Identification of residues mediating inhibition of glycine receptors by protons

Neuropharmacology 52 (2007) 1606e1615 www.elsevier.com/locate/neuropharm

Identification of residues mediating inhibition of glycine receptors by protons Zhenglan Chen, Renqi Huang* Department of Pharmacology and Neuroscience, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107, USA Received 23 August 2006; received in revised form 14 February 2007; accepted 12 March 2007

Abstract We previously identified H109 of the glycine a1 subunit as a putative proton binding site. In the present studies, we explored additional proton binding site(s) as well as the mechanism underlying modulation of glycine receptors by protons. Whole-cell glycine currents were recorded from HEK 293 cells transiently expressing wild type or mutant glycine receptors. Individual mutation of 3 of 4 remaining extracellular histidine residue into alanine (i.e., a1 H107A, H215A or H419A), reduced the receptor sensitivity to protons to a varying extent. In contrast, mutation of a1 H201A did not affect proton sensitivity. Double, triple or quadruple histidine mutation of these residues caused a further reduction of proton sensitivity, suggesting multiple binding sites for proton action on glycine receptors. Furthermore, the substitution T133A, which mediates Zn2þ inhibition, virtually abolished the proton effect on peak amplitude and current kinetics of glycine response. Replacement of T with S on position 133 partially restored receptor sensitivity to protons, suggesting the hydroxyl group of residue T133 is essential for proton-mediated modulation. In heteromeric a1b receptors, mutations b H132A and S156A, which correspond to H109 and T133 of the a1 subunit, respectively, also affected proton inhibition. In conclusion, multiple extracellular histidine residues (H107, H109, H215 and H419) and threonine residues of the a1 and b Zn2þ coordination sites are critical for modulation of the glycine receptor by protons. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: pH; Glycine receptor; Protons; Histidine; Threonine; Zinc; Copper

1. Introduction Glycine is the major inhibitory neurotransmitter in the mammalian central nervous system, predominating in the brainstem and spinal cord, where it participates in a variety of motor and sensory functions (Lynch, 2004). Glycine acts by binding to Cl conducting glycine receptors. The glycine receptor is a member of the ligand-gated ion channel superfamily including nicotinic acetylcholine (nACh), 5-HT3, GABAA and GABAC receptors. Glycine receptors are pentameric transmembrane proteins comprising homologous subunits arranged to form a channel pore. Adult synaptic glycine receptors are heteromeric proteins formed by a(1e4) and b subunits with an a:b stoichiometry of 2:3 (Grudzinska et al., 2005). The a subunits can also form * Corresponding author. Tel.: þ1 (817) 735 2095; fax: þ1 (817) 735 0408. E-mail address: [email protected] (R. Huang). 0028-3908/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2007.03.005

homomeric receptors which express during early development, largely extrasynaptically (Flint et al., 1998; Singer et al., 1998). pH in the brain changes with neural activity. In physiological conditions, a brain pH of 6.5e8.0 may exist (Kaila and Ransom, 1998). In certain pathological conditions such as stroke, seizure and ischemia, brain pH can shift up to 1 unit (Siesjo et al., 1985; von Hanwehr et al., 1986). Acidosis affects neuronal excitability, at least in part by modulation of inhibitory and excitatory neurotransmission (Kaila and Ransom, 1998). For example, extracellular acidic pH inhibits GABAA (Huang and Dillon, 1999; Krishek and Smart, 2001; Huang et al., 2004) but see (Pasternack et al., 1996; Mozrzymas et al., 2003; Feng and Macdonald, 2004), GABAC (Wegelius et al., 1996; Rivera et al., 2000) and glycine receptors (Harvey et al., 1999; Li et al., 2003; Chen et al., 2004) as well as voltage-gated Naþ, Kþ and Ca2þ channels (Daumas and Andersen, 1993; Tombaugh and Somjen, 1996).

Z. Chen, R. Huang / Neuropharmacology 52 (2007) 1606e1615

One hypothesis for pH modulation of channel/receptor function is protonation of ionizable amino acid residues that govern function. Indeed, this hypothesis has been demonstrated in a number of ligand-gated channels such as GABAA (Wilkins et al., 2002), glycine (Harvey et al., 1999; Li et al., 2003; Chen et al., 2004), purinoceptor P2X (Clyne et al., 2002), nACh and NMDA receptors (Low et al., 2000). The protonated and the non-protonated forms of amino acid residues are chemically and electrically different. Consequently, residue interaction may be significantly different at pH above or below its pKa value. Of those ionizable amino acid residues, histidine is particularly suitable to act as a proton sensor. The pKa (z6) for the imidazole group from the histidine side chain is within physiological pH. Physiological and pathological shifts of pH would dramatically alter the protonation state of histidine’s imidazole side chains. In fact it has been reported that histidine residues play a critical role in proton modulation in ATP-sensitive Kþ (KATP) channels (Piao et al., 2001; Xu et al., 2001), P2X (Clyne et al., 2002) and acid-sensing ion channels (ASICs) (Baron et al., 2001). We and others (Harvey et al., 1999; Li et al., 2003; Chen et al., 2004) have shown that protons inhibit the glycine receptor at the extracellular site. Furthermore, mutation of a conserved histidine residue (H109) at the N-terminus partially abolished proton sensitivity, suggesting that H109 may be one of Hþ coordination sites in the glycine receptors (Chen et al., 2004). The homomeric a1 glycine receptor has four extracellular histidine residues in addition to H109. We have thus evaluated the involvement of these remaining histidines in pH modulation of glycine receptors. In addition, because some residues modulate response to divalent cations as well as protons (Chen et al., 2004; Miller et al., 2005; Chen et al., 2006), we also assessed the influence of a1 T133, recently shown to be a Zn2þ binding site (Miller et al., 2005), on pH sensitivity. We found that the minimal combination of mutations that could eliminate pH sensitivity was H109A and H215A. 2. Materials and methods 2.1. Cloned receptors Wild type human glycine a1 and b cDNA were generous gifts from H. Betz. The wild type or mutant receptor cDNA was expressed in human embryonic kidney (HEK) cell lines via the mammalian expression vector pCIS2. Cells were transiently transfected using calcium phosphate precipitation or liposome (lipofectamineÔ 2000, Invitrogen, CA) transfection. Briefly, HEK cells were plated onto coverslips and transfected with wild type or mutant subunits. Typically, a1 or a1 plus b (a:b, 0.5:5 mg) cDNA (Pribilla et al., 1992) was added to cells growing exponentially on one coverslip placed in a 35 mm culture dish. After 6e8 h, cells were washed and placed in fresh culture medium. Transfected cells were used for electrophysiological analysis 24e48 h after the transfection.

2.2. Mutagenesis Mutations of receptor cDNA were performed using commercially available a QuickChange site-directed mutagenesis kit (Strategene, La Jolla, CA) with commercially produced mutagenic primers (MWG Biotech, NC). All mutants were verified by DNA sequencing (MWG Biotech, NC).

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2.3. Electrophysiology Whole-cell patch recordings were made at room temperature (22e25  C). Patch pipettes of borosilicate glass (1B150F, World Precision Instruments, Inc., Sarasota, FL) were pulled (Flaming/Brown, P-87/PC, Sutter Instrument Co., Novato, CA) to a tip resistance of 1e2.5 MU. The pipette solution contained (in mM): 140 CsCl, 10 EGTA, 10 HEPES, 4 Mg-ATP; pH 7.2. Coverslips containing cultured cells were placed in a small chamber (w1.5 ml) on the stage of an inverted light microscope (Olympus IMT-2) and superfused continuously (5e8 ml/min) with the following external solution containing (in mM): 125 NaCl, 5.5 KCl, 0.8 MgCl2, 3.0 CaCl2, 20 HEPES, 10 D-glucose, pH 7.3. Glycine-induced Cl currents from the whole-cell patch clamp technique were obtained using an Axoclamp 200A amplifier (Axon Instruments, Foster City, CA) equipped with a CV201 AU headstage. Currents were lowpass filtered at 5 kHz, monitored on an oscilloscope and a chart recorder (Gould TA240), and stored on a computer (pClamp 6.0, Axon Instruments) for subsequent analysis. Series resistance compensation (60e80%) was applied at the amplifier. To monitor the possibility that access resistance changed over time or during different experimental conditions, at the initiation of each recording we measured and stored on our digital oscilloscope the current response to a 5 mV voltage pulse. This stored trace was continually referenced throughout the recording. If a change in access resistance was observed during the recording period, the patch was aborted and the data were not included in the analysis. Cells were voltage-clamped at 60 mV. Heteromeric a1b glycine receptors are 17-fold less sensitive to picrotoxin than homomeric a1 receptors (Pribilla et al., 1992). Thus the incorporation of b subunits with a1 subunits was confirmed by assessing picrotoxin sensitivity. If 100 mM picrotoxin caused 30% or less inhibition of the current activated by EC30 glycine, it was assumed that heteromeric a1b receptors were expressed.

2.4. Experimental protocol pH of external solutions was altered by addition of NaOH or HCl, and routinely checked before and during experiments. Glycine was prepared in the extracellular solution, and was applied from independent reservoirs by gravity flow for 10 s to cells using a Y-shaped tube positioned within 100 mm of the cells. With this system, the 10e90% rise time of the junction potential at the open tip is 12e51 ms (Huang and Dillon, 1999). Once a control glycine response was determined, the effect of pH on the response was examined. To assess the pH effect, cells were first bathed in medium that was set to the test pH, then glycine, dissolved in the same test pH solution, was applied to the cells. Because external solution of low pH elicits a transient inward current through ASICs (Krishek et al., 1996; Zhai et al., 1998; Huang and Dillon, 1999), glycine application at various pH test values was made after the transient current was fully inactivated. Glycine applications were separated by at least 3 min intervals to ensure both adequate washout of glycine from the bath and recovery of receptors from desensitization, if present.

2.5. Data analysis Data analysis and curve fitting were performed with Origin 5.0 (Microcal Software, Northampton, MA) or Clampfit 6.0 (Axon Inc). Glycine concentrationeresponse profiles were constructed with at least seven concentrations of glycine and were fitted to the following equation: I/Imax ¼ [glycine]n/ (EC50n þ [glycine]n), where I and Imax represent the normalized glycineinduced current at a given concentration and the maximum current induced by a saturating concentration of glycine, respectively, EC50 is the 50% effective glycine concentration, and n is the Hill coefficient. Current activation time was assessed using the time to rise from 10% to 90% of current amplitude activated by saturating [glycine]. Desensitization was described by the percentage of desensitization using the formula %D ¼ 100  (Ipeak  I10s)/Ipeak, where Ipeak is the current at the peak response to saturating [glycine], and I10s is the residual current at the end of 10 s glycine application. Deactivation kinetics were measured with current evoked by 10 s application of EC30 [glycine] which induced minimal desensitization. The decay of current back to baseline after removal of glycine was fitted to a single exponential decay function: I10s  exp(t/t) þ C, where I10s is the current

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Z. Chen, R. Huang / Neuropharmacology 52 (2007) 1606e1615

amplitude at the end of 10 s glycine application, t is time, t is the deactivation time constant, and C is baseline current, using a Simplex iterative procedure where the sum of squared errors was minimized. A minimum of three individual experiments were conducted for each paradigm. All data are presented as mean  SEM. Student’s t-test (paired or unpaired) or one-way ANOVA test was used to determine statistical significance ( p < 0.05).

3. Results

3.2. pH sensitivity in the multiple histidine mutants Of the four extracellular histidines we have identified, mutation of none individually was sufficient to abolish the pH sensitivity. Therefore, we examined the effects of simultaneously mutating multiple histidines. In view of the fact that mutation H201A did not have an effect on pH sensitivity in the present studies, or on Zn2þ/Cu2þ modulation in previous studies (Harvey et al., 1999; Chen et al., 2006), we did not introduce H201 in the combinations of mutation. In the double receptor mutants H107/H109A or H215/H419A, the proton modulation

M1 M2 M3

S-S

M4

N

C * CTKHYN 201

* GAHFHEITT 107 109 112

pH

B

7.3

8.4

7.3

* ARFHLER 215 6.4

7.3

* RREDVHNQ 419 5.4

7.3

WT

3.1. Effect of individual extracellular histidine mutation on pH modulation

250 pA 20 s

pH

7.3

8.4

7.3

6.4

7.3

5.4

7.3

H215A 500 pA 20 s

C

160 140

Relative current

Since the action site(s) for proton is extracellular (Li et al., 2003; Chen et al., 2004), we focused on the role of extracellular histidine residues in proton modulation. Five extracellular histidine residues can be found in a glycine a1 subunit. Of those, H109, H201, H215 on the N-terminus and H419 on the C-terminus are conserved in all glycine a1e3 subunits whereas H107 is only present in the a1 subunit (Fig. 1A). In our previous studies, substitution of H109 with alanine in the a1 subunit greatly reduced but did not completely eliminate proton sensitivity (Chen et al., 2004), suggesting additional potential binding site(s) for Hþ. We therefore systemically mutated other extracellular histidine residues to alanine. Since the pH effect on glycine receptors depends on glycine concentration (Harvey et al., 1999; Chen et al., 2004), we used an equipotent glycine concentration (zEC30) to evaluate the pH sensitivity between wild type and mutant receptors. Individual mutation of H107, H201, H215 or H419 to alanine caused a slight shift of glycine EC50 (Table 1) compared to wild type. Fig. 1B, C shows the influence of individual mutation of the extracellular histidine residue on proton sensitivity in glycine a1 receptors. As reported in our previous paper (Chen et al., 2004), acidic pH strongly inhibits while alkaline pH slightly potentiates glycine currents activated by EC30 in the wild type glycine receptors. Mutation H201A failed to affect proton sensitivity, suggesting that H201 is not involved in proton modulation. However, mutation of other histidine residues, i.e., H107A, H215A and H419A, influenced the proton sensitivity to a varying extent. Mutation H215A or H419A abolished the potentiating effect of alkaline pH (8.4) and significantly reduced the inhibitory effect of acidic pH (6.4 and 5.4) (Fig. 1). In contrast, mutation H107A only decreased the sensitivity to an extremely acidic pH (5.4) (Fig. 1C), suggesting that the role of these histidines in proton modulation may be different.

α1

A

WT H107A H109A H201A H215A H419A

120 100 80

** **

* *

** *

60

*

40

*

* *

20 0

pH 8.4

pH 6.4

pH 5.4

Fig. 1. Histidines in the extracellular domains of glycine a1 receptors are critical for the proton-mediated modulation. (A) Schematic of the glycine a1 subunit showing the positions of histidine in the extracellular domain that were investigated. The subunit consists of a long N-terminus (N), four transmembrane domains (M1e4) and the three loops and one short C-terminus (C). The asterisks indicate the histidine residues conserved in all glycine a1e3 subunit and an arrow indicates the threonine (T112) that is important to strychnine-, Zn2þ- and Hþ-mediated inhibition (Laube et al., 2000; Nevin et al., 2003; Chen et al., 2004). Of those histidine residues, extracellular H109, H201, H215 at the N-terminus and H419 at the C-terminus are conserved in a1e3 subunits and H107 is only present in a1 subunits. (B) The whole-cell currents activated by EC30 glycine were recorded at control (pH 7.3), pH 8.4, 6.4 and 5.4 in wild type a1 and a1(H215A) receptors. Note that the response to pH was greatly reduced in a1 H215A. (C) Summary of the pH effect on glycine activated currents in wild type, mutant receptors. All the currents were normalized to the control response at pH 7.3 (assigned as 100%). The column represents the average from at least three cells (see Table 1). *p < 0.05, unpaired t-test, compared to wild type. For comparison, the data of wild type represented here include partial data published in our earlier paper (Chen et al., 2004). The data of H109 is also replotted from the same paper.

of glycine currents behaved similar to that of single mutation H109A or H215A (Table 1). The mutation H109A combined with mutation H215A or H419A resulted in further reduction of the sensitivity to acidic pH compared to that of single mutation. However, triple (H107/H109/215A) or quadruple (H107/ H109/H215/H419A) histidine mutation produced the receptors

Z. Chen, R. Huang / Neuropharmacology 52 (2007) 1606e1615

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Table 1 Glycine and proton sensitivity in wild type and mutant glycine receptors Receptor

EC50 (mM)

EC30 (mM)

pH sensitivity (% of current at pH 7.3) pH 8.4

pH 6.4

pH 5.4

a1 a1(H107A) a1(H109A) a1(H201A) a1(H215A) a1(H419A) a1(H107/H109A) a1(H109/H215A) a1(H107/H109/H215A) a1(H107/H109/H215/H419A) a1(H109/H419A) a1(H215/H419A) a1(T133A) a1(T133S) a1(H107/H109/H215/H419/T133A) a1b a1b(H132A) a1(H107/H109/H215/H419A)b a1(H107/H109/H215/H419A)b(H132A) a1(T133A)b a1b(S156A) a1(T133A)b(S156A)

23  2.1 (11) 39  5.6 (6) 24.2  2.4 (3) 43  7.7 (3) 34  6.1 (5) 34  2.5 (5) 92  12 (6) 51  7.2 (4) 123  18.9 (6) 92  11 (4) 29  1.4 (5) 16  3.0 (5) 83  6.3 (5) 124  6.3 (5) 173  3.6 (6) 18  3 (10) 123  17.7 (4) 190  11 (4) 484  21 (7) 317  34 (5) 91  4.6 (5) 314  43 (6)

15 28 15 34 25 25 60 30 90 65 20 12 65 85 120 10 90 120 300 300 65 210

119  6.5 (9e11) 142  12 (6) 78  5.9 (5e6) 125  4.5 (3e5) 73  8 (9e13) 90  3.8 (8e9) 145  10 (5) 82  5.1 (9) 84  5.4 (5) 70  4.0 (10) 55  5.3 (9) 72  3.9 (11) 113  7.0 (9e10) 123  9.0 (5) 92  3.8 (9e10) 117  6.4 (7e12) 143  13 (5e6) 102  4.6 (6) 70  4.0 (5) 147  7.8 (11e12) 124  12 (6) 138  7.6 (8)

30  2.6 30  3.3 61  6.4 31  8.1 78  5.7 77  2.9 59  9.6 103  6.4 97  3.3 91  2.2 107  5.0 72  4.7 79  5.1 64  1.7 97  5.2 34  4.0 39  5.4 44  5.1 91  2.2 58  4.5 25  2.4 98  9.6

6.7  1.8 28  4.3 46  4.5 4.3  0.9 45  8.9 17  1.7 51  8.9 76  6.4 77  4.9 69  4.7 35  4.7 5  1.4 187  15.4 50  6.1 29  6.3 3.5  1.0 11  0.9 30  1.8 69  4.7 77  11 1.0  0.6 134  19

Values are means  S.E.M. EC50 values were achieved from concentrationeresponse data fitted with a logistic equation. pH modulation was investigated using a glycine concentration corresponding to the respective EC30 value. Glycine currents activated by EC30 glycine were normalized to control current at pH 7.3 (assigned as 100%). The numbers in parentheses represent the cell number. The glycine EC50 and pH sensitivity in the wild type a1, a1b and mutant a1 H109A were taken from our previous paper (Chen et al., 2004). The glycine EC50 in H201A, H215A, H419A, H107/H109A and T133A taken from another previous paper (Chen et al., 2006).

which had pH sensitivity similar to that of the double H109/ H215A mutation ( p > 0.05, compared to H109/H215A (Fig. 2, Table 1).

WT H107/H109A H109/H215A H107/ H109/H215A H109/H419A H215/H419A H107/H109/H215/H419A

180 160

3.3. Effect of T133 mutations on pH sensitivity

*

Relative current

140

From the data above, the receptors with mutation of all extracellular histidine residues still retained pH sensitivity at pH 5.4 (Fig. 2). This indicates that residues other than histidine are involved in pH modulation. An extracellular threonine residue conserved in all glycine a subunits (T133 in a1) has been recently identified to be one of the inhibitory Zn2þ coordination sites (Miller et al., 2005). As it has been shown that an individual residue can influence both Zn2þ and pH sensitivity (Chen et al., 2004), we examined whether T133 also participates in the inhibition by protons. Fig. 3 shows that exchanging T with neutral residue A at the position of a1 T133 resulted in dramatic impact on the sensitivity to acidic pH. At pH 5.4, mutant a1 T133A receptors completely lost proton-mediated inhibition, and in fact displayed a significant potentiation of the glycine response by 87% above the control. The response of glycine receptors to pH 6.4 reduced from 30  2.6% of the control current in wild type to 79  5.1% in a1 T133A. The response to alkaline pH (8.4), however, was not affected by the mutation (113  7% in a1 T133A, p > 0.05, compared to the wild type). Next, we examined whether the hydroxyl group at position 133 is important to pH modulation by replacing T with another hydroxylated amino acid serine. As shown in

120

*

* *

100

*

* * 80 60

* *

* *

*

*

*

*

* *

40 20 0

pH 8.4

pH 6.4

pH 5.4

Fig. 2. Effect of multiple histidine mutations on proton sensitivity of glycine a1 receptors. Proton modulation was investigated using a glycine concentration corresponding to the respective EC30 values. The wild type data are replotted from Fig. 1 and served as the control for direct comparison (likewise afterwards). Proton sensitivity was further reduced by double (H107/H109A, H109/H215A) mutations ( p < 0.05, unpaired t-test, compared to H109A or H107A). Receptors containing double (H109/H215A), triple (H107/H109/ H215A) or quadruple (H107/H109/H215/H419A) mutation lost proton sensitivity to physiological pH (6.4e8.4). *p < 0.05, unpaired t-test, compared to wild type. Glycine EC50 values, testing glycine concentration (EC30) and cell number are shown in Table 1.

Z. Chen, R. Huang / Neuropharmacology 52 (2007) 1606e1615

1610

pH

7.3

5.4

7.3

6.4

7.3

8.4

3.4. Effect of mutations on the maximum current

7.3

80

From the data above, mutations of four extracellular histidine residues (H107, H109, H215 and H419) and T133 have significant impact on pH modulation of the current elicited by EC30 [glycine]. It has been reported that the inhibition of glycine current by proton is competitive in wild type glycine receptors (Harvey et al., 1999; Li et al., 2003; Chen et al., 2004). In order to examine whether mutations alter the competitive behavior of the sensitivity of glycine receptor to protons, we tested the pH effect on the currents elicited by saturating concentrations of glycine (1e3 mM) in the mutant receptors (i.e., T133A, H107/H109/H215/H419A, H107/H109/ H215/H419/T133A) which displayed significant reduction of pH sensitivity. In the wild type a1 receptors, at the saturating concentration of glycine, changes in pH (pH 8.4e5.4) had no significant effect on the current amplitude ( p > 0.05, n ¼ 8e 19, paired t-test, compared to control). This is consistent with the competitive inhibition by protons reported previously (Harvey et al., 1999; Li et al., 2003; Chen et al., 2004). The competitive manner of pH inhibition was not altered by histidine mutations or T133A mutation as protons had little effect on the current activated by saturating [glycine] in the mutant receptors (Fig. 4).

40

3.5. Effect of protons on current kinetics

T133A

pH

7.3

5.4

7.3

6.4

7.3

8.4

7.3

T133S 2000 pA 20 s

B

Relative current

200

WT T133A T133S

160

120

0

pH 8.4

pH 6.4

pH 5.4

Fig. 3. Effect of T133 mutations on proton sensitivity in glycine a1 receptors. T133 was mutated to neutral residue A or hydroxylated residue S. The wild type and alanine mutation are also displayed for comparison. Glycine EC50 values and cell number are shown in Table 1. Note that mutation T133A completely abolished proton-induced inhibition while T133S mutation partially restored the sensitivity to protons. *p < 0.05, unpaired t-test, compared to the wild type or mutant T133A.

Fig. 3 and Table 1, compared to alanine substitution, the sensitivity to acidic pH was partially restored by serine substitution. These data suggest the hydroxyl group at 133 is essential to pH sensitivity. Mutational studies have so far revealed that pH modulation is mediated by multiple extracellular residues. Furthermore these residues may play different roles with the respect to proton modulation. Histidine residues seem to have relatively more impact on the physiological pH (pH 6.4 and pH 8.4) as seen in mutant a1(H107/H109/H215/H419A) receptors whereas T133 has more influence on extreme acidic condition (i.e., pH 5.4). Thus we tested the proton response in the receptors in which four extracellular histidine residues and T133 were simultaneously mutated. Mutation H107/H109/H215/H419/ T133A caused a 7.5-fold shift of glycine EC50 (from 23 mM in wild type to 173 mM in mutant receptors) (Table 1, p < 0.01, unpaired t test). The mutation completely abolished the pH sensitivity at pH 8.4 and 6.4. However, the inhibitory response to pH 5.4 was further enhanced compared to T133A or multiple histidine mutations (Table 1).

We attempted to characterize the influence on current kinetics by protons as well as by different mutations. Activation (10e90% rise time of maximal currents), desensitization (percentage desensitization of maximal current, %D) and deactivation (time constant of decay phase of current evoked by EC30 [glycine]) were estimated in the wild type receptors and three receptors containing the mutations that produced significant α1 α1(T133A) α1(H107/H109/H215/H419A) α1(H107/H109/H215/H419/T133A)

120 100

Relative current

A

80 60 40 20 0

pH 8.4

pH 6.4

pH 5.4

Fig. 4. Effect of protons on currents activated by saturating glycine concentrations. Whole-cell currents activated by saturating glycine concentrations (1 or 3 mM) were recorded at different pH (pH 5.4e8.4) from wild type and mutant receptors. All the currents were normalized to the control response at pH 7.3 (assigned as 100%). The column represents the average from 8 to 19 cells. Compared to significant effect of pH on currents activated by EC30 glycine (Table 1), protons had little effect on the maximal currents.

Z. Chen, R. Huang / Neuropharmacology 52 (2007) 1606e1615

impact on proton sensitivity (i.e., a1(T133A), a1(H107/H109/ H215/H419A) or a1(H107/H109/H215/H419/T133A)). Although the limits on the speed of solution exchange (the quickest exchange time is approximately 12 ms of 10e90% rise time) prohibited us from measuring the kinetics parameters that are comparable with those reported previously with an ultrafast piezo-based perfusion system (Lewis et al., 2003; Mohammadi et al., 2003), our system is still sufficient to detect relative changes in current kinetics by protons or mutation. On the other hand, however, due to the relatively slow exchange time, we could potentially miss fast events/components of receptor kinetics and thus underestimate the modifications of the current kinetics by protons. Nevertheless, our data serve as a qualitative description on the influence of kinetics by protons. As shown in Fig. 5, in the wild type a1 receptors, protons modified the activation and desensitization of the current activated by saturating [glycine]. The activation time increased to 134  13% (n ¼ 5) and 150  9.6% (n ¼ 17) of the control at pH 6.4 and 5.4, respectively ( p < 0.05,

a

pH 7.3 pH 5.4 pH 7.3 (wash out)

*

500 pA 20 ms

compared to the control, paired t-test. Fig. 5Ab). The desensitization in response to saturating [glycine] was slightly reduced at pH 5.4 (%D decreased to 91  4.5% of control, n ¼ 20, p < 0.05, paired t-test, Fig. 5B). But the deactivation time constant was not significantly altered by pH (Fig. 5C). The receptors containing mutation T133A (a1T133A and a1(H107/H109/H215/H419/ T133A) significantly increased the activation time and the deactivation time constant at control conditions. The 10e90% rise the time increased from 37  2.4 ms in the wild type to 58  3.9 ms in T133A and to 153  18 ms in T133A combined with histidine mutations, respectively ( p < 0.05, unpaired t-test, compared to the wild type). Likewise, these T133A-containing receptors deactivated more slowly than the wild type at pH 7.3. The deactivation time constant of current evoked by EC30 [glycine] was changed from 407  55 ms in the wild type to 1586  427 ms in T133A, and to 1114  427 ms in T133A together with histidine mutations, respectively ( p < 0.05, unpaired t-test, compared to the wild type). Mutation T133A had dramatic impact not only on

240

b

220

Normalized activation time

A

1611

200 180

α1 α1(T133A) α1(H107/H109/H215/H419A) α1(H107/H109/H215/H419/T133A)

*

**

160

*

140

*

120 100 80 60 40 20 0

B

C 180

Normalized %D

160 140

**

120 100

*

80 60 40 20 0

pH 8.4

pH 6.4

pH 5.4

180

Normalized deactivation time

α1 α1(T133A) α1(H107/H109/H215/419A) α1(H107/H109/H215/H419/T133A)

200

160

pH 8.4

pH 6.4

pH 5.4

α1 α1(T133A) α1(H107/H109/H215/H419A) α1(H107/H109/H215/H419/T133A)

140 120 100 80 60 40 20 0

pH 8.4

pH 6.4

pH 5.4

Fig. 5. Effect of protons on current activation (A), desensitization (B) and deactivation (C) of wild type and mutant glycine a1 receptors. All the data were normalized to the control values at pH 7.3 (assigned as 100%). Each data point represents at least 5 cells. (Aa) The current traces on an expanded time scale showing that activation was reversibly decreased by pH 5.4 in glycine a1(H107/H109/H215/H419/T133A) mutants. The currents activated by 3 mM glycine recorded from the same cell in control (pH 7.3), pH 5.4 (marked with ‘‘*’’) and washout (pH 7.3). Peak currents were normalized to the same peak amplitude to allow for comparison of their activation. (Ab) Mean values of normalized activation (10e90% rise time) measured from wild type and mutant receptors at different pH. Protons significantly decreased the activation in wild type and mutant and histidine mutants while mutation T133A blocked this effect. (B) Protons significantly decreased the desensitization in wild type whereas mutation T133A enhanced the desensitization at pH 5.4. *p < 0.05; **p < 0.01, paired t-test, compared to the control. (C) The deactivation of current activated by EC30 glycine was assessed with deactivation time constant (see Section 2) and normalized to the control. Protons had little influence on deactivation kinetics in wild type and mutant receptors.

Z. Chen, R. Huang / Neuropharmacology 52 (2007) 1606e1615

modulation of peak amplitude as described above, but also on modification of current kinetics by protons. Fig. 5 shows that mutation T133A prevented modulation of activation (Fig. 5Ab) and desensitization kinetics by acidic pH (Fig. 5B). Multiple extracellular histidine mutations did not affect pH-induced modifications of activation (Fig. 5Ab). However, the activation in T133A combined with histidine mutations caused a further increase in activation time at pH 5.4 (Fig. 5A), which is consistent with the observation that the effect of pH 5.4 on peak amplitude activated by EC30 glycine was further enhanced in this receptor (Table 1).

A

α1 α1β α1β(H132A) α1(H107/H109/H215/H419A) α1(H107/H109/H215/H419A)β α1(H107/H109/H215/H419A)β(H132A)

160 140

Relative current

1612

120 100

*

80 * 60 40

3.6. Role of b subunit

3.7. Effect of mutations on Zn2þ-mediated modulation The residues H107, H109 and T133 have been identified as binding sites for the Zn2þ modulatory effect on glycine receptors, suggesting that both protons and Zn2þ may share a similar mechanism (Nevin et al., 2003; Chen et al., 2004; Miller et al., 2005). In the present studies, compared to the wild type, the

20 0

B

pH 8.4

pH 6.4

pH 5.4

240 α1 α1β α1β(S156A) α1(T133A) α1(T133A)β α1(T133A)β(S156A)

200

Relative current

Adult glycine receptors are predominantly a1b heteromers (Laube et al., 2002). Heteromeric a1b glycine receptors have proton sensitivity similar to homomeric a1 receptor (Fig. 6) (Chen et al., 2004). The b subunit has a histidine residue at position 132 (H132) which corresponds to a1 H109, and a hydroxylated residue (S156) homologous to a1 T133. To investigate whether these two extracellular residues on the b subunit participate in pH modulation, we mutated H132 to A on the b subunit. As Fig. 6A shows, mutation b H132A did not affect pH sensitivity at pH 8.4 and 6.4. However, mutation b H132A caused a small fraction of reduction in sensitivity to pH 5.4, from 3.5  1.0% of the control in wild type to 11  0.9% of the control in mutant receptors ( p < 0.05, unpaired t-test, Fig. 6A, Table 1). To examine whether the b subunit is able to rescue the proton sensitivity of the mutant a1 subunit, we coexpressed the wide type b subunit with a1(H107/H109/ H215/H419A) which displayed strongly reduced proton sensitivity (see Fig. 2). Coexpression with the b subunit only rescued partial sensitivity to pH 5.4 (relative current from 69  4.7% in mutant a1 to 30  1.8% in a(H107/H109/H215/H419A)b, p < 0.01, unpaired t-test, Fig. 6, Table 1). Exchanging the wild type b subunit with mutant b H132A (i.e., a1(H107/ H109/H215/H419A)b(H132A)), proton sensitivity to pH 5.4 was significantly reduced (Fig. 6A, Table 1). Similar to b H132, S156 on the b subunit also plays a certain role in pH modulation of heteromeric glycine receptors. As shown in Fig. 6B, compared to homomeric mutant a1(T133A) receptors, pH sensitivity was partially restored by coexpression of wild type b subunit. However, mutation b S156A alone (i.e., a1b(S156A)) caused a slight increase in proton sensitivity. Double mutant a1(T133A)b(S156A) yielded receptors which displayed pH sensitivity similar to a1(T133A) (Fig. 6B, Table 1). These data suggest that these two extracellular residues on the b subunits (H132 and S156) play a role in modulation of glycine a1b receptors at extreme acidic pH (pH 5.4).

*

160

* 120

* *

*

80

40

0

pH 8.4

pH 6.4

pH 5.4

Fig. 6. Effect of mutation H132A (A) or S156A (B) on the b subunit on proton sensitivity. (A) The a1 and a1b receptor had similar sensitivity to protons. Mutation b H132A caused a small but significant reduction of response to pH 5.4 from 3.5% in wild type a1b to 11% in a1b(H132A) (*p < 0.05, compared to wild type a1b) while sensitivity at pH 8.4 and pH 6.4 was not affected. Coexpression of the wide type b subunit with a1(H107/H109/H215/H419A) rescued partial sensitivity to pH 5.4 (*p < 0.05, compared to mutant a1). Compared to a1(H107/H109/H215/H419A)b, mutation a1(H107/H109/ H215/H419A)b(H132A) yielded additional reduction of sensitivity to pH 5.4 (*p < 0.05, unpaired t-test). (B) Mutation b S156A alone (i.e., a1b(S156A)) caused an increase in proton sensitivity compared to wild type a1b receptors. Coexpression of wild type b with mutant a1 T133A subunit partially rescued proton sensitivity (*p < 0.05, unpaired t-test, compared to homomeric a1 T133A). Double mutant a1(T133A)b(S156A) further reduced proton sensitivity (*p < 0.05, compared to a1(T133A)b). These data suggest that these two extracellular residues on the b subunits (i.e., H132 and S156) play a role in modulation of glycine a1b receptors by acidic pH.

mutant a1 H107/H109A and a1(T133A)b receptors displayed a significant increase in the potentiating effect of alkaline pH (i.e., low [Hþ], Fig. 2, Table 1), raising a possibility that the potentiating effect of low concentration of Zn2þ may also be modified by the mutations. As the inhibitory component of Zn2þ action starts to be visible at concentrations higher than 10 mM (Laube et al., 1995), we estimated the efficacy of Zn2þ -induced potentiation with 1 and 3 mM Zn2þ on the

Z. Chen, R. Huang / Neuropharmacology 52 (2007) 1606e1615 α1 α1(H107A) α1(H107/H109A) α1β α1(T133A)β

300

Relative current

250 200 150 100 50 0

1 μM Zn2+

3 μM Zn2+

Fig. 7. Potentiating effect of Zn2þ on wild type and mutant glycine receptors. Zn2þ (1 or 3 mM) was co-applied with EC30 [glycine] for 10 s. The currents were normalized to the control before Zn2þ application (assigned as 100%). Compared to the wild type a1 or a1b receptors, the potentiating effect of Zn2þ was unaffected by introduction of mutation H107/H109A or T133A.

glycine response (activated by EC30 glycine). The Zn2þ potentiating effect in wild type a1 was apparently greater than that in a1b receptor (Fig. 7). However, neither H107/H109A nor T133A mutation significantly changed the sensitivity to low [Zn2þ] in homomeric or heteromeric receptors (Fig. 7). 4. Discussion In the present studies, in addition to H109, we identified three additional histidine residues (H107, H215 and H419) and one threonine residue (T133) in the extracellular domains necessary for proton inhibition of the glycine a1 subunit. The histidine residue (H132) and a hydroxylated residue (S156) at the b subunit have a limited impact on pH sensitivity of glycine a1b receptors. The pH sensitivity within physiological range (pH 6.4e8.4) requires at least two histidine residues (H109/H215) in the a1 subunit. Histidine is an ideal candidate for sensing extracellular pH changes because the pKa value of its imidazole ring is within the physiological pH range. In our previous studies, mutation of H109 at the N-terminus greatly reduced proton sensitivity (Chen et al., 2004). Here, we have identified three additional extracellular histidine residues (i.e., H107, H215 and H419) that are critical to proton inhibition in homomeric glycine a1 receptors. Our single histidine mutations reveal that the role of the extracellular histidine residues is not equal in modulation of glycine receptor by protons. Also, the contribution of the histidine residues is different at different pH levels. H107 is only present in the a1 subunit and seems to be associated with receptor sensitivity to an extremely acidic pH (5.4). In contrast, H109, H215 and H419 play a role in sensitivity to pH ranging from 5.4 to 8.4 (Fig. 1). As revealed from multiple histidine substitutions, H109 and H215 appear to be the major players responsible for the sensitivity to

1613

physiological pH as mutation of both histidine residues markedly diminished proton-mediated inhibition (Fig. 2). The exact mechanism by which protons inhibit glycine receptors is not clear. Notably, both divalent cations and protons are capable of interacting with the imidazole group of the histidine side chain. The studies from our lab and others indicate that histidine residues are major determinants of proton- and divalent cation (Zn2þ and Cu2þ)-mediated modulation of glycine receptors (Harvey et al., 1999; Nevin et al., 2003; Chen et al., 2004; Miller et al., 2005; Chen et al., 2006). Our data support a broad view that proton inhibition of glycine receptors is at least partially mediated through low affinity sites for Zn2þ inhibition rather than high affinity sites for Zn2þ potentiation since the mutant receptors with reduced pH and Zn2þ inhibition (a1(H107/H109A), a1(T133A)b) still retain a Zn2þ potentiating effect similar to the wild type. Furthermore, in the present studies, double mutation H107/H109 did not further decrease proton sensitivity compared to single mutation, suggesting that proton action may have a common mechanism/pathway at positions 107 and 109. H107 and H109 have been previously identified as coordinated inhibitory Zn2þ sites (Harvey et al., 1999; Nevin et al., 2003; Miller et al., 2005). It has been proposed that Zn2þ is likely bound to pairs of histidines from 107 and 109 of an adjacent a1 subunit to restrict the intersubunit movements (Nevin et al., 2003). It is reasonable to hypothesize that protons inhibit glycine receptor through a mechanism similar to Zn2þ. In contrast to H107 and H109, H215 and H419 form inhibitory action sites for Cu2þ but not for Zn2þ (Harvey et al., 1999; Chen et al., 2006). Double mutation H109A/H215A or triple mutation H107A/H109A/H215A yielded additional reduction of proton sensitivity, which supports the idea that H215 may influence glycine receptors through a mechanism different from H107/H109. The simplest interpretation of our results is that protons and Cu2þ may inhibit glycine receptor function through a common pathway at H215 and H419 as discussed in our previous paper (Chen et al., 2006). In the present studies, mutation of a1 T133 that was previously identified as a Zn2þ binding site (Miller et al., 2005), dramatically affected the sensitivity to acidic pH, especially to pH 5.4. However, on one hand, protons are unlikely to modulate channel function by protonation of T133 since threonine is not a titratable residue. In addition, introduction of mutation T133A to the receptor containing four histidine mutations (H107/H109/H215/H419A) did not eliminate the inhibitory response to acidic pH, which does not support the role of T133 as a binding site. On the other hand, substitution of Twith another hydroxylated residue, serine, partially recovered the sensitivity to protons. Based on the acetylcholine binding protein (AChBP), T133 is predicted to be located at the interface of the a-subunit and has been proposed to be a Zn2þ binding residue of the inhibitory site (Miller et al., 2005). It may be implied that protons more likely influence the hydrogen bond between T133 and the adjacent residue and in turn modulate receptor function. In view of the fact that protons competitively inhibit current peak and slow current activation, we also consider the hypothesis that protons inhibit the receptors at the glycine binding site. However, there is no direct evidence to support the

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Z. Chen, R. Huang / Neuropharmacology 52 (2007) 1606e1615

hypothesis. No evidence suggests that the residues identified to be critical to pH modulation (i.e., H107, H109, H215, H419, T112 and T133) are in the glycine binding pocket (Chen et al., 2004; Lynch, 2004). Furthermore, the observation that mutation T133A completely reversed the effect of protons on peak current as well as on kinetics of macroscopic current is also against this hypothesis although we cannot rule out an allosteric interaction of protons with the glycine binding site. Our studies also support the hypothesis that the sites for proton action are predominately located in the a1 subunit of heteromeric glycine receptors. The stoichiometry of the heterooligomeric glycine receptors is 2a:3b (Grudzinska et al., 2005). Mutation b H132A which corresponds to a1 H109 only influenced the sensitivity to pH 5.4. This may be at least partially attributed to the thought that fewer (3) histidine residues in the b subunits are available to bind protons compared to 8 active histidine residues from a1 subunits. Likewise, mutation b S156A aligned to a1 T133 had minimal influence on the pH sensitivity of a1b receptors. The fact that coexpression of mutant a1 subunit with wild type b subunit (i.e., a1 (H107/ H109/H215/H419A)b and a1(T133A)b) resulted in partial rescue of proton sensitivity supports the notion that the b subunit contributes to proton inhibition in heteromeric glycine receptors. This is also consistent with our previous observation that the b subunit influences pH sensitivity of a1b glycine receptors through a threonine residue (b T135) corresponding to a1 T112 (Chen et al., 2004). In summary, we have demonstrated that protons inhibit glycine receptors through multiple extracellular histidine residues (H107, H109, H215 and H419) and T133 in the a1 subunit. At least two histidine residues (H109/H215) are required to constitute the sensitivity to physiological pH. Our data also suggest that the proton-dependent modulation is mediated by multiple mechanisms, partially sharing Zn2þ- and Cu2þ-mediated inhibition pathways. Acknowledgements This research was supported by American Heart Association Texas Affiliate (RQH), and Faculty Research Grant from University of North Texas Health Science Center (RQH). We thank Dr Glenn Dillon for helpful comments on the manuscript, and Dr Heinrich Betz for providing the wild type glycine a1 and b subunit cDNA. References Baron, A., Schaefer, L., Lingueglia, E., Champigny, G., Lazdunski, M., 2001. Zn2þ and Hþ are coactivators of acid-sensing ion channels. J. Biol. Chem. 276, 35361e35367. Chen, Z., Dillon, G.H., Huang, R., 2004. Molecular determinants of proton modulation of glycine receptors. J. Biol. Chem. 279, 876e883. Chen, Z., Dillon, G.H., Huang, R., 2006. Identification of residues critical for Cu2þ-mediated inhibition of glycine alpha1 receptors. Neuropharmacology 51, 701e708. Clyne, J.D., LaPointe, L.D., Hume, R.I., 2002. The role of histidine residues in modulation of the rat P2X2 purinoceptor by zinc and pH. J. Physiol 539, 347e359.

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von Hanwehr, R., Smith, M.L., Siesjo, B.K., 1986. Extra- and intracellular pH during near-complete forebrain ischemia in the rat. J. Neurochem. 46, 331e339. Wegelius, K., Reeben, M., Rivera, C., Kaila, K., Saarma, M., Pasternack, M., 1996. The rho 1 GABA receptor cloned from rat retina is down-modulated by protons. Neuroreport. 7, 2005e2009. Wilkins, M.E., Hosie, A.M., Smart, T.G., 2002. Identification of a beta subunit TM2 residue mediating proton modulation of GABA type A receptors. J. Neurosci. 22, 5328e5333. Xu, H., Wu, J., Cui, N., Abdulkadir, L., Wang, R., Mao, J., Giwa, L.R., Chanchevalap, S., Jiang, C., 2001. Distinct histidine residues control the acid-induced activation and inhibition of the cloned KATP channel. J. Biol. Chem. 276, 38690e38696. Zhai, J., Peoples, R.W., Li, C., 1998. Proton inhibition of GABAactivated current in rat primary sensory neurons. Pflugers Arch. 435, 539e545.