Molecular determinants for modulation of persistent sodium current by G-protein betagamma subunits

Abstract

Voltage-gated sodium channels are responsible for the upstroke of the action potential in most excitable cells, and their fast inactivation is essential for controlling electrical signaling. In addition, a noninactivating, persistent component of sodium current, I(NaP), has been implicated in integrative functions of neurons including threshold for firing, neuronal bursting, and signal integration. G-protein betagamma subunits increase I(NaP), but the sodium channel subtypes that conduct I(NaP) and the target site(s) on the sodium channel molecule required for modulation by Gbetagamma are poorly defined. Here, we show that I(NaP) conducted by Na(v)1.1 and Na(v)1.2 channels (Na(v)1.1 > Na(v)1.2) is modulated by Gbetagamma; Na(v)1.4 and Na(v)1.5 channels produce smaller I(NaP) that is not regulated by Gbetagamma. These qualitative differences in modulation by Gbetagamma are determined by the transmembrane body of the sodium channels rather than their cytoplasmic C-terminal domains, which have been implicated previously in modulation by Gbetagamma. However, the C-terminal domains determine the quantitative extent of modulation of Na(v)1.2 channels by Gbetagamma. Studies of chimeric and truncated Na(v)1.2 channels identify molecular determinants that affect modulation of I(NaP) located between amino acid residue 1890 and the C terminus at residue 2005. The last 28 amino acid residues of the C terminus are sufficient to support modulation by Gbetagamma when attached to the proximal C-terminal domain. Our results further define the sodium channel subtypes that generate I(NaP) and identify crucial molecular determinants in the C-terminal domain required for modulation by Gbetagamma when attached to the transmembrane body of a responsive sodium channel.

Figure 1.

Normalized current traces elicited by a step stimulus to 0 mV from a holding potential of -70 mV from representative tsA-201 cells transfected with rNa_v1.2a (A) or with hNa_v1.1 (B). The inset in A (oval) shows the end of the pulse on a 15-fold expanded vertical axis. C, Individual data points (circles) and mean values ± SEM (open squares) of I_NaP as a percentage of I_NaT in tsA-201 cells transiently transfected with rNa_v1.2a (n = 36) or hNa_v1.1 (n = 20; p < 0.01). The horizontal lines represent the first and 99th percentiles of the range.

Figure 2.

Changes in I_NaP produced by Na_v1.2 induced by cotransfection of G-protein β and γ subunits. A, B, Current traces from representative tsA-201 cells transfected with rNa_v1.2a alone (A) or cotransfected with Gβ₂ and Gγ₃ (B) elicited by a depolarization to 0 mV from a holding potential of -70 mV. C, Individual data points (circles) and mean values ± SEM (open squares) of I_NaP as a percentage of I_NaT in tsA-201 cells transfected with rNa_v1.2a and the indicated plasmids. The whiskers represent the first and 99th percentile values. Mean values were the following, for cells transfected with (from left to right): CD8 receptor as reporter gene (I_NaP = 4.5 ± 0.7%; n = 36); rNa_v1.2a, Gβ₂, Gγ₃, and CD8 receptor (I_NaP = 11.8 ± 2.3%; n = 18); rNa_v1.2a with YFP and CFP as reporter genes (I_NaP = 6.8 ± 1.0%; n = 20); rNa_v1.2a, Gβ₂ expressed in a bicistronic vector with YFP and Gγ₃ expressed in a bicistronic vector with CFP (I_NaP = 16 ± 1.0%; n = 26); rNa_v1.2a and Gβ₂ expressed in a bicistronic vector with YFP (I_NaP = 17 ± 2%; n = 27); rNa_v1.2a and Gγ₃ expressed in a bicistronic vector together with YFP (I_NaP = 7 ± 1%; n = 11). Because the CD8 control group and the CFP YFP control group did not differ significantly, they were pooled for additional statistical comparisons (mean of pooled data, I_NaP = 5.3 ± 0.6%; n = 56).

Figure 3.

Properties of I_NaT and I_NaP in tsA-201 cells transfected with Na_v1.2 alone or cotransfected with Gβ₂γ₃. A, Mean voltage dependence of activation for cells transfected with rNa_v1.2a alone (open circles) and cells cotransfected with rNa_v1.2a and Gβ₂γ₃ (filled squares). The solid lines are the Boltzmann fits that gave the following parameters (see Materials and Methods for definitions): Na_v1.2 alone, n = 9, V_½ = -5.9 ± 0.2 mV, k = -7.3 ± 0.2 mV; Na_v1.2a plus Gβ₂γ₃, n = 13, V_½ = -7.2 ± 0.2 mV, k = -6.6 ± 0.2 mV. B, Currents elicited by slow voltage ramps from a representative cell transfected with rNa_v1.2a alone (smaller current) or cotransfected with Gβ₂γ₃ (larger current). C, Voltage dependence of inactivation from representative cells with the mean level of I_NaP determined with 100 ms prepulses to a variable voltage followed by a test depolarization to 0 mV for Na_v1.2a alone (open circles) or cotransfected with Gβ₂γ₃ (filled squares). Normalized test pulse current is plotted as a function of prepulse potential. Mean parameters derived from fits of Boltzmann functions to the data were: rNa_v1.2a alone, n = 7, V_½ = -44.1 ± 0.2, k = 7.3 ± 0.1, C = 0.023 ± 0.002; rNa_v1.2a plus Gβ₂γ₃, n = 13 (fit with the sum of 2 Boltzmann components), V_½, 1 = -45.1 ± 0.4, k₁ = 8.3 ± 0.4, A2 = 0.35, V_½, 2 = -10.0 ± 0.7, k₂ = 6.0 ± 0.7, C = 0.117 ± 0.003).

Figure 4.

I_NaP conducted by hNa_v1.1 in two stable cell lines and its modulation by Gβ₂γ₃. I_NaP was measured in two cell lines, CL1 and CL2, stably expressing hNa_v1.1 and in CL2 transiently transfected with G-protein β₂ and γ₃ subunits. A, Current traces from representative cells during depolarizations to 0 mV from a holding potential of -70 mV from CL2 alone (A) or CL2 cotransfected with Gβ₂ and Gγ₃ subunits (B). C, I_NaP as a percentage of I_NaT in the indicated cells. Values for individual cells are plotted as small symbols; open squares indicate mean ± SEM. Horizontal bars represent the first and 99th percentiles of the range.

Figure 5.

Properties of transient and persistent current in Na_v1.1 alone and cotransfected with Gβ₂γ₃ as expressed in CL2. A, Mean voltage dependence of activation for CL2 stably expressing hNa_v1.1 (open circles) and CL2 cells transiently transfected with Gβ₂γ₃ (filled squares). The solid lines are fits of a Boltzmann equation to the mean data. For CL2 cells expressing Na_v1.1 (n = 18), V_a = -7.5 ± 0.5 mV and k = -7.3 ± 0.4 mV; for CL2 cells cotransfected with Gβ₂γ₃ (n = 6), V_a = -3.5 ± 0.5 mV and k = -6.4 ± 0.4 mV. B, Currents elicited by slow (70 mV/s) voltage ramps from a representative CL2 cell (smaller current) or a representative CL2 cell cotransfected with Gβ₂γ₃ (larger current). The ramps shown are from cells with I_NaP that is somewhat larger than the means of the control and Gβγ-transfected groups to more clearly demonstrate the voltage-dependent properties of I_NaP. C, Voltage dependence of inactivation from representative cells, selected to have approximately the mean level of I_NaP, determined with 100 ms prepulses to a variable voltage followed by a test depolarization to 0 mV for CL2 alone (open circles) or CL2 cotransfected with Gβ₂γ₃ (filled squares). Normalized test pulse current is plotted as a function of prepulse potential. Mean parameters derived from fits of Boltzmann functions to the data were as follows: for CL2, V_½ = -37.6 ± 0.1 mV, k = 6.2 ± 0.1 mV, C = 0.029 ± 0.003, n = 11; for CL2 cotransfected with Gβ₂γ₃, V_½ = -34.6 ± 0.2, k = 7.2 ± 0.2, C = 0.063 ± 0.003, n = 4.

Figure 6.

A, Normalized superimposed current traces from two representative tsA-201 cells transfected with rNa_v1.4 alone or cotransfected with Gβ₂ and Gγ₃. Currents were elicited by a depolarization to 0 mV from a holding potential of -70 mV. B, Normalized superimposed current traces from two representative tsA-201 cells transfected with rNa_v1.5 alone or cotransfected with Gβ₂ and Gγ₃. Currents were elicited by a depolarization to -10 mV from a holding potential of -70 mV. C, Bar graph showing I_NaP as percentage of I_NaT for cells transfected with the indicated sodium channel α subunit alone (black bars) or in combination with Gβ₂ and Gγ₃ (open bars). For rNa_v1.2a: control (pooled data), I_NaP = 5.3 ± 0.6%, n = 56; plus Gβ₂γ₃, I_NaP = 16 ± 1.0%, n = 26. For rNa_v 1.4: control, I_NaP = 1.9 ± 0.4%, n = 7; plus Gβ₂γ₃, 2.9 ± 0.6%, n = 8. For rNa_v1.5: control, 2.6 ± 0.5%, n = 10; plus Gβ₂γ₃, 3.7 ± 0.7%, n = 5. Error bars represent SEM.

Figure 7.

I_NaP in sodium channels containing mutations affecting the QXXER motif and their modulation by G-protein βγ subunits. Bar graph showing I_NaP as percentage of I_NaT for cells transfected with the indicated sodium channel α subunit alone (black bars) or in combination with Gβ₂ and Gγ₃ (open bars). For rNa_v1.2a, rNa_v1.4, and rNa_v1.5, see Figures 2 and 6 for mean values and errors. For Na_v1.2a/1.5CT: control, I_NaP = 1.4 ± 0.3, n = 7; Gβ₂γ₃, I_NaP = 21.8 ± 8.1, n = 5. For Na_v1.2a/1.4 CT: control, I_NaP = 8.7 ± 3.2, n = 8; Gβ₂γ₃, I_NaP = 12 ± 2, n = 12. For rNa_v1.2/1.4QxxER: control, I_NaP = 3.0 ± 0.7, n = 9; Gβ₂γ₃, I_NaP = 16 ± 6, n = 5. For rNa_v1.2a/AAMEAA: control, I_NaP = 5.9 ± 0.9, n = 10; Gβ₂γ₃, I_NaP = 20 ± 3, n = 7. Error bars represent SEM. Asterisks indicate significant modulation by GB₂γ₃ (p < 0.05).

Figure 8.

I_NaP in truncated Na_v1.2a channels and its modulation by G-protein β₂γ₃ subunits. Left, Diagram of the CT of the truncated channel constructs. The rectangle indicates the position of the QXXER motif. Right, Bar graph showing I_NaP as percentage of I_NaT for cells transfected with the indicated sodium channel α subunit construct alone (black bars) or in combination with Gβ₂ and Gγ₃ (open bars). For rNa_v1.2/K1890: control, I_NaP = 7.7 ± 0.8%, n = 9; plus Gβ₂γ₃, I_NaP = 9 ± 2%, n = 6. For rNa_v1.2/A1909: control, I_NaP = 3.6 ± 0.7%, n = 16; plus Gβ₂γ₃, I_NaP = 3.5 ± 0.6%, n = 8. For rNa_v1.2/S1929: control, I_NaP = 7 ± 1%, n = 8; plus Gβ₂γ₃, I_NaP = 9 ± 1%, n = 10. For rNa_v1.2/T1951: control, I_NaP = 6 ± 1%, n = 10; plus Gβ₂γ₃, I_NaP = 10 ± 2%, n = 9. For rNa_v1.2/S1977: control, I_NaP = 6.1 ± 0.9%, n = 21; plus Gβ₂γ₃, I_NaP = 10 ± 2%, n = 10. For rNa_v1.2/K1998: control, I_NaP = 4 ± 1%, n = 7; plus Gβ₂γ₃, 9 ± 1%, n = 13. For rNa_v1.2/1891Δ1977: control, I_NaP = 2.2 ± 0.3%, n = 14; plus Gβ₂γ₃, I_NaP = 14 ± 4%, n = 8. Asterisks indicate significant modulation of a construct by Gβγ (p < 0.05); # indicates modulation by Gβγ that is significantly reduced when compared with modulation of the full-length channel (p < 0.05). Error bars represent SEM.

Figure 9.

Modulation of rNa_v1.2a/1891Δ1977 channels by G-protein βγ subunits. A, B, Examples of representative current traces during depolarizations to 0 mV from a holding potential of -70 mV from a cell expressing rNa_v1.2a/1891Δ1977 channels alone (A) and from a cell coexpressing rNa_v1.2a/1891Δ1977 and Gβ₂γ₃ (B). C, D, Currents in response to voltage ramps at 70 mV/s from -60 to +40 mV from the same cells shown in A and B, respectively. E, Mean voltage dependence of activation for cells transfected with rNa_v1.2a/1891Δ1977 channels alone (open circles) and cotransfected with Gβ₂γ₃ (filled squares). The solid lines are fits of a Boltzmann equation to the mean data. For rNa_v1.2a/1891Δ1977: control, V_½= -5.6 ± 1.10 mV, k = -8.6 ± 0.47 mV, n = 9; plus Gβ₂γ₃, V_½ = -0.1 ± 1.55 mV and k = -8.5 ± 0.48 mV, n = 7. F, Voltage dependence of inactivation from a representative cell transfected with rNa_v1.2a/1891Δ1977 channels alone (open circles) and cotransfected with Gβ₂γ₃ (filled squares). Representative cells were chosen to have approximately the mean level of I_NaP. Mean parameters derived from fits of Boltzmann functions to the data were for rNa_v1.2a/1891Δ1977: control, V_½= -54.5 ± 0.15 mV, k = 6.3 ± 0.13 mV, C = 0.04 ± 0.002, n = 8; plus Gβ₂γ₃ (fit with 2 Boltzmann components), V_½, 1 = -54.5 ± 0.52 mV, k₁ = 4.0 ± 0.80 mV, A2 = 0.43, V_½, 2 = -50.8 ± 2.89, k₂ = 15.7 ± 2.67 mV, C = 0.03 ± 0.008, n = 3.

Scheme 1