Effect of protein kinase-C activation on the Mg2+-sensitivity of cloned NMDA receptors

002%3908(95)00177-8

Neuropharmacology, Vol. 35, No. 1, pp. 29-36, 1996 Copyright 0 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0028-3908/96 $15.00 + 0.00

Effect of Protein Kinase-C Activation on the Mg2+sensitivity of Cloned NMDA Receptors D. A. WAGNER and J. P. LEONARD* Department of Biological Sciences, Laboratory for Molecular Biology, and Committee on Neuroscience, University of Illinois at Chicago, Chicago, U.S.A. (Accepted 16 October 1995) Summary-The mechanisms responsible for protein kinase-c (PKC) mediated potentiation of NMDA receptors are poorly understood. One hypothesis is that PKC-activation reduces the receptor’s characteristic voltage-dependent Mg2+-blockade. Experiments performed on Xenopus oocytes expressing cloned NMDA receptors demonstrated that PKC-activation induced no change in the sensitivity of cl/s3 and cl/a4 receptors to Mg’+-blockade and, even though PKC-activation did induce a small shift in Mg2+sensitivity for the [l/s1 and cl/&2 receptors, the change seen was not large enough to account for an appreciable increase in NMDA receptor activity. Baseline Mg2+-sensitivities and levels of PKC-mediated potentiation were also quantified for each of the di-heteromeric NMDA receptors. The order of Mg2’-sensitivity is jl/&l (most sensitive) > [l/&2 > [l/&4 > [l/&3 (least sensitive). PKC-activation caused a 2-fold increase in
receptor (cloned), Xenopus oocytes, PKC-potentiation,

Mg2+-blockade.

Ionotropic glutamate receptors mediate most of the fast excitatory neurotransmission in the vertebrate central nervous system (Mayer and Westbrook, 1987a). Based on amino acid sequence identity and agonist selectivity these receptors are divided into three subgroups: N-methyl-D-

NMDA receptor subunits that have been cloned from mouse brain include the ligand-binding cl-subunit (Yamazaki et aZ., 1992) and four structurally related Esubunits, (al, ~2, ~3, ~4) (Meguro et al., 1992; Kutsuwada et al., 1992; Ikeda et al., 1992). Oocytes injected with 51

aspartate (NMDA), a-amino-3-hydroxy-Smethyl-isoxazoleproprionate (AMPA), and kainate (Hollmann and Heinemann, 1994). NMD,4 receptors are notable because they are highly permeable to Ca2+ and experience voltage dependent Mg2+-blockade (Nowak et al, 1984; Mayer and Westbrook, 1987b). There is strong evidence that this unique combination of properties allows NMDA recep-

cRNA express functional homomeric NMDA receptors while the epsilon subunits, when expressed alone, do not form functional NMDA receptor channels. However, when each of the epsilon subunits is co-expressed with [l, functional heteromeric NMDA receptors are formed and each cl/a combination possesses unique properties (Kutsuwada et al., 1992). These subunits have distinct

tors to play a key role in many crucial

expression (Watanabe

patterns in both developing and mature brains et uZ., 1993) which could potentially provide functional diversity to NMDA receptors in vivo. NMDA

uZ., 1984), and excitotoxicity (Rothman and Olney, 1987; Koh and Choi, 1988).

receptor subunits homologous to those found in mouse have also been cloned from rat. These are called NMDARl (homologous to Cl) (Moriyoshi et al., 1991) and NMDAR2A-NMDAR2D (homologous to al-a4) (Ishii et uZ., 1994). In addition, eight splice variants of the

neural processes including development (Constantine-Paton et al., 1990) synaptic plasticity and learning (Collingridge et al., 1983; Gustafsson et uZ., 1987; Wigstrom et al., 1986; Harris et

ligand binding NMDARl subunit (NMDARl.lANMDARl.4B) have been cloned (Sugihara et al, 1992;

*To whom correspondence should be addressed, at: UIC Biological Sciences, 845 W. Taylor St., Chicago IL 606077060, USA.

Hollmann et al., 1993). Two properties of the NMDA receptor 29

that provide

an

30

D. A. Wagner and J. P. Leonard

example of how the epsilon subunits supply functional diversity to NMDA receptors are sensitivity to Mg’+blockade and susceptibility to PKC-mediated potentiation of currents. Di-heteromeric receptors containing the ~1 or ~2 subunit @‘l/al and cl/e2) are both very sensitive to Mg2+-blockade and support PKC potentiation while diheteromers containing the ~3 or ~4 subunit (cl/s3 and cl/ ~4) are much less sensitive to Mg2+-blockade and cannot be potentiated by activation of PKC (Yamazaki et al., 1992; Kutsuwada et al, 1992; Mori et al., 1993). PKC-activation has been shown to enhance NMDA currents in varied systems including oocytes expressing total rat brain mRNA (Kelso et aZ., 1992) and in rat trigeminal neurons (Chen and Huang, 1991). The mechanisms underlying PKC-potentiation of NMDA receptors are not completely understood but, in rat trigeminal neurons PKC-activation appears to both increase the NMDA receptor’s Pope,, value (the percentage of time that the receptor is open in the presence of agonist) and reduce the receptor’s sensitivity to Mg*+blockade (Chen and Huang, 1992). On the other hand, in cultured rat striatal neurons PKC-activation does not appear to reduce the NMDA receptor’s sensitivity to Mg2+-blockade (Murphy et al., 1994). In order to clarify the role of Mg2+-blockade in PKCpotentiation of NMDA receptors we have measured the effect that PKC-activation has on the Mg2+-sensitivity of the four ‘di-heteromeric’ NMDA receptor subtypes (ill al, [l/&2, jl/e3, [l/&4). Our results indicate that reduction of Mg2+-blockade does not play a substantial role in PKC-potentiation of cloned NMDA receptors. In addition, by quantifying both Mg’+-sensitivity and PKCpotentiation for all four heteromers, we have further defined the functional diversity that is supplied to NMDA receptors by the epsilon subunits.

METHODS

Preparation of RNA and oocytes Plasmids pBKSA[l (Yamazaki et al., 1992), pBKSAa1, pBKSAe2, pBKSAa3 (Meguro et al., 1992), and pSP64Ts4 (Ikeda et al., 1992) containing cDNAs encoding NMDA subunits isolated from mouse brain were provided by M. Mishina. cDNA was prepared by in vitro transcription (mCAPTM mRNA transcription kit; Stratagene) from plasmid linearized just 3’ of the clone with the appropriate restriction endonuclease and transcribed with T3 RNA polymerase except for ~4 which was transcribed with SP6 RNA polymerase. Adult female Xenopus laevis are anesthetized with 0.25% tricaine methanesulfonate prior to surgery. Following surgical removal from the frog, oocytes are denuded of overlying follicle cells by agitation for 2 hr in 2 mg/ml collagenase (type lA, Sigma) in Ca*+-free solution (82.5 mM NaCl, 2 mM KCl, 1 mM MgC12, 5 mM HEPES [pH 7.51). Selection of stage V and stage VI oocytes begins when 50% of the cells are denuded. Oocytes are injected with

25-100 ng of cRNA’s in an approximately 2:l molar ratio of [l to each epsilon subunit. Before recording, oocytes are incubated at room temperature for 14 days in the following medium: 96 mM NaCl, 2 mM KCI, 1.8 mM CaC12, 1 mM MgC12, 5 mM HEPES, 2.5 mM pyruvate, 100 &ml gentamicin (pH 7.5), and 5% horse serum (Irvine Scientific) to increase oocyte viability (Quick et al., 1992). Recording solutions All experiments are performed in nominally Ca2+-free solutions in order to minimize the contribution of endogenous Ca2+-dependent Cl- channels to the NMDA response (Leonard and Kelso, 1990). The standard recording solution, barium gocyte saline (BOS), was nominally Ca2+ and Mg2+ free and contained 96 mM NaCl, 2 mM KCI, 5 mM HEPES (pH 7.5), and 2.8 mM BaC12 (as a divalent cation replacement). Agonist containing solutions had 100 PM NMDA and 10 PM glycine in BOS. 12,13-phorbol ester dibutyrate (PDBu) was prepared from frozen 1 mM stock solutions in dimethylsulfoxide (DMSO). Final concentrations: PDBU 20 nM, DMSO 0.01%. Mg2+ recording solutions were prepared via serial dilution of BOS containing 5 mM Mg2+ into Mg2+-free BOS. Electrophysiological

recordings

Currents were recorded via two-electrode voltageclamp techniques using a Warner Instrument OC-725 Oocyte Clamp and were collected and analyzed using PCLAMP software (Axon Instruments). Electrodes were filled with 3 M KC1 and had resistances of 0.5-2 M. NMDA responses were recorded while the oocyte membrane was voltage-clamped at -80 mV. Currents were evoked by bath perfusion of NMDA agonist solution for 15 set, followed by a washout with standard recording solution (BOS). Dose-response data was recorded by varying the concentration of Mg2+ in the recording saline between 0 and 5000 PM in a ‘pyramid’ sequence (low Mg2+ high Mg2+, high Mg2+ low Mg2+) in order to minimize variability caused by temporal instability of current amplitude sometimes seen in [l/&l and [l/a2 receptors. In addition, recordings made post-PDBu were finished within 10 min of PDBu treatment in order to avoid washout of the PKC effect. ZIVcurves were recorded using a voltage ramp protocol that runs linearly from -90 mV to +40 mV in 2 sec. Recordings made in the absence of agonist were averaged and subtracted from recordings made in the presence of agonist to give an NMDA Z/V curve. Recording saline was Ba2+ and Ca2+-free with 2.8 mM Mg2+. Traces filtered at 30 Hz prior to plotting for clarity, perfectly overlapped with unfiltered traces. Calculation of ZC5& ICsus for Mg2+-blockade were calculated by linear

PKC and Mg2+-sensitivity of NMDA receptors

A

31

generated by fitting the data points with polynomial or cubic spline equations.

NMDA

RESULTS Subunit-specific PKC effects

B NMDA

500 450 400 350 300 250 200 150 100

50 0 Fig. 1. PDBu application (20 nM for 10 min, in bath) variably potentiates the NMDA current of oocytes expressing channels composed of different subunits. (A) cl/&2 currents are enhanced by PDBu. (B) [l/‘&4 currents are not potentiated by PDBu treatment. (C) Summary of PDBu effects on all di-heteromers expresses in percent control +_ SEM. [l/&l = 182 f 12%, n = 13; [l/&2 = 438 + 40%, n = 13; n = 11.; (l/&4 = 86 + 5%, n = 11. [l/&3 = 96 * 4%, VHold= - 80 mV, 100 PM NMDA, 10 FM Gly.

interpolation between the Mg” concentrations that bracketed 50% block. The values calculated this way were not significantly different from those calculated using the Michael&Menton type equation, I = lOO%* ([M$+Wi + Wg*+l)(where Ki is the concentration of that blocks 50% of NMDA current), or those Mg

Experiments were performed in order to quantify the subunit specific effects of PKC activation. In these experiments oocytes expressing heteromeric NMDA receptors ([l/&l, j1/&2, jl/~3, or [l/&4) were treated with a 10 min application of the PKC activator 12,13-phorbol ester dibutyrate (PDBu, 20 nM, bath applied) which results in near maximal potentiation of NMDA currents (Kelso et al., 1992). Potentiation was calculated by dividing the mean amplitude of 2-3 recordings made immediately prior to PDBu treatment by the mean amplitude of W recordings made near the peak of potentiation and expressed as percent of control. Representative current traces for [l/&2 and (l/&4 receptors recorded at -80 mV both before and after PDBu treatment are shown in Fig. l(A, B). Because the amplitudes of NMDA currents in oocytes expressing either [l/81, (l/&2, or cl/~4 receptors could be inconsistent (usually getting smaller with time) PDBu was applied after the currents had stabilized (10-15 min after the start of recording). NMDA currents in oocytes expressing cl/~3 receptors were very stable and PDBu was usually applied to them within 5 min after the start of recording. In oocytes voltage clamped at - 80 mV, PKCactivation induced a 2-fold potentiation of [l/cl currents (182 f 12% control, mean + SEM, n = 13) and a greater than 4-fold potentiation of [l/82 currents (438 f 40% control, n = 13) while cl/&3 and 4’1/&2> > [l/&4 > cl/&3 (least sensitive). This ordering is consistent with earlier reports (Kutsuwada et al., 1992; Monyer et al., 1994), however, this is the first demonstration that (11~4 and cl/&3 receptors are differentially sensitive to Mg*+-blockade (P < 0.0005, independent t-test). PKC-activation does not alter the ordering of Mg*+-sensitivities of the di-heteromers but it does induce a 2-fold reduction in the Mg*‘-sensitivity of both [l/&l and [l/&2 NMDA receptors. This effect, which is only half the size of that reported in trigeminal neurons, is not likely to significantly contribute to PKC potentia-

32

D. A. Wagner and J. P. Leonard

B

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Fig. 2. Normalized Mg2+ dose-response data for NMDA currents recorded before and after PDBu application. PDBu induced small but significant shifts in the Mg*+-sensitivity of [l/&l (A) and [l/s2 (B) receptors but had no significant effect on Mg’+-blockade of 4X/&3(C) and [l/a4 (D) receptors. A, pre PDBu; 0, post PDBu. During all dose-response experiments oocytes were held at - 80 mV. Note that due to the considerable differences in Mg’+sensitivity between the subunits the x-axis for each graph is scaled differently.

tion of [l/s1 and jlls2 NMDA receptors (see Discussion). PDBu treatment had no effect on the Mg*+sensitivity of cl/~3 and {l/a4 receptors. PKC effect on the voltage dependence of M$+-blockade We have shown that PKC activation has little effect on the Mg*+-sensitivity of NMDA receptors in oocytes voltage-clamped at -80 mV. However, a shift in the voltage dependence of Mg*+-blockade could also influence the amplitude of NMDA currents. Therefore, experiments that study the effect of PDBu treatment on the voltage dependence of Mg*+-blockade were

performed. I/V curves recorded from NMDA receptors in Mg*+-containing salines have a negative slope region that is due to the voltage dependence of Mg’+-blockade. Because of this, any shift in the shape of the I/V curve or the voltage at which maximum inward current is observed would indicate a change in the voltage dependence of Mg*+-blockade. I/V curves recorded from oocytes expressing the di-heteromeric NMDA receptors both before and after PDBu treatment demonstrate that PKC activation does not affect either the overall shape of the curve, (Fig. 3), or the voltage at which inward NMDA current peaks (Table 2). The fact that there is no

33

PKC and Mg*+-sensitivity of NMDA receptors Table 1. PKC-effect on Mg*+-sensitivity of heteromeric NMDA receptors IC, PM k SEM Receptor

?I

ll/El

12 8 5 5

Pre-PDBu 2.8 10.7 349 147

+ * + +

0.5 3.1 30 28

Post-PDBu 6.7 19.0 373 194

f + * f

1.8 5.3 41 70

f-test P value PcO.04 P < 0.02 P > 0.6 P > 0.3

Table 2. PKC-effect on voltage of maximum inward NMDA current I-maxi, (mv) + SEM n

Receptor ills1 WE2 LWE3 We4

Pre-PDBu -21.1 -21.9 -32.9 -33.3

4 4

f f + It

0.7 0.7 0.4 1.5

Post-PDBu - 19.7 -22.9 -34.0 -31.7

f 0.6 f 1.8 If: 1.2 * 1.7

I-test P value P>O.l P > 0.5 P > 0.3 P > 0.6

0

1

1 (nA)

-60

(-30)

-60

’ (4

1 (nA) -300

(-67)

1 -240

(nA)

(-300)

Fig. 3. I/V curves recorded from the di-heteromeric NMDA receptors both before and after PDBu treatment show that PIE-activation does not change the voltage dependence of these receptors. Post-PDBu currents for @‘el, (11.52 and [l/.54 receptors were normalized to pre-PDBu levels in order to better compare the shape of the two curves. Recordings were performed in nominally C!a’+- and Ba*+-free saline containing 2.8 mM Mg*+. The voltage ramp protocol ran linearly from -90 mV to +40 mV in 2 sec.

34

D. A. Wagner and J. P. Leonard

significant difference between the pre- and post-PDBu voltages at which maximum inward current was seen and the almost perfect overlap of the normalized Z/V curves demonstrates that PKC activation has no effect on the voltage dependence of Mg2+-blockade of the di-heteromerit NMDA receptors.

DISCUSSION Subunit specificity of PKC-potentiation In confirmation of previous reports (Mori et al., 1993; Kutsuwada et al., 1992) we have shown that [l/al and cl/ ~2 receptors can be potentiated by PKC activation while [l/a3 and [l/s4 cannot. Careful measurement of the level of potentiation experienced by each heteromer has revealed that [l/c2 receptors can be potentiated more than twice as much as jl/el receptors. This data reveals a new mechanism by which epsilon subunits provide functional diversity of NMDA receptors. Mori et al. (1993) have reported that PKC activation reduces the amplitude of [l/a3 and [l/a4 currents to 70 and 50% of control respectively. We saw no change in amplitude of [l/s3 either or [l/a4 currents recorded before and after PDBu treatment. We did however notice that (l/e4 currents tended to get smaller with time, stabilizing approx 20 min after recording began. If PKC activation took place before these currents plateaued it would appear to inhibit NMDA currents which may be why others have reported this phenomenon. Subunit specificity of baseline i@+-sensitivity Although the Mg2+-sensitivity of various heteromeric NMDA receptors cloned from both rat and mouse has been reported by several sources (Monyer et al., 1994; Ishii et al., 1994; Mori et al., 1992; Kutsuwada et al., 1992) this is the first time it has been systematically quantified for all four di-heteromeric NMDA receptors. With respect to Mg2+-blockade, NMDA receptors have frequently been divided into two groups in which cl/al and ll/e2 receptors are more sensitive to M 2+ and [l/.53 and [l/a4 receptors are less sensitive to Mg9+. However, we show here that each of the heteromers possesses a unique sensitivity to Mg2+ with cl/&l receptors more sensitive than [l/e2 receptors and {l/e4 receptors more sensitive than [l/e3 receptors. Theoretically, this difference could confer a unique voltage sensitivity to each heteromer, providing yet another mechanism by which Esubunits can contribute to the functional diversity of NMDA receptors. In addition, the voltage-dependence could be even more finely tuned by expression of receptors with two or more different types of e-subunit (Sheng et al., 1994). The difference between the voltage-sensitivity of [l/al and [1/~2 receptors compared to cl/e3 and ill.54 receptors is quite obvious when comparing the voltage of maximum inward current (see Table 2) and has been previously demonstrated using rat subunits (Monyer et

al., 1994). We were, however, not able to distinguish a significant difference in the voltage at which inward currents peak for [l/al compared to ll/e2 receptors or cl/ ~3 compared to [l/a4 receptors. This difference may be very subtle, but a shift in voltage-sensitivity of a few mV could be crucial in the integrative role played by NMDA receptors. PKC-potentiation and A@-blockade The main objective of these experiments was to determine whether a reduction in Mg*+-sensitivity is part of the mechanism involved in PKC potentiation of cloned NMDA receptors. Although we did see PKC-mediated reductions in Mg2+-sensitivity of
PKC and Mg”-sensitivity those gathered from rat trigeminal neurons as reported by Chen and Huang (Chen and Huang, 1992). In their system, PKC-potentiation induced a 4-fold reduction of Mg’+-Ki to 110 /tM which had an observable effect on both the amplitude and voltage dependence of NMDA currents. The most obvious explanation for the discrepancy between their data and ours is that they may have been recording from a receptor type that is not equivalent to any of those used here. This idea is supported by the fact that the NMDA receptors in rat trigeminal neurons have a baseline Mg’+-Ki of 27 PM which does not correspond to that for any of the cloned receptors we studied. The receptors in rat trigeminal neurons could have a different subunit makeup due to expression of a differenr cl (NRI) splice variant, a heretofore unknown f: (NR2) subunit, or more than one c: (NR2) subunit. The difference could also be species specific, attributable to the potentially different properties of the mouse NMDA subunits and their rat homologs, but to this point no such differences have been reported and amino acid sequences ;ue ~90% identical between homologous rat and mouse subunits. In any event, the present results on PKC potentiation of NMDA receptors and the fact that this phenomenon occurs in the absence of Mg2+ suggest, at best, a small role for shifting Mg’+sensitivity. with

Acknowledgemenrs-We thank Dr Masayoshi Mishina for the NMDA receptor cDNA clones. Supported by NIH NS31962.

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