Molecular Determinants for Modulation of Persistent Sodium Current by G-Protein βγ 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 βγ 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 Gβγ are poorly defined. Here, we show that I_NaP conducted by Na_v1.1 and Na_v1.2 channels (Na_v1.1 > Na_v1.2) is modulated by Gβγ; Na_v1.4 and Na_v1.5 channels produce smaller I_NaP that is not regulated by Gβγ. These qualitative differences in modulation by Gβγ 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 Gβγ. However, the C-terminal domains determine the quantitative extent of modulation of Na_v1.2 channels by Gβγ. Studies of chimeric and truncated Na_v1.2 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 Gβγ 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 Gβγ when attached to the transmembrane body of a responsive sodium channel.

Introduction

Voltage-gated Na⁺ channels are essential for neuronal excitability. They initiate fast action potentials and shape subthreshold electrical properties that contribute to important integrative functions. Voltage-gated Na⁺ channels are complexes of a poreforming α subunit and auxiliary β subunits (Catterall, 2000). The primary sequence of the α subunit contains four homologous domains (I-IV), each containing six predicted transmembrane α-helices (S1-S6). Five different α subunits are expressed in adult brain: Na_v1.1, Na_v1.2, Na_v1.3, Na_v1.5, and Na_v1.6 (Goldin et al., 2000).

In addition to the transient rapidly inactivating sodium current (I_NaT) (Hodgkin and Huxley, 1952), voltage-gated Na⁺ channels also generate a “persistent” current (I_NaP), which is maintained during long depolarizations, and whose importance in neurons has become increasingly apparent (Crill, 1996). I_NaP has been observed in multiple neuron types. It contributes to shaping repetitive firing (Azouz et al., 1997; Mantegazza et al., 1998; Parri and Crunelli, 1998; Brumberg et al., 2000), generating rhythmicity (Alonso and Llinas, 1989; Pennartz et al., 1997; Pape et al., 1998; Taddese and Bean, 2002) and amplifying both IPSPs and EPSPs (Schwindt and Crill, 1995; Stuart and Sakmann, 1995; Stuart, 1999). I_NaP has also been implicated in the generation of epileptiform activities (Kearney et al., 2001), suggesting that its regulation plays a crucial role in controlling electrical excitability (Schwindt and Crill, 1995; Stuart and Sakmann, 1995; Segal and Douglas, 1997; Stuart, 1999).

Central neuronal I_NaP is subject to modulation by intracellular signal transduction pathways. It is decreased in parallel with I_NaT by activation of muscarinic acetylcholine receptors and consequent phosphorylation by protein kinase C in hippocampal neurons (Cantrell et al., 1996). It is increased by hypoxia and nitric oxide in hippocampal neurons (Hammarstrom and Gage, 1998, 1999, 2000). In addition, I_NaP is increased when G-protein βγ subunits are coexpressed with Na_v1.2 channels (Ma et al., 1997). This increase was prevented by a putative Gβγ-binding peptide from the C terminus of the Na_v1.2 channel, suggesting that G-protein βγ subunits interact with that C-terminal site during modulation (Ma et al., 1997). Consistent with this idea, G-protein β subunits immunoprecipitate with sodium channel α subunits from cortical neuron preparations (Marin et al., 2001).

In the experiments presented here, we have compared modulation of I_NaP by G-protein βγ subunits for Na_v1.1, Na_v1.2, Na_v1.4, and Na_v1.5 channels. We show that both Na_v1.1 and Na_v1.2 conduct I_NaP (Na_v1.1 > Na_v1.2) that can be modulated by Gβγ, but Na_v1.4 and Na_v1.5 do not. Analysis of channel chimeras reveals that the transmembrane bodies of Na_v1.4 and Na_v1.5 channels are responsible for their lack of G-protein modulation rather than their C-terminal domains. However, the quantitative extent of modulation of Na_v1.2 channels is critically dependent on the C-terminal domain. Through analysis of deletion mutants, we identify new molecular determinants for modulation in the Na_v1.2 channels in the C terminus and show that the final 28 amino acids are necessary for generation of I_NaP and can substitute for the entire distal half of the C terminus in supporting regulation by Gβγ.

Materials and Methods

Isolation of cDNA for hNa_v1.1 and generation of cell lines CL1 and CL2. Six overlapping clones spanning the human Na_v1.1 coding region were isolated from human cerebellum and medulla cDNA libraries. A full-length cDNA was assembled from these partial clones using natively occurring restriction sites (BamHI, nucleotide 561; BamHI, nucleotide 2433; SphI, nucleotide 2694; NdeI, nucleotide 3786; SapI, nucleotide 5630). This was then inserted upstream of the internal ribosome entry site (IRES) element in the mammalian expression vector pCIN5 (Rees et al., 1996). Cell lines stably expressing hNa_v1.1 were isolated after transfection of human embryonic kidney (HEK) 293 cells with 2 μg of pCIN5-hNa_v1.1 and selection for 3-4 weeks in 800 μg/ml Geneticin-G418 (Burbidge et al., 2002). Clonal cell lines CL1 and CL2 were isolated after two rounds of single-cell dilution cloning.

Construction of Na_v1.2 and Na_v1.5 channel mutants. The mammalian expression plasmids, pCDM8-rH1 encoding the rat cardiac rNa_v1.5 α subunit and pCDM8-rIIA encoding the rat brain rNa_v1.2a α subunit, were described previously (Qu et al., 1994; Linford et al., 1998). cDNA encoding the full-length human brain type I Na⁺ channel hNa_v1.1 was in pCIN5-hNa_v1.1 (Clare et al., 2000). cDNA encoding the rat skeletal muscle Na⁺ channel rNa_v1.4 (Trimmer et al., 1989) (kind gift from Dr. P. Ruben, Utah State University, Logan, UT) was subcloned into pCDM8. The chimeric Na⁺ channel rNa_v1.2/Na_v1.5 C terminus (CT) was described previously (Mantegazza et al., 2001). Chimeric Na⁺ channel Na_v1.2/1.4 CT was comprised of the Na_v1.4 C-terminal cytoplasmic domain (beginning at amino acid position E1592) appended to Na_v1.2a cDNA after residue L1776. In mutant Na_v1.2/1.4QxxER, the Na_v1.2a sequence was changed from RIQMEER (R1876 to R1882) to KQTMEEK, the equivalent sequence in rat Na_v1.4. In Na_v1.2/AAMEAA mutant, the rNa_v1.2a sequence IQMEER beginning at I1875 was converted to AAMEAA. The truncation mutants Na_v1.2ΔK1890, Na_v1.2ΔA1909, Na_v1.2ΔS1929, Na_v1.2ΔT1951, Na_v1.2ΔS1977, and Na_v1.2ΔK1998, which delete segments of C-terminal cytoplasmic region of rat Na_v1.2a, have been described previously (Mantegazza et al., 2001). The Na_v1.2a deletion mutant Na_v1.2/Δ1891-1977 removed an internal segment bounded by amino acid residues V1891 and S1977. Full-length cDNA of bovine G-protein β₂ subunit was cloned into a bicistronic mammalian expression plasmid, pIRES-enhanced yellow fluorescent protein (YFP) (Clontech, Palo Alto, CA), which encodes the yellow variant of the green fluorescent protein (GFP). This construct was called pGβ₂IRES-YFP. The G-protein γ₃ subunit was cloned into a cyan variant of the same bicistronic vector and was named pGγ₃IRES-cyan fluorescent protein (CFP). All Na⁺ channel mutations generated by PCR mutagenesis were sequenced in between the restriction sites used for subcloning to verify that they were free of unintended alteration of sequences.

Expression of sodium channels. The fluorescent proteins in the bicistronic vectors or the CD8 receptor in pCD8-neomycin were used as markers of transfected cells (Margolskee et al., 1993). Plasmids were cotransfected into tsA-201 cells by CaPO₄ precipitation. Cells were grown to 75% confluence in 35 mm tissue culture dishes (Corning, Acton, MA). After addition of DNA, they were incubated at 37°C in 3% CO₂. Twelve hours after transfection, cells were removed from the dishes using 2 mm EDTA in PBS and replated at low density for electrophysiological recordings. HEK293 cell lines that expressed hNa_v1.1, CL1 and CL2, were transfected using TransFast (Promega, Madison, WI). Positive transfectants were selected visually either by anti-CD8-coated beads (Dynal, Brown Deer, WI) or by their fluorescence using a Nikon (Tokyo, Japan) Eclipse TE300 microscope for epifluorescence. Cells transfected with plasmids encoding green fluorescent proteins were selected visually using excitation/emission cubes for YFP (Chroma 41028; Chroma Technology, Rockingham, VT) and CFP (Chroma 31044v2). The tsA-201 subclone of HEK293 cells and stably transfected HEK cell lines were maintained as described previously (Herlitze et al., 1996).

Electrophysiology and data analysis. Whole-cell patch-clamp recordings were performed at room temperature using an Axopatch 200B amplifier (Axon Instruments, Union City, CA). Capacitative currents were minimized by means of the amplifier circuitry. Seventy percent prediction and 90-95% series resistance compensation were used routinely. The remaining capacity transients and leakage currents were eliminated using P/4 subtraction. The intracellular solution contained the following (in mm): 120 Cs-aspartate, 5 NaCl, 2 MgCl₂, 10 EGTA, 10 HEPES, pH 7.3 with CsOH. The extracellular solution contained the following (in mm): 140 NaCl, 2 CaCl₂, 2 MgCl₂, 10 HEPES, pH 7.4 with NaOH. pClamp 6.0.4 software and a Digidata 1200 interface (Axon Instruments) were used to generate the voltage stimuli and to acquire the current signals, which were filtered at 5 or 10 kHz. The data were analyzed using pClamp and Origin 6.0 (OriginLab, Northampton, MA) on a Pentium II-based personal computer (Intel, Santa Clara, CA). Unless indicated, the holding potential for voltage clamp recordings was -70 mV.

Conductance-voltage (G-V) relationships were calculated from the current-voltage (I-V) relationships according to extended Ohm's law G_Na = I_Na/(V - E_Na), where I_Na was the peak Na⁺ current measured at potential V, G_Na was the sodium conductance, and E_Na was the calculated Nernst equilibrium potential for Na⁺. Curves were fit using the Levenberg-Marquardt algorithm. The voltage dependence of activation and voltage dependence of fast inactivation were fitted to Boltzmann relationships of the following form: normalized G_Na = ((1 - C)/(1 + exp (V - V_½)/k)) + C, where V was the membrane potential, V_½ was the voltage of half-maximal activation or inactivation, k was a slope factor, and C was the baseline noninactivating current. For cells with large I_NaP, a second Boltzmann component was included, as follows: ((1 - C - A2)/(1 + exp ((V - V_½,1)/k₁))) +(A2/(1 + exp((V - V_½, 2)/k₂))) + C. The statistical results are given as mean ± SEM. The threshold p value for statistical significance was 0.05.

Sequence alignments were produced by ClustalW (Thompson et al., 1994) followed by manual editing to produce the alignments shown.

Results

I_NaP conducted by rNa_v1.2a and hNa_v1.1 channels

Sodium currents produced by expression of Na_v1.2 channels in tsA-201 cells have two components: a large, rapidly inactivating component followed by a persistent component that fails to inactivate by the end of 60 ms depolarizations (Fig. 1A). I_NaP was quantitated as the mean current remaining between 45 and 55 ms after the beginning of the voltage step and expressed as a percentage of I_NaT. The level of I_NaP varied considerably from cell to cell with a mean of 4.5 ± 0.7% of I_NaT (n = 36) (Fig. 1C). hNa_v1.1 channels expressed similarly in tsA-201 cells conducted significantly larger I_NaP than rNa_v1.2a (mean I_NaP = 13 ± 2%; n = 20; p <0.01) (Fig. 1 B, C), similar to the results of Smith and Goldin (1998) for expression of rNa_v1.1 in Xenopus oocytes. The variability of the fraction of I_NaP versus I_NaT among single clonal tsA-201 cells transfected with identical cDNA for either rNa_v1.2a and hNa_v1.1 channels (Fig. 1C) suggests that cellular signaling processes that differ from cell to cell can control the level of I_NaP.

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.

Modulation of I_NaP through rNa_v1.2a channels by Gβγ

I_NaP conducted by Na_v1.2 channels is increased by coexpression of G-protein βγ subunits (Fig. 2B,C) (Ma et al., 1997). Transfection of Gβ₂γ₃ increased I_NaP from 4.5 ± 0.7 to 11.8 ± 2.3% (n = 18) (Fig. 2C). The level of I_NaP obtained with cotransfection of plasmids encoding G-protein β₂ and γ₃ subunits was highly variable in different cells (Fig. 2C), and this variability complicated the study of I_NaP modulation. One possible source of variability was the requirement for cotransfection of cDNAs encoding three different proteins: the Na_v1.2 α subunit, the G-protein β₂ subunit, and the G-protein γ₃ subunit. To ensure expression of both G-protein β and γ subunits in all cells studied, we cloned the cDNAs encoding these G-protein subunits into bicistronic vectors containing an IRES followed by cDNA encoding the yellow or cyan mutants of GFP as reporter genes (Trouet et al., 1997). Control cells transfected with the same fluorescent protein vectors lacking G-protein subunits (Fig. 2C, CFP, YFP) had I_NaP that was not significantly different from cells transfected with rNa_v1.2 and CD8 (CD8, mean I_NaP = 4.5 ± 0.7%, n = 36; CFP, YFP, mean I_NaP = 6.8 ± 1.0%, n = 20; p = 0.08) (Fig. 2C). When Gβ₂-YFP and Gγ₃-CFP were cotransfected, and only cells clearly fluorescent for both markers were studied, the data were less variable than without use of bicistronic vectors (mean I_NaP = 16 ± 1.0%; n = 26) (Fig. 2C, YFP Gβ₂,/CFP Gγ₃). Variance of the experimental groups decreased from 92 with CD8 as a reporter to 34 with bicistronic Gβγ as reporter (p = 2 × 10^-8). As a result of the reduced variability using GFP reporters to monitor expression, smaller numbers of experiments were required to obtain meaningful results when Gβγ coexpression was studied. The IRES vectors were used to verify expression in all additional studies.

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).

Modulation of I_NaP through rNa_v1.2a channels by Gβ₂ subunits alone

To better characterize Gβγ modulation, we examined the ability of single G-protein β and γ subunits to increase I_NaP. Expression of only Gβ₂ with rNa_v1.2a was sufficient to increase I_NaP comparably to that caused by expression of Gβγ (mean I_NaP = 17 ± 2%; n = 27; p = 0.00005) (Fig. 2C, YFP, Gβ₂). In contrast, expression of Gγ₃ alone did not modulate I_NaP (mean I_NaP = 7 ± 1%; n = 11; p = 0.8) (Fig. 2C, CFP, Gγ₃). Expression of Gβ subunits is also sufficient for G-protein modulation of calcium channels (Herlitze et al., 1996).

Effect of Gβ₂γ₃ on voltage-dependent gating of rNa_v1.2

We compared the voltage-dependent gating of I_NaT and I_NaP before and after modulation by Gβ₂γ₃. As in previous work (Ma et al., 1997), the major effect of Gβ₂γ₃ on the properties of rNa_v1.2a was the increase in I_NaP. Little change in the voltage dependence of activation of I_NaT was observed (control, V_½ = -5.9 ± 0.2 mV; Gβ₂γ₃, V_½ =-7.2 ± 0.2 mV) (Fig. 3A). Coexpression of Gβγ strikingly increased the amplitude of I_NaP as measured in response to slowly rising voltage ramps (Fig. 3B), during which I_NaT is inactivated by the slow ramp depolarization. This protocol gives the clearest visual illustration of the large effect of Gβγ coexpression on I_NaP. The presence of I_NaP resulted in a biphasic inactivation curve, which was fit by the sum of two Boltzmann relationships (Fig. 3C). The component of current that had the more positive voltage dependence of inactivation also had slower inactivation kinetics and corresponded to I_NaP (data not shown) (Ma et al., 1997). Gβγ caused an increase in the fraction of current inactivating with the more positive voltage dependence with little change in the voltage dependence of the individual components (Fig. 3C).

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).

Modulation of I_NaP conducted by hNa_v1.1

We were concerned that the large variation in I_NaP among single tsA-201 cells expressing hNa_v1.1 channels (Fig. 1) might impair measurements of Gβγ modulation, even using our bicistronic vectors and imaging transfected Gβγ. Therefore, we examined the level of I_NaP in two clonal cell lines, derived by stable transfection of hNa_v1.1 into HEK293 cells (Clare et al., 2000). In the first cell line tested, CL1, the level of I_NaP was similar to that observed after transient transfection of hNa_v1.1 into tsA-201 cells (mean I_NaP = 13 ± 4%; n = 10) (Fig. 4C). In the second cell line, CL2, lower levels of I_NaP were observed (mean I_NaP = 2.5 ± 0.4%; n = 25) (Fig. 4A,C, CL2). These results with two clonal cell lines are consistent with the striking variation of I_NaP among single cells transfected transiently (Fig. 1C).

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.

Because of its low level of intrinsic I_NaP in control, we chose CL2 to study the effect of Gβ₂γ₃ on I_NaP. We cotransfected pGβ₂IRES-YFP and pGγ₃IRES-CFP into CL2 cells stably expressing hNa_v1.1. I_NaP with Gβ₂γ₃ was significantly higher than in untransfected CL2 cells (mean I_NaP = 11 ± 2%; n = 6; p = 10^-7 relative to untransfected CL2) (Fig. 4B,C, CL2, YFP Gβ₂, CFP Gγ₃). Thus, as for rNa_v1.2a, I_NaP conducted by hNa_v1.1 is also increased by coexpression of Gβ₂γ₃.

Effect of Gβ₂γ₃ on voltage-dependent gating of hNa_v1.1

Cotransfection of hNa_v1.1 with Gβ₂γ₃ resulted in a small positive shift in the voltage dependence of activation of I_NaT (Fig. 5A). Coexpression of Gβ₂γ₃ also substantially increased I_NaP measured using slowly rising voltage ramps (Fig. 5B). The increased persistent sodium current in cells expressing Gβ₂γ₃ caused a positive shift in the overall inactivation curve, and the baseline of noninactivating current at positive potentials was significantly increased (Fig. 5C). However, as a result of the relatively small level of I_NaP in this cell line, even in the presence of Gβ₂γ₃, the second Boltzmann component from these cells was not as clearly resolved as for Na_v1.2. As described above, the relative size of I_NaP in tsA-201 cells transiently transfected with hNa_v1.1 or in cell line CL1, in which hNa_v1.1 had been stably expressed in HEK293 cells, was much larger than in CL2. However, the voltage-dependent properties of I_NaT and I_NaP in these cells were not different from those of CL2 except for the larger amplitude of I_NaP (data not shown). Thus, for both hNa_v1.1 and rNa_v1.2a, coexpression of Gβ₂γ₃ increases both the amount of I_NaP relative to I_NaT and the fraction of current with slowed and positive voltage-dependent inactivation without major effects on the voltage dependence of activation of I_NaT.

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.

Modulation of I_NaP through rNa_v1.4 and rNa_v1.5 channels by Gβ₂γ₃

We tested whether skeletal muscle rNa_v1.4 and cardiac rNa_v1.5 channel isoforms were modulated by Gβ₂γ₃ when cotransfected in tsA-201 cells. When transfected alone, rNa_v1.4 and rNa_v1.5 give little I_NaP (I_NaP = 1.9 ± 0.4%, n = 7; and 2.6 ± 0.5%, n = 10, respectively) (Fig. 6). When Gβ₂γ₃ was coexpressed with either Na_v1.4 or Na_v1.5 channels, no significant increase in I_NaP was observed (Na_v1.4 plus Gβ₂γ₃, 2.9 ± 0.6%, n = 8; Na_v1.5 plus Gβ₂γ₃, 3.7 ± 0.7%, n = 5) (Fig. 6). Thus, little I_NaP is produced by these two isoforms, and it is not increased by coexpression of Gβ₂γ₃.

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.

Modulation of I_NaP by the CT domains of rNa_v1.4 and rNa_v1.5 channels

We proposed previously that the CT of rNa_v1.2a is a domain required to produce I_NaP in that channel (Ma et al., 1997). To test this, we used the construct Na_v1.2/1.5 CT (Mantegazza et al., 2001). In this construct, the rNa_v1.5 CT beginning with E1755 replaced the CT of rNa_v1.2a, which had been truncated before E1777. This chimeric mutation did not increase I_NaP in the absence of modulation by Gβγ (Fig. 7). However, the chimera was strongly modulated by Gβ₂γ₃ (Fig. 7). Thus, although the CT of Na_v1.5 does not produce modulation when attached to its native subunit, it produces strong modulation of rNa_v1.2a.

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).

This contrasts with the effects observed in chimeras of rNa_v1.4 and rNa_v1.2a, in which the CT of rNa_v1.2a was completely or partially replaced by the analogous sequence from Na_v1.4. In Na_v1.2/1.4 CT, the rNa_v1.4 CT beginning with E1592 replaced the CT of rNa_v1.2a, which had been truncated before E1777. Substitution of the C terminus in Na_v1.2/1.4 CT did not significantly change the level of I_NaP under control conditions. In addition, there was no increase after coexpression of Gβ₂γ₃ (Fig. 7). Thus, the Na_v1.4 C terminus does not support modulation by Gβγ. These findings are consistent with the CT domains of Na_v1.2 and Na_v1.5 containing molecular determinants required for modulation of I_NaP by Gβγ that are absent in the Na_v1.4 CT.

Requirement for the rNa_v1.2a CT and its QxxER motif for modulation by Gβγ

A short sequence motif (QxxER) in the Na_v1.2a CT was suggested initially to be an important molecular determinants of Gβγ modulation based on our finding that a peptide containing this motif effectively inhibited G-protein modulation (Ma et al., 1997). A QxxER motif is conserved in hNa_v1.1, partially conserved in rNa_v1.5, but absent in the CT of rNa_v1.4. To assess the role of the QxxER region of rNa_v1.2a in modulation by Gβ₂γ₃, we constructed two mutant channels. For the first, we used the CT of Na_v1.4 to replace the QxxER sequence in the CT of Na_v1.2a, because Na_v1.4 did not support modulation by Gβγ. In Na_v1.2/1.4QxxER, the Na_v1.4 CT from K1691 to K1697 replaced the QxxER region in rNa_v1.2a CT from R1876 and R1882. In the second construct, Na_v1.2/AAMEAA, I1875, Q1876, E1879, and R1880 in rNa_v1.2a were replaced with alanines, thus removing the specific side chains of the critical amino acids in the QxxER motif. Both Na_v1.2/1.4QxxER and Na_v1.2/AAMEAA were modulated fully by Gβ₂γ₃ (Fig. 7), indicating that this motif is not necessary for modulation of Na_v1.2 by Gβγ. Molecular determinants for modulation of Na_v1.2a sodium channel by Gβγ must reside in other regions of the Na_v1.2 CT that have amino acid sequence differences from the Na_v1.4 CT.

Requirement for the last 28 amino acids of rNa_v1.2a CT for modulation by Gβγ

To localize the molecular determinants in the CT responsible for I_NaP and its modulation by Gβγ, we used a series of rNa_v1.2a mutants in which the CT was truncated at different positions, as described previously (Mantegazza et al., 2001). Mutants that were truncated on the N-terminal side of K1890 did not express measurable sodium current or gave rise to currents that were too small to study (Mantegazza et al., 2001). Therefore, we only tested mutants in which the truncation was on the C-terminal side of K1890. Although the mean level of I_NaP in the absence of transfected Gβγ subunits varied among the truncation mutants, the differences from wild type (WT) were not significant (p > 0.05) (Fig. 8). However, the effects of cotransfected Gβγ on these mutant channels differed significantly. Coexpression of Gβ₂γ₃ did not significantly increase I_NaP when the channel was shortened beyond T1951 in mutants ΔΚ1890, ΔΚ1909, ΔΚ1929, and ΔT1951 (Fig. 8, bars lacking asterisks). In constructs with longer C termini, including ΔS1977 and ΔK1998, I_NaP was increased significantly by Gβ₂γ₃ coexpression (Fig. 8, bars with asterisks). However, the increase of I_NaP caused by Gβ₂γ₃ coexpression with ΔS1977 and ΔK1998 was not as large as for WT rNa_v1.2, as denoted by the bars labeled with #. The pattern of decreased modulation by Gβγ in these deletion constructs suggests that the amino acid residues between positions 1951 and 1977 and between position 1977 and the C terminus of the protein at position 2005 are important for modulation by Gβγ.

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.

To test the functional role of the final 28 amino acid residues directly, we constructed the mutant Na_v1.2a/Δ1891-1977, in which amino acid residues from V1891 to S1977 were deleted. This is equivalent to Na_v1.2a/ΔK1890 having the last 28 amino acids of the C terminal added directly to it. Modulation of this construct by Na_v1.2a/Δ1891-1977 was similar to modulation of the WT Na_v1.2a channel (Fig. 8). Thus, the last 28 amino acids of the CT are necessary for modulation by Gβγ and are sufficient to replace the requirement for the remainder of the distal half of the C-terminal domain.

Voltage-dependent gating and modulation of Na_v1.2Δ1891-1977

We analyzed the functional properties of Na_v1.2Δ1891-1977 more completely and compared them with WT. Coexpression of Gβ₂γ₃ increased I_NaP during single depolarizations (Fig. 9A,B) or during slow voltage ramps (Fig. 9C,D). The voltage dependence of activation of I_NaT conducted by Na_v1.2a/Δ1891-1977 was unchanged from that of wild-type Na_v1.2a, and, as for WT, cotransfection of Gβγ caused little change in the voltage dependence of activation (Fig. 9E). The voltage dependence of inactivation was shifted ∼10 mV more negative than for WT Na_v1.2a. This is consistent with previous findings implicating the C-terminal region of the sodium channel in modulating inactivation (Deschenes et al., 2001; Mantegazza et al., 2001; Cormier et al., 2002; Motoike et al., 2004). Cotransfection of Gβ₂γ₃ subunits with Na_v1.2a/Δ1891-1977 did not appreciably change the negative component of the steady-state inactivation curve but increased the amplitude of a more positive component of inactivation (Fig. 9F, closed squares), similar to Gβγ modulation of WT. Thus, although the deletion in Na_v1.2a/Δ1891-1977 causes a negative shift in the voltage dependence of inactivation of I_NaT under control conditions, I_NaP is modulated essentially normally by cotransfection of Gβ₂γ₃.

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.

Comparison of the amino acid sequences of the distal C-terminal domains of Na_v1 channels

Because the distal CT of Na_v1.1 and Na_v1.2 can support modulation by Gβγ, but the CT of Na_v1.4 cannot, it is interesting to compare the amino acid sequences of the distal CT showing the identities (black blocks) and similarities (gray blocks) to Na_v1.2a, as follows:

Scheme 1.

Na_v1.1 is quite similar to Na_v1.2 but is seven amino acids shorter. Among the 21 aligned amino acid residues, 10 are identical, two are similar, and there are no gaps. In contrast, Na_v1.4 has only five identical and one similar residues, and a four-residue gap in Na_v1.2 is required for alignment. Na_v1.5 has seven identical and three similar residues but requires gaps of four and five residues in Na_v1.2 for alignment. Thus, the similarities of the Na_v1.1 and Na_v1.2 sequences support a common function for their distal C-terminal segments, whereas the differences in sequence of Na_v1.5 and especially Na_v1.4 C-terminal sequences suggest different functions. Additional experiments will be required to determine whether these segments play important roles in some aspect of G-protein modulation of cardiac and skeletal muscle sodium channels.

Discussion

Persistent sodium current in neurons

I_NaP is implicated in many aspects of neuronal function, including shaping bursting behavior, controlling firing frequency, integrating synaptic input, and generating epileptiform hyperexcitability (see Introduction). I_NaP is thought to be conducted by the same sodium channels that generate I_NaT in some neurons (Alzheimer et al., 1993; Taddese and Bean, 2002), but the sodium channel subtypes that generate I_NaP and the mechanisms that regulate I_NaT versus I_NaP in neurons are unknown. Here, we have addressed this gap in knowledge by analyzing the level of I_NaP generated by different sodium channel subtypes, demonstrating subtype-selective effects of Gβγ subunits on I_NaP and identifying an essential molecular determinant of Gβγ modulation of Na_v1.2 channels.

Different levels of persistent sodium current conducted by sodium channel subtypes

Our results show that different sodium channel α subunits intrinsically generate different levels of I_NaP. Na_v1.1 and Na_v1.2 both conduct substantial I_NaP, but when expressed transiently in tsA-201 cells, Na_v1.1 produces significantly more I_NaP than Na_v1.2. Conversely, Na_v1.4 and Na_v1.5 conduct little I_NaP. Although the CT has been implicated in the modulation of I_NaP by Gβγ (Ma et al., 1997; this work) and in setting the different inactivation properties of Na_v1.5 relative to Na_v1.2a or Na_v1.4 channels (Mantegazza et al., 2001; Deschenes et al., 2001; Cormier et al., 2002; Motoike et al., 2004), the CT of Na_v1.4 did not reduce I_NaP produced by the Na_v1.2a in chimera Na_v1.2/1.4 CT. Thus, the intrinsic level of I_NaP is determined by the pre-CT portion of the channel rather than by the CT. However, the CT alters modulation of I_NaP when attached to a sodium channel that conducts substantial I_NaP.

Regulation of persistent sodium currents by G-proteins

Although I_NaP is important physiologically, few experimental conditions have been identified that alter I_NaP relative to I_NaT. We showed previously that coexpression of G-protein βγ subunits can substantially increase I_NaP, relative to I_NaT, and we implicated the CT of the Na_v1.2 channel α subunit in those effects by demonstrating block of I_NaP by a competing CT peptide (Ma et al., 1997). Our present results extend those findings in three important respects. First, using bicistronic vectors and fluorescence to verify expression of Gβ and Gγ, we substantially reduced cell-to-cell variability in Gβγ modulation of I_NaP, thereby providing stronger evidence that Gβγ subunits are important regulators of I_NaP. Second, with this improved assay, we identified a novel subtype specificity for Gβγ modulation of I_NaP.Gβγ subunits can regulate I_NaP of Na_v1.1 and Na_v1.2 channels but not of Na_v1.4 or Na_v1.5 channels. However, the CT of Na_v1.5, but not that of Na_v1.4, can support modulation by Gβ₂γ₃ when attached to Na_v1.2a. The ability of the Na_v1.5 CT to support modulation when incorporated in a chimera with Na_v1.2a but not in Na_v1.5 highlights the importance of the pre-CT portion of the channel in permitting modulation by Gβγ acting via the CT. Finally, our improved methods allowed analysis of deletion mutants to directly identify the site of Gβγ action in the CT. Surprisingly, these results implicate the final 28 amino acids of Na_v1.2 in Gβγ binding and regulation. We propose that this short segment forms part of the Gβγ interaction site and plays an important role in modulation of channel gating.

Gβγ subunits do not greatly alter the voltage dependence of activation or inactivation of I_NaT. In contrast, the voltage dependence of inactivation of I_NaP is shifted positively with respect to I_NaT. These results are consistent with a mechanism in which Gβγ generates I_NaP by destabilizing fast inactivation and switching a fraction of sodium channels to a noninactivating gating mode with slowed and positively shifted inactivation (Ma et al., 1997). The molecular basis for this might be Gβγ-induced destabilization of binding of the inactivation gate to its receptor.

Molecular determinants of Gβγ modulation in the C terminus

Ma et al. (1997) proposed that Gβγ bound to the CT of the Na_v1.2 channel to modulate I_NaP, because its effect was blocked by a peptide containing the QMEER sequence of the CT. Recent biochemical evidence shows that Gβγ indeed binds to Na_v1.2 sodium channel α subunits (Marin et al., 2001) and to the CT of the Na_v1.3 α subunit (Lenkowski et al., 2004). These findings are consistent with an essential role for the CT in Gβγ modulation of I_NaP. Peptides containing a similar QxxER motif reduced Gβγ modulation of other target proteins, including calcium channels (Chen et al., 1995; Herlitze et al., 1997; Zamponi et al., 1997). Such peptides are expected to bind Gβγ subunits and inhibit their action. However, we show that the QxxER motif is unnecessary for modulation of Na_v1.2 by coexpressed Gβγ. Perhaps the QxxER motif tethers and localizes G-protein βγ subunits near their site of action in high local concentration, enhancing Gβγ effects at low levels of G-protein activation.

Our results show that the last 28 amino acids of rNa_v1.2 are necessary for Gβγ modulation of Na⁺ channels. Modulation was lost when the CT of rNa_v1.2a was replaced with that of rNa_v1.4 or when the CT of Na_v1.2 was truncated at K1890. Addition of only the last 28 amino acids of the rNa_v1.2a CT to this mutant restored Gβγ modulation. Thus, these 28 residues are sufficient for modulation of rNa_v1.2a by Gβγ, and the intervening residues between K1890 and S1977 are unnecessary.

The distal CTs of sodium channels contain other motifs of potential importance in regulation of channel function, localization, and degradation. An IQ domain starting at position 1912 in Na_v1.2 has been implicated in interactions of calmodulin with several sodium channel isoforms (Deschenes et al., 2002; Tan et al., 2002; Herzog et al., 2003; Kim et al., 2004). Just N-terminal to the site of G-protein regulation, a sequence (PPSY) is implicated in interaction of Nedd4 ubiquitin ligase and is present in Na_v1.1, Na_v1.2, and Na_v1.5 but not in Na_v1.4 (van Bemmelen et al., 2004). In addition, the final three amino acids of Na_v1.4 and Na_v1.5 are potential interaction sites for PDZ (postsynaptic density-95/Discs large/zona occludens-1) proteins such as the syntrophin family of dystrophin-associated proteins, which may contribute to channel localization in cardiac and skeletal muscle (Gee et al., 1998). These processes could potentially interact with regulation by G-proteins.

Comparison of regulation of I_NaP in transfected cells and neurons

I_NaP in neurons is generally smaller than I_NaP in transfected tsA-201 cells, even without modulation by Gβγ. The ratio of I_NaP to I_NaT reported here would probably be incompatible with normal neuronal function in vivo (Kearney et al., 2001). Evidently, other regulation in neurons limits I_NaP. Nevertheless, I_NaP in neurons resembles I_NaP produced by Gβγ in transfected cells in several respects. Activation of I_NaP of sodium channels expressed in tsA-201 cells is shifted negatively relative to I_NaT (data not shown), as it is in neurons (Brown et al., 1994; Mantegazza et al., 1998; Taddese and Bean, 2002). In addition, I_NaP in both neurons and tsA cells has positively shifted voltage dependence of inactivation relative to I_NaT. Our results with transfection of single sodium channel isoforms demonstrate that I_NaP can result from modulation of the same sodium channels that produce I_NaT (Ma et al., 1997). Thus, our working hypothesis is that Gβγ modulation is an important regulatory influence on I_NaP in neurons.

The persistent current in a neuron is probably produced by a mixture of sodium channel subtypes with individual propensities for producing I_NaP. The sodium channel α subunit(s) responsible for I_NaP has not been identified unequivocally in any central neuron, but it has been proposed that Na_v1.6 is the primary contributor to I_NaP in cerebellar Purkinje neurons and in prefrontal cortex pyramidal neurons (Raman et al., 1997; Maurice et al., 2001). In addition to Na_v1.6, the other primary brain Na⁺ channel α subunit isoforms, Na_v1.1, Na_v1.2, and Na_v1.3, all can produce I_NaP. Their relative contributions could be modulated according to cell expression pattern and/or degree of neuromodulation of individual channel subtypes.

Modulation of persistent sodium current in neurons

Dynamic modulation of I_NaP would dramatically modify integrative properties of neurons. Activation of muscarinic acetylcholine receptors in hippocampal neurons reduces both I_NaP and I_NaT to comparable extents via protein kinase C signaling (Cantrell et al., 1996). There have been few reports of positive modulation of I_NaP in neurons (see Introduction). Increase of I_NaP by dopamine has been observed in prefrontal cortex (Gorelova and Yang, 2000) (but see Maurice et al., 2001). Modulation by free Gβγ in neurons might occur locally in membrane microdomains (Galbiati et al., 2001) strategically positioned to affect action potential generation in the axon hillock, action potential back-propagation at dendritic branches, or other localized electrical events. Such tightly delimited modulation might be critically important for integrative properties of the neuron but would make little contribution to whole-cell sodium current. Additional studies of sodium channel modulation by G-proteins in vitro may provide methods to probe the mechanism and significance of this form of modulation in intact neurons more incisively.