Ser-890 in the C1 cassette reduces PKC potentiation. (A) Amino acid sequence of the C1 cassette of the NR1 subunit. Serine residues 889, 890, 896, and 897 were mutated to alanines, singly or in combination. (B) Currents recorded in Ca²⁺ Ringer's solution from oocytes expressing NR1₁₁₁/NR2A, NR1₁₁₁(S_889–897A)/NR2A, and NR1₁₀₁/NR2A receptors before (Upper) and after (Lower) incubation with TPA. Potentiation of NR1₁₁₁(S_889–897A)/NR2A receptors was greater than that for NR1₁₁₁/NR2A receptors. (C) Mean PKC potentiation of wild-type (WT) and mutant receptors in Ca²⁺ Ringer's solution. TPA potentiation of wild-type NR1₁₁₁, NR1₁₁₁(S889A), NR1₁₁₁(S890A), NR1₁₁₁(S896A), NR1₁₁₁(S897A), NR1₁₁₁(S_889–897A)/NR2A, and wild-type NR1₁₀₁/NR2A was to 5.7 ± 0.5 (n = 7), 5.2 ± 0.5 (n = 3), 7.9 ± 0.4 (n = 6; *, P < 0.05 vs. NR1₁₁₁/NR2A), 4.7 ± 0.3 (n = 3), 5.0 ± 0.5 (n = 3), 8.2 ± 0.8 (n = 7; *, P < 0.05 vs. NR1₁₁₁/NR2A), and 10.0 ± 0.8 [n = 6; ***, P < 0.001 vs. NR1₁₁₁; **, P < 0.01 vs. NR1₁₁₁(S890A) and NR1₁₁₁(S_889–897A)] times control, respectively.

The C-terminal tails of NR1 and NR2 are not required for PKC potentiation. (A) Responses of NR1₁₀₀/NR2A, NR1(ΔC)/NR2A, NR1₁₀₀/NR2A(ΔC), and NR1(ΔC)/NR2A(ΔC) receptors in Ba²⁺ and Ca²⁺ Ringer's solution before and after TPA treatment. Responses of NR1(ΔC)/NR2A receptors were larger in Ca²⁺ than in Ba²⁺ Ringer's solution and increased during agonist application. Basal currents of NR1/NR2A(ΔC)and NR1(ΔC)/NR2A(ΔC) receptors were small relative to those of wild-type receptors. (B) Mean PKC potentiation of mutant and wild-type receptors illustrated in A and of NR1₁₀₀(ΔC2′)/NR2A receptors. NR1(ΔC)/NR2A receptors showed significantly less potentiation than did wild-type NR1₁₀₀/NR2A receptors (which contain the C0 region and C2′ splice cassette) in Ca²⁺ Ringer's solution (P < 0.001) but did not differ significantly in Ba²⁺ Ringer's solution. The apparent reduction in the degree of potentiation could be accounted for by the larger basal current of the truncation mutant. NR1(ΔC)/NR2A receptors showed less potentiation than did NR1₁₀₀(ΔC2′)/NR2A receptors (which contain the C0 region) in Ca²⁺ (P < 0.001) and Ba²⁺ Ringer's solution (P < 0.01). Potentiation of NR1₁₀₀/NR2A(ΔC) receptors did not differ significantly from that of wild-type receptors. The double-truncation mutant NR1(ΔC)/NR2A(ΔC) showed significantly less PKC potentiation that did either single truncation mutant [P < 0.01 vs. NR1(ΔC)/NR2A and vs. NR1₁₀₀/NR2A(ΔC)]. PKC potentiated responses of NR1₁₀₀/NR2A, NR1(ΔC)/NR2A, NR1₁₀₀/NR2A(ΔC), NR1(ΔC)/NR2A(ΔC), and NR1(ΔC2′)/NR2A receptors to 9.9 ± 0.6 (n = 24), 5.1 ± 0.5 (n = 16), 9.1 ± 0.9 (n = 7), 3.9 ± 0.5 (n = 6), and 11.8 ± 0.1.3 (n = 10) times control in Ca²⁺ and to 5.5 ± 0.5 (n = 19), 6.2 ± 0.7 (n = 11), 7.2 ± 1.1 (n = 7), 2.8 ± 0.4 (n = 6), and 10.8 ± 1.1 (n = 10) times control in Ba²⁺ Ringer's solution. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Splicing in of the C1 cassette reduces PKC potentiation. NMDA responses recorded from Xenopus oocytes expressing NR1/NR2A receptors before and after treatment with TPA at a holding potential of −60 mV. (A) Currents recorded in Ca²⁺ Ringer's solution before (Left) and after (Right) treatment with TPA (100 nM; 10 min) from oocytes expressing four receptors: NR1₁₀₀/NR2A, NR1₁₁₀/NR2A, NR1₁₀₁/NR2A, and NR1₁₁₁/NR2A. The rows show receptors that differ by presence or absence of the C1 cassette; columns show receptors that differ by presence of C2′ vs. C2. (B) Mean PKC potentiation values for eight receptors arranged in pairs; arrows show the effect of splicing in of C1. C1-containing receptors showed reduced PKC potentiation (filled circles, Ca²⁺ Ringer's solution; open circles, Ba²⁺ Ringer's solution). For NR1₁₀₀/NR2A vs. NR1₁₁₀/NR2A, potentiation was to 12.1 ± 1.0 (n = 11) vs. 4.2 ± 0.7 (n = 6) times the control response in Ca²⁺ (P < 0.001; Upper Right vs. Upper Left records in A). For NR1₁₀₁/NR2A vs. NR1₁₁₁/NR2A, potentiation was to 9.3 ± 0.9 (n = 8) vs. 4.8 ± 0.5 (n = 5) times the control response (P < 0.01; Lower Right vs. Lower Left records in A). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) The degree of PKC potentiation is not inversely related to basal current.

Alternative splicing of exon 22 has little effect on PKC potentiation. (A) Fig. 1C replotted with receptor pairs connected by arrows to indicate effect of exchange of C2′ by C2. (B) Wild-type NR1₁₀₀ and NR1₁₀₁ and mutant NR1₁₀₀(ΔC2′) subunits. (C) Currents recorded from oocytes expressing mutant NR1₁₀₀(ΔC2′)/NR2 receptors before and after TPA treatment. (D) Mean potentiation of NR1₁₀₁/NR2A, NR1₁₀₀/NR2A, and NR1₁₀₀(ΔC2′)/NR2A receptors. In Ca²⁺, potentiation of NR1₁₀₁/NR2A, NR1₁₀₀/NR2A, and NR1₁₀₀(ΔC2′)/NR2A receptors was to 10.6 ± 0.7 (n = 9), 9.9 ± 0.6 (n = 24), and 11.8 ± 0.1.3 (n = 10) times control (differences not statistically significant). In Ba²⁺, potentiation was to 5.3 ± 0.0.7 (n = 9), 5.5 ± 0.5 (n = 19), and 10.8 ± 1.1 (n = 10) times control (***, P < 0.001 vs. each wild-type receptor).