results ROMK channels are strongly dependent on intracellular pH The response of ROMK1 channels to changes in pHi in the physiological range is illustrated in Figure 1 A. Bicarbonate-induced intracellular acidification (from about pHi 7.6 to pH- 6.6) of Xenopius oocytes expressing ROMK 1 channels reversibly decreased currents mediated by ROMK1 (Figure IA) and ROMK2 channels (not shown) 4093

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Fig. 1. ROMK channels are strongly dependent on intracellular pH. (A) Intracellular acidification induced by 90 mM KHCO3 and measured by a pH-sensitive microelectrode (lower panel) reversibly inhibited ROMK1-mediated currents in Xenopus oocytes. Cuffents in response to voltage ramps from -120 to 50 mV in 20s were recorded from whole oocytes by two-microelectrode voltage clamp. (B) ROMKI- and (C) ROMK2-mediated currents exhibit similar steep pH dependence in giant inside-out patch-clamp recordings. Currents were measured at a potential of -80 mV which was stepped to 50 mV for 50 ms every 1.0 s. Experiments were performed in the presence of 1 ,uM SPM on the cytoplasmic side of the patches. Zero current level in (A-C) is given by horizontal bars; scaling of time and current is as indicated. (D) pH dependence of ROMKI- and ROMK2mediated currents on intracellular pH. Lines represent the results of equation (1) fitted to the data (mean of 10 and nine experiments, respectively). The value of pKO5 as yielded by the fit was 6.89 and 6.81 for ROMK2 and ROMK1, respectively; the Hill coefficient was 3.9 for both splice variants.

measured in response to voltage ramps (from -120 to 50 mV in 20 s). For a quantitative analysis of the sensitivity of ROMK1 channels to pHi, we used giant inside-out patches, where solutions with well-defined pH values can be applied to the cytoplasmic side of the membrane. Figure lB and C shows currents recorded at a membrane potential of -80 mV (intermittently stepped to 50 mV for 50 ms every 1.0 s) at various pHi values and indicates the slow time course of pHi-induced current decrease. The single channel conductance did not change during pHi-induced current decrease, as observed at increased gain for the last active channel at pHi 6.0 (32.2 ± 2.1 pS, n = 4; data not shown). Moreover, Figure lB and C shows the close similarity in pH dependence of ROMK1 and ROMK2 channels. To minimize the influence of run-down (Fakler et al., 1994a; McNicholas et al., 1994), each pHi was applied for only -10 s. The steady-state inhibition of the current at all pH,s was then determined by monoexponential fits to the time course of inward current at each pHi and normalized with respect to the corresponding control current at pHi 8.0 immediately before each pHi application. Fitting equation (1) (see Materials and methods) to pHi-induced current inhibition resulted in a half-maximal inhibition (pKO5) at pH 6.81 and 6.89 for ROMK1 and ROMK2 channels, respectively (Figure ID). The Hill coefficient obtained by the fitting procedure was 3.9 for both ROMK splice variants (Figure ID), indicating that the process underlying pH regulation is highly cooperative.

pH dependence is determined by an N-terminal lysine residue In contrast to the highly pH-sensitive ROMK1 channels, IRK 1 [Kir2. 1 according to Doupnik et al. (1995)] channels 4094

were found to be much less sensitive to intracellular acidification (Figure 2A). To determine the position of residues which underlie pH regulation, we tested chimeric constructs where structural domains of ROMK 1 were introduced into the pH-insensitive IRK1. As shown in Figure 2B, pH sensitivity could successfully be conferred on IRK1 by replacing its N-terminus by that of ROMK1 [IR-RO(N)]. Chimeric channels in which the core region (hydrophobic segments Ml and M2 plus P-region) of IRKI was replaced by that of ROMK1 were as pH insensitive as IRKI wild-type, while the introduction of the ROMK1 C-terminus into IRKI did not result in expression of functional channels. The results shown in Figures 1 and 2 suggest that the steep pH dependence of ROMK1 channels is mediated by residues within the N-terminal part of the channel protein. No significant differences were observed from both splice variants tested (ROMK1 and ROMK2), indicating that the alternatively spliced region at the very N-terminus does not contribute to pH regulation. We therefore aligned the N-terminal regions adjacent to the hydrophobic segment Ml from several Kir subunits (Figure 3A). While most of this sequence is highly conserved among all known Kir members, three amino acid residues which are immediately adjacent to M1 in the ROMK sequence are less conserved: threonine (T82), methionine (M8 1) and lysine (K80) in ROMK, and valine (V86), leucine (L85) and methionine (M84) in IRK 1. Mutating each of these amino acids in ROMK1 [ROMK 1 (T82V), ROMK1 (M8 1L), ROMK 1 (K80M)] showed that changing lysine 80 to methionine [ROMK 1 (K80M)] removed steep pH dependence in whole-cell (Figure 3B) and inside-out patch experiments (Figure 3C). In contrast, ROMKI(T82V) and

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due to direct competition between spermine (SPM) and hydrogen ions, since a significant block in outward current was observed when the experiment was repeated in the absence of any blocking ion on the cytoplasmic side of the patches (n = 10; Figure 4C, lower trace). Thus, hydrogen ions in micromolar concentration block the channel pore in a voltage-dependent manner, which might also underly the residual pH dependence of the inward current in IRKI wild-type channels. To investigate the role of the lysine residue further, M84 in IRKI was changed to arginine (R) which is supposed to be as well protonated at physiological pH. However, injection of IRK1(M84R)-specific cRNA into oocytes did not lead to measurable K+ currents (not shown). The lack of channel function induced by R84 might be due to the size of the arginine residue. However, replacement of M84 by phenylalanine (F), which is almost equivalent in molecular size (Chothia, 1984), resulted in functional channels which were as pH insensitive as IRKl wild-type (Figure 4E). We next introduced histidine (H; pKa 6.0) at position 84 [IRK1(M84H)], which should be positively charged at low values of pHi. As shown in Figure 4D, IRKI(M84H) channels were as pH insensitive as IRKI wild-type in the pH range between 8.0 and 6.0 (Figure 4D). =

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Fig. 2. Strong pH dependence of ROMK1 is conferred on pH-insensitive IRKI by exchange of the N-terminus. Experimental conditions were as in Figure IA. (A) Intracellular acidification had almost no effect on IRK1-mediated currents, while currents mediated by IR-RO(N) (B) chimeric channels exhibit strong pH dependence. Note the similarly slow recovery of pHi and inward current in the experiment with IR-RO(N).

ROMK1(M81L) exhibited high pH sensitivity, as seen in ROMK1 wild-type (n = 5; data not shown). Analysing the weak pH effect observed in inside-out patches of ROMKI(K80M) showed that its pKO5 was 5.3 and that the pH dependence was much less steep, yielding a Hill coefficient of 0.5 (n = 5; Figure 3D). The loss of steep pH dependence in ROMK1(K80M) channels suggests that K80 either contributes to a distinct pH regulation site or that the K80M mutation caused a structural disturbance of the channel protein resulting in loss of pH sensitivity. Mutation of residue 84 to lysine introduces pH dependence into IRK1 channels To test for the role of this lysine residue, we introduced a lysine at the homologous position in IRKI channels [IRK(M84K)]. As shown in Figure 4, the resulting channels exhibited high sensitivity to intracellular acidification in whole-cell experiments (Figure 4A) and in inside-out patch recordings (Figure 4B). Quantitative analysis of this pH dependence indicated that IRK 1 (M84K) channels were slightly more sensitive to intracellular pH than ROMK1 (pKO5 7.1; Hill coefficient 3.6), and that recovery from pHi-induced inhibition was slower and less complete than in ROMK channels. The slight increase in outward current observed in inside-out patches with IRKl wild-type channels at pHi 6.0 (Figure 4C, upper trace) is probably

The side chain of residue 84 is accessible from the cytoplasm The results presented in Figure 4 raised the question of whether the side chain of residue 84 in IRKI is accessible to the intracellular solution, permitting interactions with hydrogen ions, or whether it is buried inside the protein structure. To test for accessibility of this side chain, we introduced a cysteine residue at position 84 [IRKI(M84C)] which can react with bath applied, positively charged ethylammonium-methanethiosulfonate (MTSEA) (Akabas et al., 1992). In control experiments, cysteine oxidation induced by the intracellular application of 2.5 mM MTSEA slowly inhibited IRKI wild-type channels with a time constant between 14 and 36 s (Figure SA; n = 4). In contrast, IRK 1 (M84C) channels were inhibited much faster, with time constants between 2.7 and 2.9 s (Figure SB; n = 3). This demonstrated that the side chain of residue 84 is freely accessible to the intracellular solution and points to a direct interaction with H+. Inhibition of IRK1(M84C) channels by MTSEA suggests that IRK1 channels are non-functional whenever a positive charge occurs at the pH regulation site. However, such a hypothesis implies that lysine is not charged at pH 8.0 and that histidine is neutral at pH 6.0, in conflict with the nominal PKa values measured for the pure amino acids. However, the local environment of this position may alter the effective pKa.

Anomalous titration of residue 84 To test the hypothesis of shifted pKas for the titratable residues at position 84 in IRK1, we extended the experiments of Figure 4 to very acidic pH1 values and compared histidine with a titratable side chain (M84H) with phenylalanine (M84F), whose side chain is non-titratable (Figure 6A, B and D). In the pHi range of >5.0, both mutant channels displayed only a slight instantaneous inhibition,

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Fig. 3. pH sensitivity is removed in ROMK1 channels by changing lysine 80 to methionine. (A) Partial alignment of amino acid sequences of the different members of the superfamily of inward-rectifier K+ channels (Ho et al.. 1993; Kubo et al., 1993a,b; Ashford et al., 1994; Bond et al., 1994; Koyama et al.. 1994; Lessage et al., 1994; Zhou et al., 1994; Inagaki et al., 1995a,b; Krapivinsky et al., 1995). The stretch of sequence shown comprises parts of the first hydrophobic segment MI and the 16 amino acids N-terminally adjacent to Ml. Nomenclature adopted from Doupnik et al. (1995). (B) and (C) CulTents through ROMK1(K80M) mutant channels exhibit only weak dependence on intracellular pH in whole-cell (B) and inside-out patch experiments (C). Experimental conditions as in Figure IA and B. (D) Steep pH dependence of ROMK1 is removed by the (K80M) mutation. The line represents the fit of equation (1) to pH-induced current inhibition (pKO5 5.3; Hill coefficient 0.5); data points represent the mean of five experiments.

which was also observed in IRKI wild-type channels (Figure 4B). At more acidic pHi values, a much steeper component superimposes only in IRKl(M84H) channels (Figure 6B). Onset and recovery of this pHi-induced inhibition closely resemble those seen in IRK1(M84K) and ROMK channels (Figures 1 and 4B). Fitting the pH dependence observed for IRK1(M84F) channels yielded a Hill coefficient of 0.7 (n = 4; pKO5 4.5), a value similar to that found for ROMKl(K80M) channels. In contrast, analysis of the pHi-induced inhibition in IRKl(M84H) revealed two components: one with a Hill coefficient >3 (n = 7; component 1: pK(.5 4.7; Hill coefficient 3.1) and the other with a Hill coefficient 8.0 (Figure 6C). Fitting equation (1) to the pH dependence of IR-RO(N) yielded pK0o5 and Hill coefficient values of 8.6 and 2, respectively (n = 7). The difference between the apparent pKa1 of the lysine residue in wild-type ROMK

4096

and IR-RO(N) is likely due to contributions by other domains of the chimeric subunit, donated by IRK1, which provide a different local chemical environment.

Discussion Our results establish that pH regulation of ROMKI channels is determined by lysine 80, where hydrogen ions regulate the channel in a highly cooperative manner. Moreover, pH sensitivity can be conferred on other members of the inward rectifier family by changing the homologous residue to lysine. Besides in ROMK, a lysine residue is only found in BIR10 (Kir4.1) channels and is most likely responsible for the steep pH dependence of this inward-rectifier subtype (our unpublished results). The residues present at the homologous site in other members of the inward rectifier family are asparagine [GIRKI (Kr3. 1), GIRK2 (Kir3.2) and CIR (Kir3.4)], threonine [uKATP (KIr6. 1) and BIR (Kir6.2)] and arginine [GIRK3 (Kir3.3)], none of which have been investigated with respect to a putative effect on pH regulation. The arginine (R) residue present in the GIRK3 sequence is particularly interesting, since the IRK1(M84R) mutation did not result in expression of functional channels. Since GIRK3 also does not express as a homotetramer (Lessage et al., 1994), but most likely co-assembles in heteromultimeric channels (R.Murrell-Lagnado, personal communication), it might be interesting to elucidate the effect of the pH regulation site in heteromultimeric channels. It is tempting to speculate that the positive charge at the pHregulation site of each subunit in a particular channel contributes to the transition of the channel into an inactive state. The finding of a lysine residue responsible for pH

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Fig. 4. pH sensitivity is introduced into IRKI by changing methionine 84 to lysine. Experimental conditions were as in Figure lA and B. (A) and (B) IRKI(M84K)-mediated currents are strongly dependent on intracellular pH in whole-cell (A) and inside-out patch experiments (B). (C-E) IRKI(M84H) (D) and IRK1(M84F) (E) mutant channels show weak dependence on pH- similar to IRKI wild-type (C) in inside-out patch experiments. Experiments with IRKI wild-type were performed in the presence (upper panel in C) and absence (lower panel in C) of 1 ,uM SPM. Note the differential block in outward current at pHi 6.0 in the absence and presence of SPM.

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Fig. 5. The side chain of cysteine 84 is susceptible to MTSEA applied to the cytoplasmic side of inside-out patches. The time course of current inhibition induced by the application of 2.5 mM MTSEA (dissolved in KFR, pHi 8.0) is significantly different in IRKI wildtype (A) and IRKI(M84C) mutant channels (B). Experimental conditions were as in Figure IB, but experiments were performed in

reported that the chemical environment in a protein, such as neighboring charges or acid-base interactions, may indeed induce considerable pKa shifts of titratable residues (Dewan et al., 1993; Yu and Fesik, 1994). The idea of a chemical environment defined by the tertiary and quatemary structure of the protein may be supported by two of the findings presented here: (i) the apparent shift in pKa assumed for lysine is also reproduced for histidine at the same site; (ii) in the chimeric construct IR-RO(N), which most likely is structurally altered, the apparent shift in pKa found for lysine 80 was significantly less pronounced (8.6 instead of 6.9 determined in ROMK wild-type). The loss of cooperativity (the Hill coefficient was reduced from 3.9 to 2) that accompanied this less pronounced shift in pKa might also be due to an alteration of the protein structure. Experiments on the regulation of Kir channels by pHi were carried out in both whole oocytes and in inside-out patches. The results in both systems were different with respect to the reversibility of the pHi-dependent decrease in current. In whole oocytes, the current amplitude after wash-out of bicarbonate was at least as large as before acidification. In inside-out patches, however, acidic pHi induced a significant run-down of the current which was not reversed by subsequent alkalinization. This irreversible component of pHi-induced current decrease depends on the amino acid residue present at the pH-regulation site and on the channel subtype. After application of pHi 6.0 for 10 s, application of pHi 8.0 recovered >95% of the current amplitude in IRKI wild-type, IRK1(M84F), IRK1(M84H) and in ROMK1(K80M), while in ROMK1, ROMK2 and IR-RO(N) recovery was -75%. In IRKI (M84K), recovery was