Abstract
We report a novel coupled system of sodium-activated potassium currents (IKNa) and persistent sodium currents (INaP), the components of which are widely distributed throughout the brain. Its existence and importance has not been previously recognized. Although IKNa was known to exist in many cell types, the source of Na+ which activates IKNa remained a mystery. We now show in single membrane patches generated from the somas of rat neurons that sodium influx through INaP is sufficient for activation of KNa channels, without substantial contribution from the transient sodium current or bulk [Na+]i. INaP was found to be active at cell membrane resting potentials, a finding that may explain why IKNa can be evoked from negative holding potentials. These results show an unanticipated role for INaP in activating a negative feedback system countering the excitable effects INaP; the interrelatedness of INaP and IKNa suggests new ways neurons can tune their excitability.
Introduction
Sodium-activated potassium channels (KNa channels) were first described over 25 years ago as high conductance potassium channels activated by high concentrations of internal sodium (Kameyama et al., 1984). Despite many reports of their expression throughout the nervous system and other tissues (for review, see Dryer, 1994; Bhattacharjee and Kaczmarek, 2005), it was unclear whether KNa channels were active under normal physiological conditions. Experiments with inside-out patches appeared to show that KNa channel activity requires bulk sodium concentrations in excess of that normally present in bulk cytosol. Such experiments were taken as evidence against the participation of IKNa during normal electrophysiological activity. Rather, it was suggested that KNa channels might represent a type of “reserve conductance” active only under conditions of hypoxia when [Na+]i might substantially increase. However, recent studies have shown that KNa channels produce large outward currents in several types of neurons under normal physiological conditions, and appear to be dependent on sodium influx rather than high bulk [Na+]i (Budelli et al., 2009; Lu et al., 2010; Nuwer et al., 2010).
IKNa is a tetrodotoxin (TTX)-sensitive delayed outward current that lasts for seconds but the transient sodium current (INaT) is over within a few milliseconds; INaT thus seems an unlikely source of Na+ to activate IKNa. Here we explore the possibility that the TTX-sensitive persistent sodium current (INaP) is the source of Na+ activating IKNa. Amplitudes of INaP are tiny compared with peak amplitudes of INaT, and even prolonged INaP activity will not quickly increase bulk [Na+]i. Therefore a mechanism permitting INaP to deliver sufficient cytosolic Na+ to activate IKNa would require that channels underlying both currents be present together in single membrane patches. We show that the activity of IKNa indeed depends on the activity of INaP by recording single-channel INaP and IKNa currents in membrane patches isolated from the soma of olfactory bulb mitral cells. Single-channel currents recorded from outside-out patches revealed that treatments that eliminated INaP, also removed IKNa; conversely, enhancing INaP resulted in increased IKNa activity. This dependence of IKNa on INaP was demonstrated under conditions where INaT was absent, and bulk [Na+]i was 0, thus verifying the importance of INaP. These results focus further attention on INaP which is increasingly being recognized for its role in controlling many aspects of cell excitability (Crill, 1996; Vervaeke et al., 2006; Huang and Trussell, 2008). Although the action of INaP is usually thought of as producing an increase in cell excitability (Stafstrom et al., 1984) our results suggest an unanticipated role for INaP in activating a negative feedback system, IKNa, which provides an opposing inhibitory current.
Materials and Methods
Primary neuronal culture preparations.
Primary cultures of dissociated olfactory bulbs were prepared from either sex of neonatal Sprague Dawley rats (postnatal day 0). Dissected olfactory bulbs were incubated in 0.25% trypsin in PBS for 30 min at 37°C. For the last 5 min, trypsin solution was supplemented with 0.05% DNase. Enzyme digested tissue was then triturated in PBS containing soy bean trypsin inhibitor using Pasteur pipettes of decreasing diameter. Cells were plated on poly-lysine-coated glass coverslips in Dulbecco's Modified Eagle Medium/F-12 medium (Invitrogen) with 10% FBS and 2% B-27 supplement (Invitrogen). Three days after plating, medium was replaced with Neurobasal (Invitrogen) + 2% B-27 supplement and 5 μm arabinofuranosyl cytidine to inhibit division of glia. Whole-cell and excised patch recordings were made up to 4 and 10 d after plating, respectively.
Voltage-clamp recordings.
Tufted/mitral cells were identified on the basis of their pyramidal shape and large size (Trombley and Westbrook, 1990). Recordings were made with borosilicate glass pipettes with tip resistances of 3–6 MΩ. Outside-out patches were obtained by steadily moving the pipette away from the cell after establishing a whole-cell patch at the soma. Voltage-clamp recordings were made with an Axoclamp 200B, filtered at 2 kHz and digitized at 50 kHz with a Digidata 1440A. Internal pipette solution contained the following (in mm): 140 KCl, 10 HEPES, 5 EGTA, 1 MgCl2, pH 7.40 with KOH. Bath solutions contained the following (in mm): 150 NaCl, 10 HEPES, 10 dextrose, 5 KCl, 2 MgCl2, pH 7.30 with NaOH. For recordings of INa, the internal solution contained 130 CsCl, 10 NaCl, 1 MgCl2, 10 HEPES, 5 EGTA, pH 7.40 with KOH. The external solution for recording INa was the same as the standard external recording solution with 10 mm tetraethylammonium (TEA) · Cl, 2 mm 4 aminopyridine (4-AP) · Cl, and 0.2 mm CdCl2 replacing equimolar NaCl. Zero Na+ external solutions replaced all NaCl with choline · Cl or LiCl as indicated. TTX and veratridine were used at 1 and 50 μm concentrations, respectively. Veratridine was stored as a 50 mm stock frozen in DMSO. Experiments using veratridine included 0.1% DMSO in control external solution. No effect of this concentration of DMSO on channel activity was noted. In control experiments examining the effect of TTX on Slack-transected human embryonic kidney cells, 10 mm NaCl replaced equimolar KCl in the internal solution.
Single-channel analysis.
Data were acquired and analyzed with pClamp 10.0 and Microsoft Excel. Single KNa channel activity was analyzed and Po calculated using the Single Channel Search tool in pClamp 10.0. KNa channel activity was measured during repeated 3 s sweeps to +60 mV from a holding potential of −70 mV, using a 10 s intersweep interval. Under differing experimental conditions, no changes in the mean amplitude of detected events or distribution of peaks in histograms of event amplitude were found. The number of channels in a patch was estimated by the maximum number of simultaneously active channels (additive steps) observed over the course an experiment. The minimum estimate of channel number provided by this method was used to determine Po. In the majority of experiments, patches analyzed were estimated to contain 4–6 channels. Single INaP channel activity was measured using Threshold Search tool in pClamp 10.0. Many of the persistent TTX-sensitive openings were so brief that we could not reliably measure a mean channel open time, even when digitizing at high rates. Thus, we used the number of detectable events, and the Threshold Search tool as a means of quantification rather than channel open probability. Threshold levels were determined by measuring unitary current levels from selected “box-like” open channel events. The brevity of many events prevented use of an all points histogram to determine unitary current level. Identical threshold searches were performed for control sweeps and sweeps evoked in TTX. A small number of events crossed threshold in TTX and those values were subtracted from corresponding control values to generate the plot in Figure 5B (see Results).
Estimation of [Na+]i increase.
The volume of pipette solution was estimated to be 7.26 μl using the inner diameter of the recording pipettes (0.68 mm) and an approximation of internal solution fill height starting at the tapering of the pipette (20 mm). The number of Na+ molecules entering the patch was estimated based on a high approximation of average INa throughout the experiment: −50 pA. Using the Faraday constant, this corresponds to 5.18 × 10−16 mol/s. Given this rate of Na+ influx and the volume of pipette solution, the concentration of sodium would increase by 0.257 μm over the course of an hour-long experiment.
This is likely an overestimate, the fill height was usually >20 mm, and there is additional volume in the tapered portion of the pipette not considered here. In most excised patches, the peak amplitude of INa was much smaller than −50 pA and even smaller at the holding potential that patches were clamped at for the majority of the experiment.
Statistical analysis.
We performed statistical analyses using T.W. Kirkman's Statistics to Use (http://www.physics.csbsju.edu/stats/) software. For comparison between two groups, we used paired Student's t tests for the same procedures before and after applied treatments.
Animal welfare.
It is the policy of Washington University Medical School that all research involving animals be conducted under humane conditions, with appropriate regard for animal welfare. Washington University Medical School is a registered research facility with the United States Department of Agriculture (USDA) and is committed to complying with the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services), the provisions of the Animal Welfare Act (USDA), and all applicable federal and state laws and regulations. At Washington University Medical School an Animal Care Committee has been established to insure compliance with all applicable federal and state regulations for the purchase, transportation, housing and research use of animals. Washington University Medical School has filed appropriate assurance of compliance with the Office for the Protection of Research Risks of the National Institutes of Health.
Results
In whole-cell recordings of olfactory bulb tufted/mitral cells (T/M cells) (Fig. 1) or striatal medium spiny neurons (Budelli et al., 2009), blockade of voltage-gated sodium (NaV) channels with TTX (1 μm) reduces the amplitude of sustained outward currents. This portion of TTX-sensitive outward current removed represents a sodium-activated potassium current (IKNa) (Budelli et al., 2009). As shown in Figure 1, the kinetic properties of the TTX-sensitive outward current (IKNa) do not appear to depend on INaT; IKNa is a long-duration delayed outward current showing very little inactivation, while INaT is over in a few milliseconds. Since a key feature of IKNa is its sensitivity to TTX, an alternative to its activation by the TTX-sensitive INaT component could be its activation by the TTX-sensitive INaP which is known to accompany INaT in most neurons (Crill, 1996). Supporting this hypothesis are our observations showing that IKNa persists even when INaT is eliminated or greatly reduced by a depolarized holding potential of −50 mV (Fig. 1b).
The TTX-sensitive delayed outward current, IKNa, does not depend on the transient sodium current. a, Whole-cell currents before and after TTX (1 μm) and TTX-sensitive currents evoked by voltage steps (−90 to +40 mV; 10 mV intervals) from a holding potential of −70 mV. Note the large component of inward Na+ current present in a (arrows) but absent in b. b, Currents evoked from the same neuron as in a from a holding potential of −50 mV. Note that despite the substantial diminution of the transient sodium current by holding at −50 mV, TTX-sensitive outward currents and persistent inward currents were largely unchanged. c, d, I–V plots of sustained currents (mean current during final 50 ms of voltage step) from −70 mV and −50 mV holding potentials, respectively. Recordings were made from the soma of dissociated tufted/mitral cells of the olfactory bulb.
IKNa in detached outside-out patches
Notably, the whole-cell experiments shown in Fig. 1 were undertaken with the internal bulk concentration of sodium ion reduced to virtually 0 mm. This means that the activation of IKNa by INaP has to be accomplished by sodium ion crossing the membrane into the cell. This also implies that the local internal concentration of Na+ in the vicinity of IKNa must be raised to a sufficiently high level without any contribution from the bulk internal [Na+]. Because of the low conductance of INaP such a scenario might seem unlikely, but might be possible if channels carrying IKNa and INaP were closely associated. A rigorous test of the hypothesis of the close association and functional coupling of these two channel types would be demonstrating the dependency of IKNa on the activity of INaP in an isolated membrane patch.
To test this hypothesis, we recorded IKNa currents in outside-out patches excised from the soma of T/M cells and showed their dependence on INaP as we had in whole-cell configuration. Figure 2 shows an outside-out macro patch containing a prominent potassium current resembling those recorded in the whole-cell configuration, but lacking any indication of INaT (Fig. 2a). As we showed for whole-cell currents, outward currents were substantially reduced when external sodium was removed (Fig. 2b) but returned when [Na+]o was restored (Fig. 2c). In these experiments, no sodium was included in the internal pipette solution so that activation of KNa currents would be minimally dependent on bulk sodium and primarily determined by sodium influx. The component of K+ current dependent on Na+ influx was revealed by subtraction, and closely resembles that seen in whole-cell recordings (Fig. 2d) (amplitude of sustained outward current in external choline was reduced to 63.5 ± 3.01% of control values p < 0.001, n = 5; upon restoration of external Na+, amplitude of sustained outward current returned to 88.4 ± 7.39% of control values, p < 0.05 compared with choline, mean ± SEM, n = 5, Vm = +60 mV). We also applied TTX to macropatches and, as in whole-cell recordings, saw a significant reduction in the amplitude of total delayed outward current, revealing a large component of IKNa (amplitude of sustained outward current in TTX was reduced to 47.3 ± 12.4% of control values p < 0.05, mean ± SEM, n = 4, Vm = +60 mV). As a control for a direct effect of TTX on KNa channels, we measured whole-cell currents from a line of human embryonic kidney cells stably transfected with the Slack gene which is known to encode KNa channels, and found no change in IKNa current amplitude following application of 1 μm TTX (amplitude of sustained outward current in TTX was 101% ± 2.62% of control value. n = 3, measured at +60 mV p = 0.6). Furthermore, replacing external sodium with the NaV-channel-permeant cation, lithium, also resulted in a significant decrease in the amplitude of sustained outward currents in excised patches (amplitude of sustained outward current in external Li+ was reduced to 56.2 ± 3.67% of control values p < 0.001, n = 4; upon restoration of external Na+, amplitude of sustained outward current returned to 78.8 ± 3.01% of control values, p < 0.01 compared with Li+ mean ± SEM, n = 4, Vm = +60 mV). These results support the hypothesis that both IKNa and INaP currents were present in macropatches.
IKNa depends on sodium influx into an outside-out macropatch. a, Currents were evoked from a holding potential of −70 mV by steps from −90 to +80 mV. b, Removal of external sodium dramatically reduced magnitude of outward currents. c, Currents returned to previous levels upon restoration of external sodium. d, Delayed outward KNa currents, revealed by subtracting 0 [Na+]o currents from control currents, resemble those recorded in whole-cell configuration. e, I–V plot of currents in a–d (mean current of final 50 ms of voltage step). Note that sodium ion was absent from the internal bulk solution.
Only trace amounts of INaT were evoked by step pulses from excised patches held at −70 mV (although patches held at −90 mV exhibited an obvious INaT). Thus, it is unlikely that INaT had any substantial role in evoking the Na+-dependent outward current seen in Figure 2d. Since internal Na+ was not included in the pipette solution in these experiments, the results imply that persistent Na+ influx was responsible. However, because of the low amplitude of INap it is unlikely that there would be a sufficient influx of Na+ during the first few milliseconds of a depolarizing step pulse to substantially raise the local [Na+]i near KNa channels to a sufficiently high level to permit IKNa activation. However, as will be shown in a following section, INap was observed at all holding potentials and thus, the most likely mechanism is that a small but persistent influx of Na+ at holding or resting potentials maintains Na+ at a higher concentration at the site of KNa channels than in the bulk cell interior. Since KNa channels resemble Ca2+-activated BK channels in having a voltage-dependent component to their activation, current amplitudes increase with depolarizing step pulses, as seen in Figure 2.
Single-channel KNa currents coupled to Na+ influx
To examine the functional association of KNa channels with INaP at higher resolution, we used smaller outside-out patches where we could distinguish the activity of individual KNa channels (Fig. 3a). These channels resemble KNa channels in inside-out patches from T/M cells as previously reported to be abundantly expressed in this cell type (Egan et al., 1992). KNa channels from these native cells also resembled sodium-activated SLO2 channels expressed in heterologous systems (Bhattacharjee et al., 2003; Yuan et al., 2003; Santi et al., 2006). The excised patch in Figure 3a contains at least 8 active high conductance channels, and even though single-channel openings are easily discerned, the evoked current as a whole resembles that seen in macropatches as in Figure 2. When TTX (1 μm) was perfused on the outside of this patch and others like it, there was a substantial decline in channel activity (Fig. 3a) (Po decreased to 57.3 ± 3.00% of control value after application of 1 μm TTX, p < 0.001 n = 5, measured at +60 mV). These results suggest that, in addition to the high conductance KNa channels in the patch which are obvious, the patch also contains TTX-sensitive NaV channels which are not. We will show in a following section that single-channel INaP can be revealed when measures are taken to block K+ channels.
Single-channel KNa activity depends on TTX-sensitive persistent sodium influx into an outside-out patch. a, Currents evoked by a family of voltage steps in control conditions and in the presence of TTX (1 μm). KNa channel activation was stimulated by TTX-sensitive entry of sodium ion from the external medium; sodium ion was absent from the internal bulk solution. Upper traces show currents evoked by step pulses from −90 to +60 mV in 10 mV intervals from a holding potential of −70 mV. Lower traces display distributed traces at indicated voltages to allow resolution of individual KNa channel events. b, Currents evoked by steps to indicated voltages from a holding potential of −70 mV in control conditions and after complete replacement of external Na+ with Li+.
Like macroscopic IKNa seen in whole-cell experiments and in macropatches, TTX-sensitive single KNa channel activity was observed without the addition of Na+ to our pipette solution (Fig. 3a). Thus, even at the single-channel level it appears that TTX-sensitive sodium influx and not bulk internal Na+ was activating IKNa. This seems especially persuasive in these experiments with excised outside-out patches because the volume of solution inside the pipette is enormous relative to the miniscule fluid volume in contact with the inner surface of the membrane patch. In these experiments, bulk [Na+]i is not likely to be significantly altered (see calculations in Materials and Methods). Yet, these experiments clearly show the Na+ dependence of single KNa channels. Such activity implies that local [Na+]i near KNa channels at the intracellular membrane surface was being raised to a higher level than bulk internal Na+ by persistent TTX-sensitive Na+ influx. As previously reported in whole-cell experiments (Budelli et al., 2009), replacement of external Na+ with Li+, decreased observed single KNa channel activity in outside-out patches (Fig. 3b). (Po decreased to 66.0 ± 4.16% of control value after replacement of Na+ with Li+, p < 0.001, mean ± SEM n = 3, measured at +60 mV).
External veratridine increases local internal [Na+]
After demonstrating that removing external Na+ or blocking Na+ influx decreases KNa channel activity in isolated patches, we hypothesized that increasing Na+ influx would increase KNa channel activity. We applied veratridine (50 μm), which prolongs the open state of the sodium channel (Barnes and Hille, 1988), to outside-out patches containing active KNa channels, and observed a significant increase in KNa channel activity (Fig. 4). The activating effect of veratridine on single KNa channel activity could be abolished by the removal of external Na+ after application of veratridine. (KNa channel Po increased to 171% ± 9.44% of control value after application of 50 μm veratridine, p < 0.001, n = 10, and decreased to 55.7 ± 12.7% value following subsequent removal of external Na+, p < 0.001, mean ± SEM, n = 10, measured at +60 mV). Thus, our interpretation of these results is that veratridine increases KNa channel activity by increasing sustained Na+ influx via NaV channels. We emphasize that the effect of veratridine must be via increasing the local [Na+] in the vicinity of KNa channels and cannot be due to changing the internal bulk [Na+]. Given the small amplitude of INaP in excised patches (see below) and the volume of internal solution used in recording pipettes, increases in bulk [Na+]i are estimated to be well below 1 μm in experiments using outside-out patches (Materials and Methods), which is orders of magnitude below [Na+] necessary for KNa channel activation. Therefore, changes in activity of KNa channels upon enhancement of INaP by veratridine must be due to local, subcellular changes in [Na+].
Enhancement of INaP increases KNa channel activity. a, Application of veratridine, which prolongs the open state of the sodium channel, increases KNa channel activity. Single-channel KNa currents were evoked by steps to indicated voltages from a holding potential of −70 mV in control conditions (left), after application of veratridine (50 μm; center), and after removal of external Na+ in the continued presence of veratridine (right). b, The effect of veratridine on KNa channel activity in the presence and absence of external Na+ was quantified by calculating the Po of the channels, as described in Materials and Methods, in each condition (n = 10 patches). For each experiment, measured Po during individual sweeps was normalized against mean Po in control conditions. Bars represent relative Po in indicated conditions. Error bars represent SE. p < 0.001 comparing control to veratridine and veratridine to 0 Na+.
Persistent Na+ currents in patch recordings
While many of the recordings described above had some indication of macroscopic and single-channel INaP currents, accurate recording of INaP currents in outside-out patches required blockade of K+ channels. This is due to the small single-channel conductance of NaV-channels, simultaneous activity of Na+ and K+ currents and prominent subconductance states of KNa channels. We found that both transient and persistent TTX-sensitive sodium currents were present in outside-out patches from the soma (Fig. 5a,b). We observed that INaP at a single-channel level consists of brief openings occurring over a wider voltage range than INaT and displaying no significant inactivation over the course of 1 s voltage steps. Many of these persistent TTX-sensitive openings were so brief that we could not reliably measure a mean channel open time, even when digitizing at high rates. Thus, we used the number of detectable events as a means of quantification rather than channel open probability (Fig. 5b). Because of the brevity of openings it is likely that we failed to detect many of them. Although TTX-sensitive openings of INaP occurred less frequently at hyperpolarized voltages, they were clearly present even at −90 mV (Fig. 5b). INaP with similar voltage-sensitivity was identified in whole-cell recordings using voltage steps and slow voltage ramps (Fig. 5c,d). These results suggest INaP is a determinant of the resting potential in T/M cells. Consistent with this, the resting potential of mitral cells undergoes subthreshold oscillations in membrane voltage known to be mediated by TTX-sensitive INa (Desmaisons et al., 1999). This finding was consistent with our earlier whole-cell experiments that seemed to indicate that at cell resting potentials KNa channels were maintained in a “primed” state under normal physiological conditions, which, given the small voltage-sensitive component to KNa channel gating, produces a delayed outward current upon sufficient membrane depolarization. Such a “primed” state is likely to be the result of a tiny but persistent replenishment of Na+ in the local vicinity of KNa which maintains local [Na+]i at a higher level than bulk [Na+]i. INaT in excised patches displayed an unusually hyperpolarized steady-state inactivation curve (V1/2 = −81.7 ± 1.3 mV, peak current measured at −20 mV following 1 s prepulses of −120 to −30 mV, n = 6 patches). Consistent with the lack of apparent INaT in our recordings of KNa channel activity, only ∼10% of the sodium channels contribute to INaT evoked from a holding potential of −70 mV. Atypically hyperpolarized sodium channel inactivation (of the transient component) has recently been reported in other mammalian neurons as well (Grimes et al., 2010; Scott et al., 2010). To confirm that INa in excised patches was sensitive to veratridine, we applied veratridine to an outside-out patch in which INa was isolated. Upon application of 50 μm veratridine, we observed substantially increased INaP activity during voltage steps and a prominent, slowly decaying tail current upon repolarization (Fig. 6a,b) consistent with previous reports of its action on INa (Barnes and Hille, 1988).
Voltage-gated sodium currents present in the soma of T/M cells include a persistent component. Sodium currents were isolated by using Cs+ in the patch pipette and TEA and 4-AP in the bath to block potassium channels. a–d, Recordings were made in outside-out patches (a, b) and whole-cell configuration (c, d) with the internal sodium concentration set to 10 mm to obtain better voltage control of inward current and allow measurement of channel reversal potential. a, Single-channel sodium currents evoked by a voltage step from −90 to −40 mV have both early occurring, high open probability, transient openings, as well as low probability, persistently recurring, brief openings (asterisks). Both components were blocked by TTX (1 μm). b, Plot of the number of channel opening events versus voltage. Channel events were counted from 50 ms after the initiation of a 1 s voltage step to its end. c, Whole-cell, TTX-sensitive persistent sodium currents evoked by 500 ms steps to indicated voltages from a holding potential of −90 mV. Mean current is plotted during the last 100 ms of the pulse (arrow). Error bars represent SEM. n = 5 cells. d, Whole-cell, TTX-sensitive sodium currents evoked by a voltage ramp from −100 to +20 mV, 40 mV/s. In c and d, TTX-sensitive currents were isolated and plotted by subtracting residual currents evoked in TTX (1 μm) from currents evoked before TTX application.
Veratridine enhances INaP in outside-out excised patches. Sodium currents were isolated by using Cs+ in the internal pipette solution and TEA and 4-AP in the bath solution to block potassium channels. Recordings were made in macroscopic outside-out patches with the internal sodium concentration set to 10 mm. a, Application of 50 μm veratridine to outside-out patches increased INaP activity during voltage steps, and induced a prominent, very slowly decaying tail current upon repolarization. Inset shows same recordings at higher time resolution. Only a minor difference is seen in the amplitude of INaT before and after veratridine. Dashed lines represent 0 current level. b, Veratridine-induced INa was revealed by subtracting control current from current recorded in veratridine.
Discussion
Upon initial identification in cardiac myocytes, the suggested role for KNa channels was that of providing a reserve conductance that would protect cells during hypoxia when an elevation in [Na+]i occurred as a result of blocking the Na+/K+ ATPase (Kameyama et al., 1984). This idea arose as a result of observations of KNa channels in inside-out patches which showed that KNa channel activation required a much higher [Na+]i than that normally found in bulk cytoplasm. However, we have uncovered an unexpected phenomenon by which Na+ entering the cell via a small but persistent sodium current is surprisingly effective in activating IKNa, even in the total absence of bulk internal Na+. The seeming implausibility of such a mechanism may be partially responsible for keeping this major and widespread component of delayed outward conductance unappreciated by electrophysiologists for many years. These observations suggest a mechanism by which IKNa can commonly participate in providing a large outward conductance in many neuronal types. KNa channels are expressed throughout the brain (Bhattacharjee et al., 2002, 2005). The Allen Brain Atlas shows that one of two genes which encode KNa channels (KCNT1, also known as Slack and Slo2.2) is as widespread and abundantly expressed as KCNB1, the gene encoding the major voltage-dependent delayed rectifier (http://mouse.brain-map.org/).
The functional coupling of IKNa to INaP that we observe at the single-channel level in isolated outside-out patches suggests a highly specialized relationship between KNa and NaV channels perhaps similar to the colocalization between calcium channels and the Slo1 calcium-activated potassium channel (BK) (Marrion and Tavalin, 1998). Although the coupling of KNa to Na channels may be analogous, the two orders of magnitude difference in the required concentrations of Na+ and Ca2+ needed to activate the respective K+-channels suggests some differences. Hence, we are unlikely to observe a correlation between the opening of single NaV and KNa channels as is observed between calcium channels and single BK channels. Nevertheless there are may parallels between the two systems. In both systems the channels are activated by two major factors, an intracellular ion ligand, and voltage (albeit KNa channels have lower voltage sensitivity). In both systems, the effect of increasing the concentration of the intracellular ion ligand is to shift the conductance-voltage relation leftward to more hyperpolarized voltages. We previously demonstrated this for IKNa by loading cells with higher concentrations of Na+ and noting a leftward shift in the conductance-voltage relation of IKNa (Budelli et al., 2009).Significantly, those experiments showed that cells loaded with higher concentrations of [Na+]i still retained an obvious delayed outward component of IKNa due to the influx of Na+. Thus, the TTX-dependent influx of Na+ appears to be additive to the [Na+]i contributed by the bulk solution.
A possible mechanism for the functional coupling of KNa and INaP is their presence in a region of limited diffusion near the plasma membrane, sometimes described as a fuzzy space or unstirred layer. Physiological evidence of such a region in cardiac myocytes has been accumulating for many years (for review, see Barry, 2006). Microheterogeneity of sodium concentrations in internal submembrane space has been inferred many times (Barry, 2006), and has even been directly measured (Wendt-Gallitelli et al., 1993). Measurements using electromagnetic pulse analysis shows [Na+] near the inner membrane surface can be severalfold higher than the bulk [Na+]i and heterogeneous within a cell, ranging from 0 to 80 mm at the inner membrane surface. Thus, it is possible that a small but constant influx of Na+ through INaP is responsible for increasing [Na+] in some submembrane regions where KNa channels are located, and that limited diffusion as well as colocalization of INaP and IKNa both contribute to activation of IKNa by INaP.
This study focused on INaP as a major source of sodium for the activation of KNa channels. While our data suggest INaT is not necessary for KNa activation as a delayed outward current, we have not ruled out a contribution of INaT in all circumstances. The NaV-channels that carry INaP are likely to also carry INaT, and it is difficult to imagine a mechanism by which only persistent Na+-channel activity would activate KNa channels. However, our study indicates that the Na+-channels present in somal membrane may be largely inactivated at resting membrane potentials with regard to their capacity to carry a transient sodium current on depolarization, while maintaining the ability to carry INaP at most voltages. Activation of IKNa by INaT has been previously examined in outside-out patches of neurons from chick brainstem (Dryer, 1991). That study found that while TTX blocked INaT it had no effect on potassium currents. However, those experiments observed the effects of INaT on outward currents at −25 to −10 mV, near peak INaT amplitude. We point out that, under conditions of low [Na]i loading, we also do not detect net outward IKNa at those voltages, which we ascribe to the voltage-dependent component of IKNa activation. Thus, Dryer's experiments must be repeated at higher voltages before ruling out a role for INaT in KNa activation.
The activity of KNa channels has been shown to be sensitive to intracellular Cl− (Bhattacharjee et al., 2003; Yuan et al., 2003). We have found the magnitude of TTX-sensitive outward current in whole-cell recordings to be accordingly affected by [Cl−]i. TTX-sensitive potassium currents in recordings using a physiological [Cl−]i of 20 mm comprise ∼30% of the sustained outward current in mitral cells, compared with 57% in 150 mm [Cl−]i (Budelli et al., 2009; our unpublished data). Our current study used an elevated [Cl−]i to increase the likelihood of observing KNa channels and to more precisely measure channel Po. KNa channels have been shown to be activated by the ubiquitous enzyme cofactor nicotinamide adenine dinucleotide (NAD+) (Tamsett et al., 2009). The presence of NAD+ may increase the activity of KNa channels at physiological concentrations of internal bulk Na+.
The contribution of INaP to the activation of a prominent potassium conductance may seem counterintuitive given the established role of INaP in increasing neuronal excitability. We point out that the INaP-IKNa coupled system produces a net inward current in mitral cells between typical resting potentials and action potential thresholds (Figs. 1c,d, 2e). Nevertheless, within those voltages the activation of IKNa may mitigate depolarization and while it does not overcome INaP, could provide some negative feedback against the excitatory role of INaP, in the form of membrane accommodation. It may also act as a repolarizing current that shortens action potential duration. In addition, there is evidence for neuromodulatory regulation of both INaP (for review, see Cantrell and Catteral, 2001) and IKNa (Santi et al., 2006; Nuwer et al., 2010). Hence, the discrete or combined regulation of either component of the INaP-IKNa coupled system could provide a mechanism for long-lasting changes in excitable properties over a wide range of both subthreshold and depolarized voltages.
INaT and the upstroke of action potentials are grossly similar across neurons. In contrast, the behavior of INaP varies and contributes to diverse physiological properties such as amplification of synaptic potentials, repetitive firing of action potentials, spike timing, and even membrane resting potential (Crill, 1996; Vervaeke et al., 2006, Huang and Trussell, 2008). Indeed, INaP may be a significant determinant of some of the most subtle excitable membrane behavior such as electrical resonance generating theta frequency firing (Hu et al., 2002). In all of these electrical phenomena in which INaP participates, IKNa may now be found to be a coparticipant. Many inherited epilepsies are due to gain- or loss-of-function mutations in voltage-gated sodium channels which alter INaP. Accordingly, several antiepileptic drugs primarily target INaP; however, understanding of pathophysiological mechanisms of INaP toward epilepsy and other disorders is incomplete (Stafstrom, 2007; Saint, 2008; Waxman, 2008). For all these reasons, INaP is a topic of great research interest for clinical and basic neuroscientists. The functional coupling of INaP to KNa channels must now also be considered as the contribution of INaP to neuronal physiology continues to be investigated.
Footnotes
Support was provided by the National Institute of Neurological Disorders and Stroke Grant RO1-NS0661871-01. We thank A. Butler, M. Covarrubias, and A. Wei for their helpful comments on the manuscript. We also thank the Salkoff laboratory for their assistance during the course of the study. We thank G. Budelli for control data from the Slack HEK cell line.
The authors declare no competing financial interests.
- Correspondence should be addressed to Lawrence Salkoff, Department of Anatomy & Neurobiology, Washington University School of Medicine, 660 S. Euclid Avenue, Box 8108, St. Louis, MO 63110. salkoffl{at}pcg.wustl.edu
