β4 expression increases persistent Na current in HEK-NaV1.1 cells
To test the influence of the Na channel β4 subunit on the properties of Na currents, TTX-sensitive currents were recorded from HEK cells stably expressing NaV
1.1 with and without transfection of β4. Activation curves were obtained by evoking transient Na currents from −110 mV with depolarizing steps in 5-mV increments. (top
) illustrates currents and the activation curve from a representative control cell, along with a conductance-voltage curve from a cell transfected with β4. Voltage control was assessed in each cell (see Methods) and all cells included for analysis are shown in Supplementary Figure 1
. Neither the slope factors (k) nor the maximum conductances (Gmax
) were significantly changed by the expression of β4 ( bottom
; control vs.
β4: k = 4.9 ± 0.3 vs.
5.2 ± 0.3 mV, p
= 45 ± 8 vs.
38 ± 7 nS, p
N=15, 13). The half-maximal voltage of activation (V1/2
), however, was sensitive to β4 expression. In control cells, V1/2
was −16.8 ± 0.9 mV and β4 expression negatively shifted this value to −21.8 ± 1.0 mV (p
). Boltzmann curves with mean fit parameters (, bottom right
) illustrate the average effect of β4 expression on activation. The observation that the properties of Na currents were modified after β4 transfection suggests that β4 was successfully incorporated into channel complexes. A similar leftward-shift of the activation curve has been reported when β4 is co-expressed with NaV
1.2 in tsA-201 cells (Yu et al., 2003
Next, we measured steady-state inactivation (availability) of Na currents by applying 100-ms conditioning steps followed by steps to 0 mV. Peak currents evoked at 0 mV were normalized, plotted as availability vs. voltage, and fit with Boltzmann functions modified to incorporate a non-inactivating component. Data for a representative control cell, along with availability for a β4-transfected cell, are shown in (top). Mean values for fits and Boltzmann curves with mean fit parameters are shown in (bottom). With β4 expression, V1/2 shifted slightly negative and the slope factor increased slightly (control vs. β4: V1/2 = −42.1 ± 0.7 vs. −45.7 ± 1.7 mV, p=0.07; k = 6.4 ± 0.4 vs. 8.1 ± 1.0 mV, p=0.13; N=15, 13). The most substantial change, however, was that the β4-transfected cells had an unusually large non-inactivating component (control vs. β4: 1.8 ± 0.3% vs. 5.8 ± 1.4%, p=0.02). These results therefore suggest that β4 expression in HEK-NaV1.1 cells destabilizes fast inactivation and instead favors channel opening at 0 mV.
Given the increase in availability measured at 0 mV, we tested whether β4 influenced the amount of persistent Na current at other voltages by measuring the mean current in the last 10 ms of the 100-ms conditioning steps (). Although the transient current amplitudes were similar with and without β4 (control vs. β4, −4.3 ± 0.8 nA vs. −3.9 ± 0.7 nA at 0 mV, N=15, 13), the persistent current was increased more than threefold in the presence of β4 (control vs. β4, −46 ± 13 pA vs. −143 ± 27 pA at −10 mV). To control for variations in current density across cells, the persistent current amplitude was normalized to the peak amplitude of the transient current at 0 mV in each cell (corrected as necessary to represent maximal conductance; see Materials and Methods) and the percent persistent current was plotted against voltage. Consistent with the availability curve, β4-transfected cells showed more persistent current compared to control cells, with the increase being most pronounced at voltages between −20 and 0 mV (, open triangles, significant main effect of condition on current F(1,26)=6.9, p=0.014).
β4 expression is not sufficient to produce resurgent Na current in HEK-NaV1.1 cells
Previous work from our group demonstrated that a 14 amino-acid sequence from the β4 intracellular domain (the “β4 peptide”) can bind open Na channels of neurons in a voltage-dependent manner. Channels are blocked by the peptide at positive voltages and become unblocked upon repolarization, allowing resurgent Na current to flow, both in Purkinje neurons after enzymatic removal of their endogenous open-channel blocking protein, or in CA3 neurons, which lack an endogenous blocking protein (Grieco et al., 2005
). To test whether expression of the full-length β4 protein might replicate this behavior in HEK-NaV
1.1 cells, we stepped cells to +60 mV to maximize the possibility of voltage-dependent block, and then repolarized to potentials between −40 mV and +20 mV. As expected, in control cells, little current was detectable upon repolarization (). To verify that NaV
1.1 channels expressed in HEK cells were capable of undergoing block and unblock in a manner similar to Na channels in their native neuronal environments, we included the β4 peptide (200 μM) in the recording pipette. In the presence of the peptide, repolarization indeed evoked a current with kinetics resembling native resurgent current in neurons, but with maximal resurgent current between −10 and −20 mV rather than at −30 mV as in neurons (). This shift is consistent with the fact that the V1/2
of activation is about 15 mV more depolarized than in neurons (Raman and Bean 1997
). In contrast, with expression of the full-length β4 protein, little if any resurgent current was evident. Instead, repolarization evoked a brief tail followed by a large steady-state current (), consistent with the increased non-inactivating component observed with step depolarizations.
Figure 2 Expression of the β4 subunit increases persistent, but not resurgent, Na current. A, Voltage protocol and representative traces for each condition, as labeled. Traces were normalized to the peak current evoked at 0 mV in each cell. B, Top, Mean (more ...)
To quantify these data, the resurgent current was calculated as the difference between the maximal current (after the tail) and the persistent current (at the end of the step). At −10 mV, the current was −26 ± 9 pA in control (N=15), −166 ± 38 pA with the β4 peptide (N=10), and −30 ± 5 pA with the β4 protein (N=13). Because the mean transient current density in the cells with the peptide (−5.3 ± 0.4 nA at 0 mV) was slightly greater than in the control and β4 protein-expressing cells (same cells as in ), resurgent current amplitudes were normalized to the peak transient current at 0 mV and plotted as percent resurgent current against voltage (, top
). Cells containing the β4 peptide indeed had more relative resurgent current than control cells, consistent with an effective block and unblock of Na channels by the β4 peptide (significant main effect, F(2,35)
; Tukey’s: β4 peptide vs.
β4 protein, p
). The current was not significantly different in control and β4-expressing cells, however (Tukey’s, p
). In contrast, expression of β4 increased the persistent current flowing upon repolarization (control, −48 ± 14 pA; β4 peptide, −185 ± 74 pA; β4 protein, −131 ± 24 pA). When persistent currents in each cell were normalized to the transient current at 0 mV, larger relative currents consistently occurred in the presence of the β4 subunit (, bottom,
significant main effect, F(2,35)
Tukey’s: β4 protein vs.
; β4 peptide vs.
). Together, these data demonstrate that expression of the full-length β4 subunit with NaV
1.1 is not sufficient to generate resurgent current in HEK-NaV
1.1 cells, consistent with the recent report that β4 does not induce a resurgent current in tsA-201 cells expressing NaV
1.2 (Chen et al. 2008
). Instead, like the results obtained with step depolarizations, the results suggest that co-expression of β4 with NaV
1.1 destabilizes inactivated states at voltages between −30 and +10 mV, permitting a higher occupancy of open states than does expression of NaV
At strongly hyperpolarized potentials, however, expression of β4 did not modify the transition from inactivated to closed states. illustrates recovery at −110 mV from inactivation induced by a short step to +60 mV in control cells, cells containing the β4 peptide, and cells expressing the β4 protein. Recovery in cells expressing the β4 protein was indistinguishable from control (double exponential fit parameters to mean data from control, β4 protein, β4 peptide: τfast
= 1.4, 1.5, 1.2 ms; τslow
=16.6, 12.9, 9.1 ms; %fast
= 44, 42, 47%, N=5, 5, 4). The similarity of recovery times between the control and the β4 protein condition supports the idea that depolarizing steps favor the same fast inactivated states in both cases, and that β4 reduces the stability of inactivation only at voltages in the vicinity of 0 mV. In contrast, channels exposed to the β4 peptide recovered more rapidly than in control conditions, consistent with the idea that block by the peptide prevents fast inactivation at positive potentials, and permits reopening and deactivation at negative potentials (Raman and Bean 2001
; Grieco et al. 2005
β1 co-expression counteracts the effect of β4 on inactivation
Expression of β4 increased persistent sodium current beyond 5% of the transient sodium current, a value that is considerably greater than anything observed in central neurons, in which persistent currents have been reported to range from 0.7 to 4% of transient currents (Taddese and Bean, 2002
, Cummins et al., 1994
, Magistretti and Alonso, 1999
, Parri and Crunelli, 1998
, Maurice et al., 2001
). It therefore seemed likely that, in neurons, other factors might limit the ability of β4 to enhance persistent current. Given the widespread expression of β1 in the nervous system, we considered the possibility that β1, which accelerates inactivation in heterologous expression systems (Isom et al. 1992
; Chen and Cannon 1995
), might modify the influence of β4. To test this idea, we first transfected HEK-NaV
1.1 cells with the Na channel β1 subunit (N=9) and measured currents evoked by 100-ms step depolarizations (, top
). With β1 transfection, the amplitudes of persistent currents were similar to control (, left
). As in other heterologous expression systems, β1 expression accelerated fast inactivation, reducing the decay time constant at 0 mV from 0.72 ± 0.03 ms in control (N=26) to 0.63 ± 0.03 ms (N=9, p
, , left
). Thus, β1 and β4 have contrasting effects on the macroscopic properties of fast inactivation.
Coexpression of wild-type β1 subunit, but not the GEFS+ mutant subunit β1C121W, prevents the β4-mediated destabilization of inactivation.
This contrast is of interest because it is likely that α, β1, and β4 subunits assemble to form heterotrimeric channels: Biochemical studies indicate that the majority of Na channel α subunits in the brain associate with one non-covalently linked β subunit, such as β1, and one covalently linked β subunit, such as β4 (Reber and Catterall 1987
; Yu et al. 2003
). Therefore, to test how β1 and β4 subunits interact functionally, we transfected HEK-NaV
1.1 cells with both β1 and β4. Co-expression of β1 and β4 (N=14) produced persistent current amplitudes that overlapped with those of control cells (N=15) or cells transfected with β1 (N=9, and , right
). Moreover, the decay time constant at 0 mV was significantly faster in cells co-expressing β1 and β4 (, left
; N=14, 0.58 ± 0.03 ms, p
control), similar to β1 expression alone. These changes indicate that, despite the presence of endogenous β1B, expression of β1 has a distinct and specific effect on β4, namely, to inhibit the β4-mediated destabilization of inactivation.
An epilepsy mutation in Scn1b decreases the ability of β1 to reverse the effects of β4
These results raise the possibility that disruptions of β1, such as those that occur in epilepsy and other types of seizure disorders, might alter the regulation of β4. Specifically, a mutation of a cysteine to a tryptophan in the β1 extracellular domain (β1C121W
) leads to GEFS+ in humans (Wallace et al. 1998
). To test whether this mutation changes the influence of β1 on β4, we measured Na currents in HEK-NaV
1.1 cells, in which β1C121W
was co-expressed with β4. β1C121W
prevented the β4-induced increase in persistent current as effectively as did β1 (at −10 mV: β4 alone, 6.0 ± 2.0%; β1+β4, 1.9 ± 0.4%; β1C121W
1.9 ± 0.5%, N=13, 14, 14; , , right
). Inspection of the traces, however, indicated that the inactivation time constant was slower when β4 was coexpressed with β1C121W
than with wild-type β1, resembling the condition with β4 alone (β1C121W
+β4, 0.76 ± 0.06 ms, N=17; vs.
). This difference in the rate of entry into inactivated states was even more apparent upon examination of the percent current remaining at the end of a 5-ms step. This amplitude, which reflects a slower component of inactivation, was relatively small in control or with β1 alone, but relatively large with β4 alone (). Moreover, when β4 was expressed with β1C121W
, the current was nearly twice that with the wild-type β1 (β1+β4 vs.
+β4; 8.7 ± 1.4 % vs.
15.7 ± 2.9%; N=14, 17; p
). Thus, the GEFS+ mutation makes the β1 subunit less effective in counteracting the destabilization of inactivation by β4, however, raising the possibility that a prolongation of Na currents contributes to the alteration of neuronal firing patterns in carriers of this mutation.
β1 subunits might exert their effects on persistent current either by preventing β4 from associating with α subunits and/or by having a dominant influence on channel gating. To test the likelihood that NaV1.1, β1 and β4 form functional heterotrimeric complexes, we transfected HEK-293T cells with different combinations of subunits and assessed their association by coimmunoprecipitation. Co-transfection of NaV1.1 with V5-tagged β1 indicated that these subunits associated (), and that this association persisted but was weakened when β4 was also present (). Transfection of cells with only β1 and β4 revealed a strong interaction between these two subunits even in the absence of NaV1.1 (), suggesting that one action of β1 may indeed be to sequester β4 and limit its association with α subunits. If so, the macroscopic electrophysiological properties measured in HEK-NaV1.1 cells transfected with both β subunits may result in part from NaV1.1 monomers, thus mimicking the control condition. β4, however, interacted strongly with NaV1.1 alone (), suggesting that any free β4 would be likely to enter a channel complex. Since the macroscopic currents in cells expressing the three subunits mimic neither the control condition nor the condition with a single β subunit, it seems likely that a nonnegligible subset of channels contain NaV1.1, β1, and β4, and that these heterotrimers contribute to the overall electrophysiological phenotype. Consistent with this idea, with all three subunits present, an interaction of β4 with NaV1.1 was evident (not shown).
Figure 4 Association of Nav1.1, β1, β1C121W, and β4 subunits. Co-immunoprecipitation experiments of Na+ channel α and β subunits were performed on transfected HEK-293T cells. All molecular weight standards are indicated (more ...)
Next, we repeated these experiments with β1C121W
substituted for the wild-type β1 subunit. The mutant subunit associated with NaV
1.1 and, in contrast to wild-type β1, this association remained strong in the presence of β4 (, ). Conversely, the association of β1C121W
and β4 in the absence of the α subunit appeared less robust than with wild-type β1. This result suggests that the β1-β4 interaction is mediated by the extracellular immunoglobulin domains (), and is consistent with previous results showing that β1C121W
does not function as a cell adhesion molecule (Meadows et al., 2002
). Considered in the context of the electrophysiological experiments, which demonstrated reduced persistent current but slowed inactivation rates relative to control, these results support the idea that heterotrimeric channels comprising α, β1C121W
, and β4 do indeed assemble, and suggest that wild-type β1 need not prevent β4 association with the α subunit in order to oppose the effects of β4 on gating.
The extracellular domain of β1 regulates persistent current
Since the C121W mutation is in the extracellular domain, these data suggested that this region of β1 is necessary for the normal regulation of inactivation. We therefore tested whether the suppression of persistent current and promotion of inactivation could be achieved without the intracellular domain of β1 by expressing a “β1/4” chimeric subunit, which consisted of the extracellular and the transmembrane domains of β1 and the intracellular domain of β4. In cells expressing β1/4 (N=7), both the persistent current amplitudes and the time course of inactivation were indistinguishable from β1-expressing cells (, top left
, and ). The simplest interpretation of these results is that the wild-type extracellular domain directly modulates the stability of inactivation. The extracellular domain, however, also contains sites required for interactions with the α subunit, thereby determining the position of β subunits in the channel complex (McCormick et al. 1998
). Since the sites of α-β interaction are likely to differ for β1 and β4, an alternative interpretation is that the β1/4 chimera inhibits the channel openings that are favored by β4 simply by wrongly positioning the β4 intracellular domain.
Chimeric β subunits suggest that the extracellular domain regulates persistent current amplitude.
To address this possibility, we co-expressed β1/4 and β4. With both subunits present, heterotrimeric channels are predicted to have two β4 intracellular domains, one in the site normally occupied by the β1 cytoplasmic tail, and one in the normal position for β4. Under these conditions, the persistent current remained at control levels, and was indistinguishable from coexpression of β1+β4 (N=11; , lower panel
). These results support the idea that the extracellular domain of β1 largely governs persistent current in NaV
1.1, as it does with other α subunits (Chen and Cannon 1995
; McCormick et al. 1998
). The slow phase of inactivation, reflected by the percentage of current remaining at 5 ms, was also restored to control levels, as it was with β1+β4. The fast inactivation time constant was not consistently reduced to levels achieved by expression of β1+β4, however, (), leaving the possibility open that intracellular domains also contribute to the regulation of inactivation (e.g. Spampanato et al., 2004
Next, we tested whether the β2 subunit, which resembles β4 both in sequence similarity and in its disulfide linkage to Na channel α subunits (Yu et al. 2003
), might also resemble β4 in its influence on Na current. Indeed, expression of β2 in HEK-NaV
1.1 cells increased persistent current amplitudes (N=12), although to a lesser extent than β4 (, top right
). Expression of β2 also increased the percent current remaining at 5 ms, while leaving the inactivation rate unaffected relative to control (). A β2/4 chimera (N=9), composed of the extracellular and the transmembrane domains of β2 and the intracellular domain of β4, behaved in much the same way as β2 and β4 (, top right
, and ). These data indicate that β2, β4, and β2/4, whose extracellular domains are expected to bind in a similar way to the α subunit, influence inactivation in a qualitatively similar manner.
Together, the data indicate that persistent current amplitudes, as well as the percent current remaining at 5 ms, can be either relatively large, occurring with β4, β2, or β2/4, or relatively small, occurring with β1, β1/4, β1+β4, or β1/4+β4, as well as with NaV1.1 alone. For convenience (with no mechanistic implication), the former group will be referred to as the “disulfide-linked” group and the latter (excluding the control) as the “wild-type β1extra” group. The β1C121W+β4 condition presents an anomaly that will be considered separately.
β subunits affect both window current and the percent non-inactivating current
To explore the basis for the differences in persistent current between the disulfide-linked and wild-type β1extra groups, we examined the activation and inactivation curves. These allow an estimation of the size of the window current between the curves, as well as the percentage of current that does not inactivate even at the most depolarized potentials. We began by analyzing the availability curves recorded in all conditions. As in , data from each cell were fitted with Boltzmann functions to obtain values of V1/2, k, and percent non-inactivating current. The mean values for the non-inactivating current fell into two groups. The wild-type β1extra group as well as control cells had < 2.3% current, whereas the disulfide-linked group as well as β1C121W+β4 had >3.4% current (). These data suggest that the disulfide-linked subunits actively increase the equilibrium occupancy of the open state, whereas the subunits with the wild-type β1 extracellular domain counteract this effect. With the exception of β1C121W+β4, this grouping parallels the amplitudes of persistent currents measured at negative voltages.
Changes in activation and inactivation parameters increase the window current in HEK-NaV1.1 expressing β4 and β2 but not β1.
Next, we tested whether β subunits expressed in HEK-NaV1.1 cells modified the window current. In general, the window current may be increased by a negative shift in the activation curve, a positive shift in the inactivation curve, and/or a flattening of the slope of either curve. As shown in , the V1/2 of inactivation was relatively insensitive to β subunit expression. In nearly all conditions, the mean V1/2 fell between −41 and −44 mV. The value for β4 alone was slightly negative to this range (−45.7 ± 1.7 mV) and for β1C121W+β4 was slightly positive to this range (−39.7 ± 1.6 mV). The slope factor of the curve, k, however, was indeed affected by the different β subunits. The four conditions with more non-inactivating current had shallower slopes, with k values ≥ 7.5 mV (disulfide-linked and β1C121W+β4), while the five conditions with less non-inactivating current had steeper slopes, with k values ≤ 6.5 mV (wild-type β1extra and control, ). Consequently, the inactivation k was correlated with the non-inactivating current (R2 = 0.79). Because the inactivation V1/2 is relatively constant across conditions, shallower slopes widen the voltage range over which window current can flow. For instance, with a V1/2 of −42 mV, shifting the slope factor from 6.25 mV to 7.5 mV doubles the availability at −15 mV. The overlay of availability curves with the mean fit parameters of β1 and β4 illustrates this effect (). These data therefore suggest that persistent current across a range of potentials may be promoted in the disulfide-linked subunits by both a weaker voltage-sensitivity of inactivation and a greater equilibrium stability of the open state.
Across conditions, the complement of β subunits affected the parameters of activation as well. The slope factors covered a relatively wide range of values but were not correlated with the amount of non-inactivating current (R2=0.11, ). The steepest slopes occurred with the chimeras (β2/4, β1/4) and the co-expressed subunits (β1+β4, β1/4+β4), suggesting that, at least in some contexts, the intracellular domain of β4 makes channel opening more sensitive to voltage. This effect is particularly noticeable when β1 is compared to β1/4 (p=0.07) or β2 is compared to β2/4 (p=0.02). An exception, however, is the moderate k value of β4 alone (5.2 ± 0.3), indicating that the effects of the intracellular and extracellular domains are not altogether independent of their context in a full protein. Excluding the data for β1C121W+β4, the V1/2 for activation was negatively correlated with the percent non-inactivating current (R2=0.67, ). The V1/2 value was most negative for β4 (−21.8 mV). Also, when β4 was co-expressed with either β1 or β1/4, the V1/2 tended to shift negative relative to the value for β1 or β1/4 alone. A hyperpolarization of V1/2 also occurred when the β4 tail was added to β2 to make the β2/4 chimera. Thus, expressing the β4 intracellular domain positioned correctly, i.e., on a disulfide-linked subunit, promoted channel opening at more negative potentials. The resulting shift in the activation curve is expected to expand the window in which persistent current can flow ().
Co-expressing β1C121W and β4 provided an exception to the generalizations that pertained to the other subunits. β1C121W and β4 generated channel complexes that resembled the disulfide-linked group in their large non-inactivating components and shallow slope of the availability curves, as well as in their large percent current remaining after 5 ms. Nevertheless, they generated small persistent currents as did the wild-type β1extra group. The activation parameters, however, offered a likely explanation for the peculiarities of the mutant subunit. The activation curves had properties at the extreme of the distribution, with the largest k (5.8) and most positive V1/2 (−12.5 mV). The depolarizing shift and flattening of the activation curve are expected to diminish persistent current by narrowing the window in which it flows (). Thus, in HEK-NaV1.1 cells, the GEFS+ mutation in β1 makes it even more effective than the wild-type subunit at stabilizing closed over open states, such that larger depolarizations are necessary to open the channel. At voltages positive enough to activate the channels, however, the mutation renders the β1C121W subunit unable to counteract the β4-induced favoring of open over inactivated states ().
Together, these data suggest the following: First, expression of wild-type β1 favors inactivated states, and this effect is dominated but not wholly controlled by the extracellular domain. Second, expression of β4 favors open states, and this effect is dominated but not wholly controlled by the intracellular domain. Third, expression of the β1 GEFS+ mutant with β4 generally weakens the overall voltage-sensitivity of gating, so that channels remain closed rather than open at moderately negative voltages, and they remain open rather than inactivated at more positive voltages.
Na currents in neurons made to over-express β4
The results in HEK-NaV
1.1 cells raise the question of which effects of β subunits are evident in neuronal environments, where Na channel gating is also influenced by factors such as additional associated proteins and post-translational modifications. To address this issue, we over-expressed the β4 protein in cultured pyramidal neurons from the CA3 region of the hippocampus (). We selected these neurons because they normally lack β4 (Yu et al. 2003
) but express high levels of β1 and β2. They also express β3, which, like β1, binds non-covalently to α subunits (Oh et al. 1994
; Whitaker et al. 2000
; Morgan et al. 2000
). Na channels in CA3 neurons are therefore expected to comprise α subunits (NaV
1.1, 1.2, or 1.6) with β1+β2 or β3+β2, and these heterotrimers likely interact with other neuronal proteins. We reasoned that transfection of neurons with β4 might allow some fraction of channels to incorporate β4 instead of β2, and/or generate a subset of channels that exist as α+β4 heterodimers.
Over-expression of β4 produces a small but consistent increase persistent current in cultured CA3 hippocampal neurons.
Since the complex morphology of cultured neurons made space clamp of transient currents difficult at the foot of the activation curve, we restricted our analysis to transient currents at 0 mV, where conductance is maximal and less affected by voltage escape, and to small, slow currents evoked by repolarization, where voltage clamp is optimal. We first assayed steady-state inactivation after 100-ms conditioning steps in control and transfected neurons (). Expression of β4 produced small changes in the V1/2 of inactivation and steady-state components of the availability curve (). The V1/2 was −51.8 ± 2.1 mV in control neurons (N=5) and −57.6 ± 1.4 mV with expression of β4 (N=5, p=0.12), while the non-inactivating component was 1.0 ± 0.2% in control neurons and 1.9 ± 0.3% with β4 (p=0.15). Although neither change was statistically significant, the tendency for a negative shift in inactivation and an increase in the non-inactivating component resembles the changes seen with transfection of only β4 into HEK-NaV1.1 cells. Furthermore, expression of β4 produced a significant change in the slope factor, which was 5.0 ± 0.3 mV in control and became 6.1 ± 0.2 mV with transfection of β4 (p=0.025, ). Again, the decrease in the steepness of the inactivation curve resembles the effects obtained with transfection of β4 alone into HEK-NaV1.1 cells.
Next, to measure persistent and (if any) resurgent current, cells were held at −90 mV, and a step depolarization to +30 mV was applied, followed by a repolarizing step to −30 mV. Currents were measured relative to the transient current amplitude at 0 mV (). Control neurons showed little if any resurgent current, and expression of β4 did not generate a larger resurgent component (control, β4: 0.9 ± 0.2%; 1.1±0.2%, p
N=14, 16). Persistent currents were also small in all neurons, but were consistently larger in β4-overexpressing neurons, doubling from 1.1 ± 0.2% in control to 2.2 ± 0.6% with β4. The cumulative probability plot illustrates that the expression of β4 increased the likelihood of a larger persistent component ( Mann-Whitney U test, Z
). Like the change in the availability curve, the change in persistent current resembles the changes observed when HEK-NaV
1.1 cells were transfected with β4 alone. It is therefore possible that transfection of CA3 cells led to the assembly of α+β4 heterodimers. Alternatively, since β3 subunits have been implicated in increasing persistent currents in expression systems (Qu et al. 2001
), it may be that α+β3+β4 channels were also formed and that β3, unlike β1, permits β4-mediated destabilization of inactivation.
Na currents in neurons expressing β4 but lacking β1 and/or β2
The changes in Na currents were smaller in hippocampal neurons than in the HEK-NaV
1.1 cells. These differences may result from a population of channel complexes that failed to incorporate β4, the expression of other proteins that modulate the effects of β4, and/or the antagonism of β4 effects by endogenous β1. To explore how the removal of β1 affects channels that normally include β4, we recorded from neurons acutely isolated from Scn1b
(β1) null and Scn1b/Scn2b
(β1/β2) double null mice as well as from littermate wild-type or Scn2b
null controls. For these experiments, we selected cerebellar Purkinje neurons, which normally express high levels of β4 as well as β1 and β2 (Yu et al. 2003
) but lack β3 (Morgan et al. 2000
). A complicating factor in these experiments is the variety of proteins, in addition to β subunits, that are known to modulate Na channels in real neurons (Abriel and Kass 2005
). These include GTP-binding protein βγ subunits (Mantegazza et al. 2005
; Kahlig et al., 2006
; Ma et al., 1997
), calmodulin (Mori et al. 2000
; Deschenes et al. 2002
; Herzog et al. 2003
; Kim et al. 2004
; Young and Caldwell 2005
), and FGF-homologous factor (Wittmack et al. 2004
; Lou et al. 2005
), as well as the endogenous blocking protein of Purkinje cells (Grieco et al., 2005
). Nevertheless, we reasoned that comparing the Na currents in the wild-type and null mice might indicate whether the influence of β1 that was present in HEK-NaV
1.1 cells might also be evident in Purkinje neurons.
We began by measuring activation curves and comparing parameters of the Boltzmann fits (). Wild-type and Scn1b
null mice showed no significant differences in the V1/2
or k of activation (control vs. Scn1b
= −31.9 ± 1.0 vs. −34.0 ± 1.4 mV, p
, k = 5.7 ± 0.3 vs. 6.0 ± 0.4 mV, p
N=8, 6). The k values were also statistically indistinguishable in the Scn2b
nulls and the double nulls (6.1 ± 0.2 vs. 6.2 ± 0.3 mV, p
, N= 9, 19). In the double nulls, however, activation was shifted significantly negative (from −32.3 ± 1.2 to −36.1 ± 1.3 mV, p
). Considering the observation that cells that normally lack β3 do not up-regulate β3 after loss of β1 (Lopez-Santiago et al. 2007
), the Scn1b
nulls should reveal the properties of α+β2 and α+β4 channels, and the double nulls should isolate the properties of α+β4 channels. The minor negative shift in the former and the significant negative shift in the latter illustrate that cells in which β4 is the only available β subunit activate more readily in widely differing contexts, from HEK cells to neurons.
Early persistent currents are increased in Scn1b null and Scn1b Scn2b double null Purkinje neurons.
Next, we compared inactivation in the presence and absence of β1. Scn1b null Purkinje cells showed a small but significant negative shift in the V1/2 of the availability curve, from −65.1 ± 1.0 mV (wild-type) to −68.9 ± 1.0 mV (Scn1b null, p=0.02) and no effect on k or the percentage of non-inactivating current (wild-type, Scn1b null; N=9, 7; k: 6.1 ± 0.2, 5.8 ± 0.1; p=0.3; % non-inactivating: 0.71 ± 0.18, 0.63 ± 0.14, p=0.7, ). Larger changes were evident in the comparison of double null Purkinje cells to the Scn2b null littermate control neurons. The percentage of non-inactivating current was increased in the double nulls (from 0.92 ± 0.23 to 1.56 ± 0.14%, p=0.003). Inactivation was also significantly hyperpolarized in the double null Purkinje cells (from −64.2 ± 1.0 mV to −70.5 ± 1.0 mV, p<0.001, N=9, N=22), without a concomitant increase in k (5.9 ± 0.1, 5.8 ± 0.1, ). This negative shift in V1/2 is greater than that seen in HEK cells and CA3 cultures transfected with β4. In the absence of the β4-induced increase in the slope factor that was present in those cell types, the negative shift in inactivation is likely to suppress the window current at negative voltages. Not surprisingly, therefore, the persistent current measured 90–100 ms after step depolarizations to voltages below 0 mV was not increased in either the single Scn1b or double nulls (p>0.1 at all potentials between −40 and −10 mV, not shown).
Despite the lack of change in persistent current, inspecting the families of traces suggested that inactivation was not identical in cells from the four genotypes. For example, the time constant of decay at 0 mV tended to be longer in the double null cells (). Because of this apparent slowing of inactivation, we also measured the “early” persistent current as the percent current remaining 25–30 ms after the depolarization. This duration is >5-fold longer than the dominant time constant of inactivation in all genotypes, but inactivation has not yet reached a steady-state by this time (). Plots of this early persistent current normalized to the peak transient current vs. voltage illustrated that the absence of β1 increased the amount of early persistent current, both in Scn1b null (N=11) vs. wild-type control cells (N=11, , top) and in double null (N=30) vs. Scn2b null cells (N=11, , bottom). As expected from the larger negative shift of the activation curve and the slower rate of inactivation, this effect was greater in the double null neurons, in which the persistent current at −30 mV was increased by 60%. In fact, persistent currents greater than 3% were seen in 30% of the double null neurons but none of the cells from β2 null littermates (Mann-Whitney U test, Z=−2.12, p=0.034).
Together, the data indicate that the properties of Na channel complexes differ widely in HEK-NaV1.1 cells, cultured CA3 neurons, and isolated Purkinje neurons, even with predicted similarities in β subunit expression. In Purkinje neurons, loss of β1 and β2 hyperpolarizes the V1/2 of inactivation relative to control more than in HEK-NaV1.1 cells (control vs. β4-transfected) or even cultured CA3 neurons (control vs. β4-transfected) without an increase in the slope factor, producing a small window current. These differences are likely to result from cell-specific factors, such as α-subunit identity, associated proteins, and post-translational modifications. Nevertheless, in neurons, as well as in HEK-NaV1.1 cells, if β4 is the only β subunit present, channels activate more readily, inactivation proceeds relatively slowly, and the non-inactivating component of the availability curve is increased. Moreover, all these characteristics are apparently counteracted by expression of β1.