Nav1.5 underlies the ‘third TTX-R sodium current’ in rat small DRG neurons

Molecular Brain Research 106 (2002) 70–82 www.elsevier.com / locate / bres

Research report

Na v 1.5 underlies the ‘third TTX-R sodium current’ in rat small DRG neurons M. Renganathan a,b,c , S. Dib-Hajj a,b,c , S.G. Waxman a,b,c , * a

b

Department of Neurology, Yale Medical School, New Haven, CT 06510, USA Paralyzed Veterans of America /Eastern Paralyzed Veterans Association, Neuroscience Research Center, Yale Medical School, New Haven, CT 06510, USA c Rehabilitation Research Center, Veteran Administration Connecticut Healthcare Center, West Haven, CT 06516, USA Accepted 30 July 2002

Abstract In addition to slow-inactivating and persistent TTX-R Na 1 currents produced by Na v 1.8 and Na v 1.9 Na 1 channels, respectively, a third TTX-R Na 1 current with fast activation and inactivation can be recorded in 80% of small neurons of dorsal root ganglia (DRG) from E15 rats, but in only 3% of adult small DRG neurons. The half-time for activation, the time constant for inactivation, and the midpoints of activation and inactivation of the third TTX-R Na 1 currents are significantly different from those of Na v 1.8 and Na v 1.9 Na 1 currents. The estimated TTX Ki (2.1160.34 mM) of the third TTX-R Na 1 current is significantly lower than those of Na v 1.8 and Na v 1.9 Na 1 currents. The Cd 21 sensitivity of third TTX-R Na 1 current is closer to cardiac Na 1 currents. A concentration of 1 mM Cd 21 is required to completely block this current, which is significantly lower than the 5 mM required to block Na v 1.8 and Na v 1.9 currents. The third TTX-R Na 1 channel is not co-expressed with Na v 1.8 and Na v 1.9 Na 1 channels in DRG neurons of E18 rats, at a time when all three currents show comparable densities. The physiological and pharmacological profiles of the third TTX-R Na 1 current are similar to those of the cardiac Na 1 channel Na v 1.5 and RT-PCR and restriction enzyme polymorphism analysis, show a parallel pattern of expression of Na v 1.5 in DRG during development. Taken together, these results demonstrate that Na v 1.5 is expressed in a developmentally regulated manner in DRG neurons and suggest that Na v 1.5 Na 1 channel produces the third TTX-R current.  2002 Elsevier Science B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Na 1 channels Keywords: Embryonic dorsal root ganglia; Primary sensory neurons; Tetrodotoxin resistant; Cardiac Na 1 channel; Na v 1.8 Na 1 channels; Na v 1.9 Na 1 channels

1. Introduction The electrophysiological properties of neurons are dependent upon the complement of sodium currents that they express [27,30]. Sensory neurons of the dorsal root ganglia (DRG) express multiple Na 1 currents [1,10,12,13,20,29,36]. Based on the kinetics of activation, inactivation and sensitivity to tetrodotoxin (TTX), the sodium currents in neurons of adult rat DRG have been *Corresponding author. Department of Neurology, LCI 707 Yale School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA. Tel.: 11-203-785-6351; fax: 11-203-785-7826. E-mail address: [email protected] (S.G. Waxman).

broadly classified as fast-inactivating TTX-sensitive (TTXS), slow-inactivating TTX-resistant (TTX-R) and persistent TTX-R. The majority of small diameter DRG neurons express all of these Na 1 currents [12,13,33]. The complex composition of the Na 1 currents of adult rat DRG neurons matches the presence of at least six Na 1 channels in these cells: Na v 1.1 (type I), Na v 1.6 (NaCh6), Na v 1.7 (PN1), Na v 1.8 (SNS), Na v 1.9 (NaN) and Na x (Na G ) [8,15]. The fast TTX-S Na 1 current appears to be a composite, produced by several channels including Na v 1.1, Na v 1.6 and Na v 1.7. Sodium channel Na v 1.8 produces the slow-inactivating TTX-R current [1,38] and plays an important role in the generation of action potentials in small DRG neurons [34], while sodium channel Na v 1.9

0169-328X / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 02 )00411-4

M. Renganathan et al. / Molecular Brain Research 106 (2002) 70–82

[15] produces a persistent TTX-R current [12] and may contribute to setting resting membrane potential of neurons and to modulating excitability [26]. An additional, fast-inactivating TTX-R Na 1 current has been recorded from a limited number of small [37,42] and medium-sized [42] DRG neurons but the channel that produces it has not yet been identified. Cardiac sodium channel, Na v 1.5 (rH1), produces a fast-inactivating TTX-R Na 1 current in native myocytes, neuroblastoma cells and when expressed in cell lines [23,32,40,51]. Several studies using independent molecular approaches to screen for Na v 1.5 transcripts did not detect them in adult rat DRG neurons [7,8,17], while a recent abstract describes trace levels of Na v 1.5 transcripts in adult rat DRG [Chaplan et al. Society for Neuroscience, abstract number 418.19, 2000]. Na v 1.3 (brain type III) expression in DRG neurons has been shown to decrease as development proceeds, reaching very low levels of expression shortly after birth [3,8,21,49]. We hypothesized that the fast TTX-R current and the channel that produces it might be expressed at higher levels at earlier developmental stages, similar to Na v 1.3. To test this hypothesis, we used patch-clamp analysis to search for the expression of the fast-inactivating TTX-R Na 1 current in rat and mouse DRG neurons and, in parallel, searched for Na v 1.5 transcripts using molecular biological techniques. Here we show that the fast-inactivating TTX-R Na 1 current is present in a large number of embryonic DRG neurons. The electrophysiological characteristics, TTX sensitivity, and cadmium sensitivity of the fast-inactivating TTX-R current are similar to those reported for Na v 1.5. We also show that Na v 1.5 transcripts are present in embryonic DRG neurons with their level falling as development continues, in parallel with the attenuation of the fast inactivating TTX-R currents. The electrophysiological and pharmacological properties of the fast-inactivating TTX-R current, and the parallel pattern of expression of Na v 1.5 transcripts, indicate that the cardiac sodium channel Na v 1.5 is expressed in DRG neurons where it underlies the third TTX-R Na 1 current.

2. Materials and methods

2.1. Animal care Experiments were carried out in accordance with NIH and institutional guidelines for the care and use of laboratory animals. Three groups of Sprague–Dawley (embryonic day 15–18: E15 to E18; neonatal: P0–P7; adult: 6-weeks old) rats and one group of P0–P2 Na v 1.8 null mice were used in this study. Animals were housed under a 12 h light–dark cycle in a pathogen-free area with free access to water and food at the Veterans Affairs Connecticut Healthcare Center, West Haven.

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2.2. Culture of dorsal root ganglia ( DRG) neurons Neonatal animals were decapitated by knife with a single stroke. Adult rats were rendered unconscious by exposure to CO 2 and decapitated. L4 and L5 lumbar DRG were freed from their connective sheaths in sterile calciumfree saline solution. Cell cultures were prepared as previously described [33]. Briefly, the L4 and L5 DRG ganglia were harvested, treated with collagenase and papain, dissociated in DMEM and Ham’s F12 medium supplemented with 10% fetal bovine serum, and plated on polyornithine and laminin coated glass coverslips. Neurons were placed in a 5% CO 2 –95% air incubator at 37 8C and, 1 h after isolation, were fed with fresh culture medium. Na 1 current properties in DRG neurons were investigated 2–8 h after isolation. Short-term culture allowed the cells sufficient time to adhere to the glass coverslips. DRG neurons displayed only short (,10 mm) axonal processes during the short period of incubation, facilitating voltageclamp.

2.3. Electrophysiological recordings Coverslips were mounted in a small flow-through chamber on the microscope stage and were continuously perfused with bath–external solution (see below) with a push–pull syringe pump (WPI, Saratoga, FL, USA). Cells were voltage-clamped via the whole-cell configuration with an Axopatch-200B amplifier (Axon Instruments, Foster City, CA, USA) using standard techniques. For currents .20 nA, we switched to the 50 MV feedbackresistor ( b 50.1) which can pass up to 200 nA. Micropipettes (0.6 to 1 MV) were pulled from borosilicate glasses (Boralex) with a Flaming Brown P80 micropipette puller and polished on a microforge and coated with a mixture prepared of three parts of finely shredded parafilm and one part each of light and heavy mineral oil (Sigma, St. Louis, MO, USA) to reduce the pipette capacitance. Capacity transients were canceled and series resistance was compensated (90%) as necessary. The pipette solution contained (in mM): 140 CsF, 1 EGTA, 10 NaCl, and 10 HEPES, pH 7.3 and adjusted to 310 mOsmol / l with glucose. The bath solution contained (in mM): 140 NaCl, 3 KCl, 1 MgCl 2 , 1 CaCl 2 , and 20 HEPES, pH 7.3, and adjusted to 320 mOsmol / l with glucose. The pipette potential was zeroed before seal formation and voltages were not corrected for liquid junction potential. Leakage current was digitally subtracted on-line using hyperpolarizing control pulses, applied before the test pulse, of onesixth test pulse amplitude (P/ 6 procedure). Whole-cell currents were elicited from a holding potential of 2130 mV and filtered at 5 kHz and acquired at 50 kHz using CLAMPEX 8.1 software (Axon Instruments). For current density measurements membrane currents were normalized to membrane capacitance which was calculated as the integral of the transient current in response to a brief

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hyperpolarizing pulse from 2120 mV (holding potential) to 2130 mV. All experiments were performed at room temperature (21–25 8C).

A1 2 A2 y 5 ]]]]p 1 A 2 1 1 (x /x 0 )

2.4. Separation of Nav 1.8 and Nav 1.9 TTX-R Na 1 currents using prepulse inactivation

was used to fit cadmium block of the third TTX-R Na current. Ki for TTX was determined from the following equation

1

Prepulse inactivation takes advantage of the differences in the inactivation properties of the Na v 1.8 and Na v 1.9 TTX-R Na 1 currents [12]. TTX (250 nM) was included in the bath solution to isolate Na v 1.8 and Na v 1.9 TTX-R Na 1 currents from fast TTX-S Na 1 currents (which are eliminated by 250 nM TTX). Na 1 currents were evoked from a holding potential of 2130 mV to test pulses ranging from 2100 to 60 mV. Na v 1.9 TTX-R Na 1 current was obtained by subtracting the current obtained following a 240 mV prepulse (500 ms duration), which elicits only Na v 1.8 TTX-R Na 1 current, from the current obtained with more hyperpolarized prepulse (2130 mV) which elicits both Na v 1.8 and Na v 1.9 TTX-R Na 1 currents. Conductance was determined as I /(V 2VR ), where I is the peak inward current at test pulse voltage V, and VR is the calculated reversal potential. Normalized conductance 1 G /Gmax 5 ]]]]]]] 1 1 exp [(V1 / 2 2V ) /k] (G /Gmax ) was fit with a single Boltzmann relationship of the form where V is the test pulse voltage, V1 / 2 is the voltage for half-maximal activation in mV, and k is the slope factor. Inactivation curves were measured using 500-ms prepulses to the indicated potentials followed by a test pulse to 0 mV for the third and Na v 1.8 TTX-R Na 1 currents. For Na v 1.9 TTX-R Na 1 current the test pulse was 250 mV, where no activation of Na v 1.8 TTX-R Na 1 current occurs. Peak test pulse current was plotted as a function of prepulse potential, normalized and fit with a single Boltzmann function 1 I /Imax 5 ]]]]]]] 1 1 exp [(Vpp 2Vh ) /k h ] where Vpp is the prepulse potential, Vh is the midpoint potential and k h is the slope factor. Inactivation kinetics were evaluated by fitting the decay of the current with a single exponential for Na v 1.8 and Na v 1.9 TTX-R Na 1 currents and with two exponentials for the third TTX-R Na 1 currents, according to the equation

OA e n

f(t) 5

i

2t / t 1

1C

t 51

where A is amplitude, t is the interval in ms, t is the time constant and C is the offset. Logistic dose–response equation (Microcal Origin 6.1, Northampton, MA, USA)

It 1 [t 1 ] 2 It 2 [t 2 ] K1 5 ]]]]] It 2 2 It 1 where It 1 and It 2 are current amplitudes at two different toxin (t 1 and t 2 ) concentrations.

2.5. Data analysis Patch-clamp data were analyzed using a combination of 8.1 (Axon Instruments) and ORIGIN (Microcal Software). For statistical analysis, Student’s unpaired t-test was used. We used ANOVA followed by posthoc analysis (Tukey test) to determine the statistical significance of the difference between Na v 1.8, Na v 1.9 and Na v 1.8 Na 1 current half-maximal activation time (t 1 / 2 ) and inactivation time constants, and V1 / 2 values. All P values were significant at 0.05 level. Data are presented as mean6S.E.M. CLAMPFIT

2.6. RNA isolation Total cellular RNA from E15, E17, P0 and P7 DRG and trigeminal ganglia, and cardiac muscle from adult Sprague–Dawley rats was isolated using RNeasy minicolumns (Qiagen, Valencia, CA, USA). First strand cDNA was reverse transcribed in a 50 ml final volume using 1 mM random hexamer (Roche, Indianapolis, IN, USA), 500 units SuperScript II reverse transcriptase (Life Technologies) and 100 units of RNase Inhibitor (Roche). The buffer consisted of 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl 2 , 10 mM DTT and 5 mM dNTP. The reaction was allowed to proceed at 37 8C for 90 min, 42 8C for 30 min and then terminated by heating to 95 8C for 5 min. Control templates were prepared in identical fashion except for the omission of the reverse transcriptase enzyme (2RT).

2.7. Polymerase chain reaction We used a RT-PCR / restriction enzyme polymorphism assay [7] to amplify products primarily from Na v 1.5 [35] and Na v 1.6 [41] that might have been present in the cDNA pool. The predicted lengths of amplified products, 518 and 507 bp, and subunit-specific restriction enzyme recognition sites, Acc I and Sph I, are characteristic for subunits Na v 1.5 and Na v 1.6, respectively [7]. PCR amplification was performed in a 30 ml volume using 1 ml of first strand cDNA, 0.8 mM of each primer and 1.75 units of expand long template DNA polymerase enzyme mixture (Roche). Control PCR reactions in which

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the –RT template was used produced no amplification products (data not shown). As described previously [14], amplification was carried out in two stages using a programmable thermal cycler (PTC-100, MJ Research, Cambridge, MA, USA).

2.8. Restriction enzyme analysis The identity of the a-subunits expressed in the DRG, trigeminal ganglia and heart were determined by restriction enzyme analysis of their PCR products. For restriction enzyme analysis, 1 / 10 of the PCR reaction is digested for 1 h at the recommended temperature and the products resolved by electrophoresis in a 1.7% agarose gel. Fragment sizes were determined by comparison to a 100-bp ladder molecular weight marker (Pharmacia, Peapack, NJ, USA). DNA was visualized by ethidium bromide fluorescence and the gel images were digitized using a Kodak Image Station 440 (Kodak, Rochester, NY, USA).

3. Results

3.1. Third TTX-R Na 1 current in neonatal DRG neurons Whole-cell patch-clamp recordings demonstrate the presence of a slow inactivating TTX-R (Na v 1.8) Na 1 current and a persistent TTX-R (Na v 1.9) Na 1 current from cultured neonatal (P0) rat DRG neurons when held at 2130 mV in the presence of 250 nM TTX (Fig. 1A). A combination of Na v 1.8 and Na v 1.9 Na 1 currents was recorded from |66% of small neurons (73 of 111, Fig. 1A). In this set of experiments, 68 cells exhibited both Na v 1.8 Na 1 currents and Na v 1.9 Na 1 currents and 5 cells had only Na v 1.8 Na 1 currents. When the cells are held at more depolarized potentials, such as 240 mV, Na v 1.9 Na 1 currents are attenuated by ultraslow inactivation [12] and only Na v 1.8 Na 1 currents are observed (Fig. 1B). If the currents obtained with the depolarized holding potential (Fig. 1B) are digitally subtracted from the currents obtained with 2130 mV holding potential (Fig. 1A), the Na v 1.9 Na 1 currents can be seen in relative isolation (Fig. 1C). The average Na v 1.8 and Na v 1.9 Na 1 current amplitudes were 16.8561.28 nA (n573) and 8.7861.45 nA (n568), respectively. The mean capacitance of the cells which expressed Na v 1.8 and Na v 1.9 Na 1 currents was 16.3561.6 pF. The average Na v 1.8 and Na v 1.9 Na 1 current densities were 1.0660.20 pF and 0.5560.08 nA / pF, respectively. Na 1 current densities were determined by dividing the peak current amplitude by cell capacitance. A third TTX-R Na 1 current with fast activation and inactivation was also observed in 20% (22 of 111) P0 neurons (Fig. 1D). Cells that expressed the third TTX-R Na 1 currents did not express Na v 1.8 or Na v 1.9 Na 1 currents. The average third TTX-R Na 1 current amplitude was 2.2260.31 nA. The mean capacitance of the cells

Fig. 1. Multiple TTX-R Na 1 currents are expressed in neonatal small rat DRG neurons. Sodium currents were recorded from a P0 small DRG neuron using a holding potential of 2130 mV. TTX (250 nM) was used to block fast TTX-sensitive Na 1 currents. Both Na v 1.9 and Na v 1.8 Na 1 currents were elicited in response to 100-ms test pulses ranging from 2100 to 160 mV. The capacitance of the DRG neuron was 16.90 pF. (B) The same neuron, when conditioned at a prepulse of 240 mV for 500 ms before test potentials from 2100 to 160 mV, elicited only Na v 1.8 Na 1 current. (C) Subtraction of current traces shown in (B) from the current traces shown in (A) yields Na v 1.9 Na 1 currents in relative isolation. (D) A third TTX-R Na 1 current is recorded from a different P0 small DRG neuron in the presence of 250 nM TTX using a holding potential of 2130 mV; cell capacitance was 17.95 pF.

which expressed the third TTX-R Na 1 current was 16.2461.32 pF, not significantly different from cells that expressed Na v 1.8 and Na v 1.9 Na 1 currents (P.0.05). The mean current density of the third TTX-R Na 1 current was 0.15160.02 nA / pF (n522), significantly smaller than Na v 1.8 and Na v 1.9 Na 1 current densities (P,0.05). The third TTX-R Na 1 current density was determined by dividing the peak current amplitude observed at | 225 mV by cell capacitance. A similar TTX-R Na 1 current with fast activation and inactivation properties has been previously reported in rat DRG neurons [37,42].

3.2. Third TTX-R Na 1 current has faster activation and inactivation time constants than Na v 1.8 or Na v 1.9 Na 1 currents The third TTX-R Na 1 current displays faster activation and inactivation than Na v 1.8 and Na v 1.9 Na 1 currents at 0 mV (Fig. 2A). The half-maximal activation time (t 1 / 2 ) and inactivation time constants for the third, Na v 1.8 and

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Fig. 2. Third TTX-R Na 1 current displays faster activation and inactivation than Na v 1.8 and Na v 1.9 Na 1 currents. Normalized current traces of the third, Na v 1.8 and Na v 1.9 TTX-R Na 1 currents at 0 mV test pulse are shown. Half-maximal activation (B) and inactivation time constants (C) were determined for the full voltage range of activation. The potentials between 270 and 260 mV and 45 and 60 were not used due to smaller currents and the absence of inactivation at these potentials. Inset shows the voltage dependence of slow inactivation time constant for the third TTX-R Na 1 currents.

Current

Half-maximal activation time (ms) at three test potentials

1

Na v 1.8 Na 1 Na v 1.9 Na Third TTX-R Na 1 Na v 1.5 Na 1

Inactivation time constant (ms) at three test potentials

Activation

215 mV

0 mV

15 mV

215 mV

0 mV

15 mV

0.7560.1 1.0060.16 0.1360.01 0.4 5

0.3260.03 0.4360.05 0.0960.006 0.15 and 0.25 1,5

0.1760.02 0.2560.02 0.0660.006 0.1 1,2

9.3661.5 12.4161.43 0.2060.03* 0.9 and 2.1 5,4

3.4760.52 5.7160.57 0.1160.008* 0.75 1,2

1.3960.2 3.1160.37 0.0860.06* 0.75 and 0.9 1,2

Inactivation

V1 / 2 (mV)

Slope (mV/ e-fold potential change)

V1 / 2 (mV)

Slope (mV/ e-fold potential change)

215.762.6 257.562.2 242.4761.5 229 to 249 1 – 5

6.461.5 5.8460.8 7.360.4 5 to 7 4,5

233.766.5 255.261.3 283.0761.55 266 to 292 1 – 5

7.262.4 6.560.4 9.0460.37 5 to 7 4,5

*, Indicates t, fast. Values for Na v 1.5 Na 1 current parameters were taken from the following references: 1 Bennett [5]; 2 Brown et al. [9]; 3 O’Leary [32]; 4 Satin et al., [39]; 5 Gu et al., [23].

IC 50 TTX (mM)

46610 47613 2.1160.34 1 to 6 1 – 5

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Table 1 Current properties of Na v 1.8, Na v 1.9, third TTX-R, and Na v 1.5 Na 1 channels

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Na v 1.9 Na 1 currents were determined for the voltage range shown in Fig. 2B and C. The half-maximal activation time (t 1 / 2 ) for the third TTX-R Na 1 current decreased with depolarizing test pulses and were significantly faster than Na v 1.8 and Na v 1.9 Na 1 current half-maximal activation time at all test potentials (n510; P,0.05; ANOVA, Tukey test). The inactivation curves of the Na v 1.8, and Na v 1.9 TTX-R Na 1 currents were fit with a single exponential, whereas the third TTX-R Na 1 current was fit with two exponentials, i.e. fast and slow. The slow component was a minor fraction (10–15%) of the third TTX-R Na 1 current. The fast inactivation time constants for the third TTX-R Na 1 current decreased with depolarizing test pulses and were significantly faster than those of Na v 1.8 and Na v 1.9 Na 1 currents at all test potentials (n510; P,0.05; ANOVA, Tukey test). The slow inactivation time constant for the third TTX-R Na 1 current was |20 ms at 250 mV and reduced to |15 ms at 40 mV (Fig. 2C, inset). The half-maximal activation time (t 1 / 2 ) and inactivation time constants for the third TTX-R and Na v 1.8 and Na v 1.9 Na 1 current at three test potentials, i.e. 215, 0, 15 mV are given in Table 1. These results demonstrate that the third TTX-R Na 1 current is distinctly faster in activation and inactivation than the Na v 1.8 and Na v 1.9 Na 1 currents that are present in DRG neurons, and it will be referred to as the fast TTX-R Na 1 current hereafter.

3.3. Current–voltage relationship and voltagedependence of activation and inactivation of the fast TTX-R Na 1 current The current–voltage relationship of the fast TTX-R Na 1 current is shown in Fig. 3A (n538, the current–voltage relationship did not change with development, therefore the data were pooled from E15 to adult animals). The current is detectable at 270 mV, peaks at 225 mV and reverses at 157 mV which is close to the calculated Nernst potential, 167 mV. Na v 1.9 Na 1 current also noticeably activates at | 280 mV and peaks at | 250 mV [12,16,33], while Na v 1.8 Na 1 current activates at 240 mV and peaks at | 220 mV [1,12,13,33,42]. The voltage-dependence of activation (open circles) and inactivation curves (closed circles) for the fast TTX-R Na 1 current are shown in Fig. 3B. Distinct midpoint voltage (V1 / 2 ) and slope (k) for activation of the fast, Na v 1.8 and Na v 1.9 TTX-R Na 1 currents were obtained from fitting the conductance–voltage curve with the Boltzmann equation (Table 1). The V1 / 2 and k values of Na v 1.8 Na 1 currents are similar to the results reported earlier [13,37,42]. The V1 / 2 and k values of Na v 1.9 Na 1 currents are similar to those observed in neonatal DRG neurons [46]. The V1 / 2 for activation of the fast TTX-R Na 1 current is significantly more hyperpolarized than Na v 1.8 Na 1 currents and significantly more depolarized than Na v 1.9 Na 1 currents (P,0.05; ANOVA, Tukey test) with no significant difference in slope values (Table 1).

Fig. 3. Current–voltage relationship and voltage-dependent activation and inactivation of fast TTX-R Na 1 current. (A) Normalized current– voltage relationships for the peak fast TTX-R Na 1 current in rat DRG neurons (E15 to adult) are shown (n538). Error bars indicate S.E.M. (B) Voltage-dependent activation (normalized conductance, open circles) and inactivation (fraction available, closed circles) were determined as mentioned in Methods. Lines connecting the symbols are Boltzmann fits to the data.

The midpoint voltages (V1 / 2 ) and slope for inactivation of fast TTX-R Na 1 current and Na v 1.8 and Na v 1.9 Na 1 currents were obtained from fitting the fraction available for opening as a function of voltage with the Boltzmann equation (Table 1). The differences in V1 / 2 values for the inactivation between the fast TTX-R Na 1 current, Na v 1.8 and Na v 1.9 currents are statistically significant (P,0.05; ANOVA, Tukey Test). Together with the time constants for activation and inactivation, the difference between V1 / 2 values for activation and inactivation of the fast TTX-R Na 1 current compared to Na v 1.8 and Na v 1.9 Na 1 currents strongly suggests the presence of a fast TTX-R Na 1

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and slope values for activation and inactivation of fast TTX-R Na 1 current in mouse DRG neurons were similar to those of rat DRG neurons. Furthermore, the fast-inactivating TTX-R Na 1 current was recorded from the majority of rat DRG neurons at age E15, a time when neither Na v 1.8 nor Na v 1.9 expression is detectable [4]. Taken together these results demonstrate that neither of these two channels underlies the fast-inactivating TTX-R Na 1 current. Fig. 4. Fast TTX-R Na 1 current is present in small DG neurons isolated from Na v 1.8 null mice. Recordings from a representative small P0 DRG neuron isolated from a Na v 1.8 (2 / 2) mouse show the presence of the fast TTX-R current (n55). Current traces were elicited from a holding potential of 2130 mV and in the presence of 250 nM TTX.

channel, distinct from Na v 1.8 and Na v 1.9, in small DRG neurons.

3.4. Fast TTX-R Na 1 current is present in Nav 1.8 (2 / 2) null mice Although the difference in the electrophysiological properties suggests the presence of three TTX-R Na 1 channels in rat DRG neurons, it could be argued that Na v 1.8 or Na v 1.9 Na 1 currents might be modulated to produce the fast-inactivating TTX-R Na 1 current. To test the possibility that Na v 1.8 Na 1 channels might underlie the fast-inactivating TTX-R Na 1 currents, we asked whether this current is present in P0 DRG neurons of Na v 1.8 null (2 / 2) mice [2], in which functional Na v 1.8 Na 1 channels are absent. As seen in Fig. 4, the fastinactivating TTX-R Na 1 current is clearly present in Na v 1.8 null DRG neurons (n55), indicating that the Na v 1.8 Na 1 channel does not produce the fast TTX-R Na 1 current. The time constants, the midpoint voltages (V1 / 2 ),

3.5. Fast TTX-R Na 1 current is more sensitive to TTX than Na v 1.8 and Na v 1.9 Na 1 currents To further establish that the fast TTX-R Na 1 current is different from Na v 1.8 and Na v 1.9 TTX-R Na 1 currents, we exposed P0 DRG neurons which expressed Na v 1.8 and Na v 1.9 Na 1 currents, and neurons which expressed the fast TTX-R Na 1 currents to 5 and 10 mM TTX concentrations (Fig. 5). TTX (250 nM) was used to block TTX-S current and to isolate the three TTX-R Na 1 currents. Both Na v 1.8 and Na v 1.9 TTX-R Na 1 currents were resistant to 5 and 10 mM TTX concentrations, displaying reduction in amplitude of 10 and 30% of Na v 1.8 currents, and 7 and 20% of Na v 1.9 currents, respectively (Fig. 5A and B). The estimated Ki values for Na v 1.8 and Na v 1.9 TTX-R Na 1 currents are 46610 mM (n54) and 47613 mM (n54). These values are similar to the earlier results which established that Na v 1.8 (IC 50 560 mM) [45] and Na v 1.9 (Ki 540 mM) [12] Na 1 currents are resistant to high concentrations of TTX. The fast TTX-R Na 1 current, however, was completely blocked by 10 mM TTX (Fig. 5C). The estimated Ki for the fast TTX-R Na 1 current is 2.1160.34 mM (n58). The similarity of the TTX sensitivity of the fast TTX-R Na 1 current to the TTX sensitivity of Na v 1.5 current [11,23,40,50] suggests that fast TTX-R Na 1 current in DRG may be produced by Na v 1.5 Na 1 channels.

Fig. 5. Fast TTX-R Na 1 current is more sensitive to TTX than Na v 1.8 and Na v 1.9 Na 1 currents. TTX (250 nM) was initially used to block fast TTX-sensitive Na 1 currents, and increasing concentrations of TTX were used to determine Ki for the fast TTX-R, Na v 1.8 and Na v 1.9 Na 1 currents. Peak Na v 1.8 Na 1 currents at 220 mV (A), Na v 1.9 Na 1 currents at 250 mV (B) and the fast TTX-R Na 1 currents at 220 mV (C) after exposure to 5 and 10 mM TTX are illustrated.

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Fig. 6. Dose–response curve of Cd 21 inhibition of fast TTX-R Na 1 current. TTX (250 nM) was used to block fast TTX-sensitive Na 1 currents prior to examining sensitivity of the fast TTX-R Na 1 current to Cd 21 . Cd 21 inhibition of fast TTX-R Na 1 current was estimated by normalizing the peak current amplitude (at 220 mV) measured in the presence of varying concentrations of Cd 21 to peak current amplitude observed in control conditions (n54); 0.75 and 1 mM Cd 21 block of fast TTX-R Na 1 current was measured in two DRG neurons (*).

3.6. Cd 21 sensitivity of the fast TTX-R current is similar to native TTX-R cardiac Na 1 channel Fig. 6 shows the dose response curve for the Cd 21 block of the fast TTX-R current in the presence of 250 nM TTX. The fast TTX-R Na 1 current was blocked by Cd 21 with an IC 50 value of |250 mM (Fig. 6; n54). Cardiac Na 1 channels from a number of mammalian species, have been reported previously to be blocked by Cd 21 with an IC 50 range of |50 to 270 mM [25,39,44,47,50]. The IC 50 value in our study is closer to the upper end of the range and may have been caused, at least in part, by the presence of 250 nM TTX in the bath solution. TTX and Cd 21 share a common binding site on Na 1 channels [19] and the presence of TTX can competitively inhibit Cd 21 binding, which may be a reason for the higher IC 50 value observed in our studies. The fast TTX-R current, like cardiac Na 1 channels [25,39,50], is almost completely blocked by a cadmium concentration (1 mM), which is not high enough to block Na v 1.8 [36] or Na v 1.9 TTX-R Na 1 currents [Leffler et al., unpublished observations]. These results demonstrate that Cd 21 sensitivity of the fast TTX-R Na 1 current is similar to that of cardiac Na 1 channels but is different from those of Na v 1.8 and Na v 1.9 TTX-R Na 1 currents.

3.7. Expression of cardiac Na 1 channel Nav 1.5 parallels the expression of the fast TTX-R Na 1 current The TTX sensitivity, cadmium sensitivity and electrophysiological properties of the fast-inactivating TTX-R Na 1 current strongly suggest that the fast TTX-R Na 1 current might be encoded by the cardiac channel Na v 1.5.

Although Na v 1.5 transcripts have not been detected previously in adult rat DRG using independent molecular techniques [7,8,17], trace levels of Na v 1.5 transcripts have been recently reported in these neurons [Chaplan et al., Society for neuroscience, abstract number 418.19, 2000]. We therefore investigated the presence of Na v 1.5 transcripts in rat DRG at different developmental stages, using an assay that utilizes the PCR amplification of fragments from domain 1 (D1) of Na v 1.5 and Na v 1.6, and the verification of the expression of these channels by length and restriction enzyme polymorphism [7]. The amplified fragments span multiple exon–intron boundaries so that the amplicons cannot be mistaken for amplified genomic sequences. The results of the restriction enzyme analysis of amplification products from DRG and trigeminal ganglia at different developmental stages and from adult heart tissue are shown in Fig. 7. The presence of a single band in the control samples (no enzymatic treatment) is consistent with previously published results [7] and is indicative of the specificity of this primer pair to Na v 1.5 and Na v 1.6 under the PCR conditions used in this study. The amplified product co-migrates with the 500 bp size marker, in agreement with the predicted length of 518 bp and 507 bp PCR products of Na v 1.5 and Na v 1.6, respectively. Except for the sample from adult heart, most of the input DNA of E17-P7 DRG and trigeminal ganglia samples was cleaved with Sph I, indicating the presence of significant levels of Na v 1.6 in the respective cDNA pools. The samples from E15 and E17 DRG were also cut with Acc I to produce two fragments (173 and 345 bp) consistent with the expected Na v 1.5 products. Similar products are detected from the adult heart sample cut with this enzyme. The fraction of PCR product cut by Acc I restriction enzyme is

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Fig. 7. Restriction enzyme analysis of RT-PCR products reveals the presence of Na v 1.5 mRNA transcripts. Marker lanes contain a 100-bp ladder. PCR amplification products from E15, E17, P0 and P7 DRG and trigeminal ganglia, and adult heart were incubated with buffer alone (2) or with Sph I and Acc I as indicated. The enzyme Acc I cleaves Na v 1.5 PCR products into 345 and 173 bp fragments. The enzyme SphI cleaves Na v 1.6 PCR products into 381 and 126 bp.

clearly reduced after E15. Little or no Na v 1.5 amplification products are detected by this assay in P0 DRG neurons. These results clearly demonstrate the presence of Na v 1.5 mRNA in embryonic DRG but a reduction of its levels with development. The temporal expression pattern of Na v 1.5 is consistent with the presence of robust fast TTX-R currents in embryonic DRG neurons and the rare detection of the fast TTX-R Na 1 currents in adult DRG neurons.

3.8. Developmental expression of fast TTX-R Na 1 current in rat DRG neurons To further test the hypothesis that the fast TTX-R current in rat DRG is produced by Na v 1.5 Na 1 channels, we asked whether the temporal expression pattern of the fast TTX-R Na 1 current in DRG neurons parallels the expression pattern of Na v 1.5 channels. We therefore investigated the presence of fast-inactivating TTX-R Na 1 currents in embryonic day 15 (E15, n518), 17 (E17, n58) and 18 (E18, n534), early postnatal 0 (P0, n5111), 1 (P1, n565) and 2 days (P2, n545) and adult (6 weeks old, n568) rats (Fig. 8A, circles). About 80% of E15 DRG neurons expressed the fast TTX-R Na 1 currents. The percentage of neurons expressing the fast TTX-R Na 1 current monotonically decreased rapidly (50% in E17 neurons; 40% in E18 neurons; ,20% after birth; 10% in P1 neurons; 5% in P2 neurons and 3% in adult DRG neurons). These results demonstrate that the expression of the fast TTX-R Na 1 current is highest in embryonic neurons and decreases with age. For comparison, the expression of Na v 1.8 (squares) and Na v 1.9 (triangles) Na 1 currents in embryonic, postnatal and adult stages are also shown in Fig. 8A. Na v 1.8 and Na v 1.9 Na 1 currents are absent in E15 (n518) and E17 (n58) neurons and appear

in E18 neurons. A small percentage of E18 DRG neurons expressed Na v 1.8 (26%, n534) and Na v 1.9 Na 1 currents (17%, n534) with the percentage increasing to |60% (n5111) in P0 neurons and to 80% (n568) in adult neurons (Fig. 8A). At each of these developmental stages, neurons that expressed the fast TTX-R Na 1 current did not co-express Na v 1.8 or Na v 1.9 Na 1 currents, with the exception of only one E18 neuron that expressed Na v 1.8 Na 1 currents and also co-expressed the fast TTX-R Na 1 current. Developmental changes in the channel densities of the three distinct TTX-R Na 1 currents are illustrated in Fig. 8B. Current density was determined by dividing the peak current amplitude by cell capacitance. The peak current amplitude for fast TTX-R, Na v 1.8 and Na v 1.9 Na 1 currents was observed at 225, 220 and 250 mV, respectively, and these values did not change significantly with development. The density of the fast TTX-R Na 1 current in cells that express this current is relatively constant, remaining between 0.0960.01 and 0.2560.01 nA / pF at all developmental stages. These results, taken together with the percentage of cells expressing fast TTX-R Na 1 current (Fig. 8A), demonstrate that the number of neurons which express fast TTX-R Na 1 current decreases with age while the fast TTX-R Na 1 current density remains constant. By contrast, Na v 1.8 and Na v 1.9 Na 1 currents first appeared in E18 neurons at densities of 0.1360.04 nA / pF (n59) and 0.0760.02 nA / pF (n59), respectively. After birth, Na v 1.8 Na 1 current densities increased to|1 to 1.5 nA / pF, the current density seen in adult stages. The density of Na v 1.9 Na 1 currents also increased after birth, rising to 0.5 nA / pF in P0 neurons and |1 nA / pF in P1, P2 and adult neurons. The density of the fast TTX-R Na 1 current is significantly lower (|5 fold) than those of Na v 1.8 and Na v 1.9 Na 1 currents in P0 to adult neurons. The increase in Na v 1.8 and

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Fig. 8. TTX-R Na 1 currents are developmentally regulated in DRG neurons. (A) Percentage of neurons expressing the fast TTX-R, Na v 1.8 and Na v 1.9 Na 1 currents were determined by dividing the number of neurons expressing these Na 1 currents by total number of neurons examined (n is given in the text). (B) The current densities of the fast TTX-R, Na v 1.8 and Na v 1.9 Na 1 currents in neurons that expressed these currents. Current density was determined by dividing the peak current amplitude by cell capacitance for cells expressing each current.

Na v 1.9 Na 1 channel density with development is consistent with the developmental increase in the expression of Na v 1.8 and Na v 1.9 Na 1 channel mRNAs [4].

4. Discussion In this study we have demonstrated developmental regulation of expression of a fast inactivating TTX-R Na 1 current in rat small DRG neurons. Furthermore, we show

that Na v 1.5 mRNA is expressed in immature DRG neurons, and is downregulated with development, in parallel with the temporal expression pattern of the fast TTX-R current. This TTX-R Na 1 current has faster activation and inactivation kinetics than those of the previously identified Na v 1.8 slow-inactivating TTX-R Na 1 currents, and Na v 1.9 persistent TTX-R Na 1 currents in DRG neurons, but are similar to those of the cardiac channel Na v 1.5. We also demonstrate that the fast-inactivating TTX-R current exhibits TTX and cadmium sensitivity similar to those reported for Na v 1.5 channels. Thus, Na v 1.5 appears to produce the fast-inactivating TTX-R Na 1 current in DRG neurons. The fast-inactivating TTX-R Na 1 current was recorded in P0 DRG neurons of Na v 1.8 null mice [2] and from the majority of DRG neurons at age E15, a time when neither Na v 1.8 nor Na v 1.9 is present at detectable levels [4]. These results indicate that neither Na v 1.8 nor Na v 1.9 underlies the fast-inactivating TTX-R Na 1 current. Consistent with this conclusion, the V1 / 2 for the activation and inactivation of the fast TTX-R Na 1 current are significantly different from the V1 / 2 for Na v 1.8 and Na v 1.9 Na 1 currents but are similar to those of a TTX-R current previously reported in DRG neurons [42], which has been also called TTX-R3 [37]. The time constants of activation and inactivation of the fast TTX-R Na 1 current are close to those of TTX-R3 and are 3- and 5-fold faster than those of Na v 1.8 and Na v 1.9 Na 1 currents at 0 mV test potential. The relatively hyperpolarized V1 / 2 of inactivation, and the faster activation and inactivation of the fast TTX-R Na 1 current are all similar to those of the cardiac Na v 1.5 Na 1 currents [25,32,36,40]. The TTX and cadmium sensitivity of the fast TTX-R Na 1 current are also similar to those reported for Na v 1.5 channels. The fast TTX-R Na 1 current is completely blocked by 10 mM TTX, similar to Na v 1.5 Na 1 channels [23,25,39,50]. In contrast, 10 mM TTX blocks only |20% of Na v 1.8 and Na v 1.9 Na 1 currents [12,45]. The fast TTX-R current, like Na v 1.5 Na 1 currents [25,40], is blocked by a cadmium concentration (1 mM), which is not high enough to block Na v 1.8 slowly-inactivating TTX-R [36] or Na v 1.9 persistent TTX-R Na 1 currents [Leffler et al., unpublished observations]. On the basis of the similarity of the voltage-dependence, kinetics, and sensitivity to TTX and cadmium of Na v 1.5 and the fast TTX-R current, we carried out RT-PCR and restriction enzyme analysis to determine whether DRG neurons express Na v 1.5 mRNA. The analysis shows that Na v 1.5 mRNA is present in embryonic rat DRG neurons and, moreover, that the developmental expression pattern of the fast-inactivating TTX-R current is paralleled by the pattern of Na v 1.5 mRNA expression. Amplification products of Na v 1.5 were detected in E15 samples but declined sharply by P0. Although the average density of the fast TTX-R Na 1 current in cells that expressed it did not change substantially with development, the number of small DRG neurons expressing this current declined from

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80% at E15 to only 3% of adult neurons. The correspondence in the temporal pattern of expression of Na v 1.5 mRNA expression and of fast-inactivating TTX-R Na 1 current provides additional evidence that Na v 1.5 channels underlie the fast TTX-R Na 1 current in small DRG neurons. Previous studies from our laboratory and others [12,13,29,36] did not detect a fast-inactivating TTX-R Na 1 current in adult mouse and rat DRG neurons. Similarly, Akopian et al. [2] did not observe a fast-inactivating TTX-R Na 1 current in adult DRG neurons from Na v 1.8 null mice. However, Rush et al. [37] clearly described a current (TTX-R3), with electrophysiological properties similar to those we observed, in DRG neurons. The present study, which analyzed a large number of cells at various developmental stages, revealed the presence of fast-inactivating TTX-R Na 1 current in only 3% of adult small DRG neurons. The low incidence of expression of this current in adult DRG neurons provides a reasonable explanation for the lack of observation of the Na v 1.5 transcripts and current in the earlier studies. DRG neurons are electrically nonexcitable before E16, but they show spontaneous activity between E17 and E20, prior to developing normal stimulus evoked activity after birth [22]. Spontaneous activity may play an important role in neural development [28,43]. Since the input resistance of the embryonic neuron is higher than in adult neurons [31], even small Na v 1.5 currents may contribute to the spontaneous electrical activity of DRG neurons between E16 and E20. In this regard, it will be interesting to determine whether there are abnormalities of structure or function of spinal sensory pathways in association with mutations of Na v 1.5 which cause the long QT syndrome and other cardiac disorders [6,48]. Na v 1.5 transcripts have also been reported in developing and adult rat and human brain [18] and in limbic regions of rat brain [24]. A heart-like Na 1 current was reported in neurons of the entorhinal cortex [50]; however, the underlying channel is less sensitive to Cd 21 and more sensitive to TTX than the Na v 1.5 channel. Although details of the function of Na v 1.5 channels in neurons are not clear at this time, the present results enlarge the family of sodium channels which are known to be expressed in DRG neurons, and demonstrate that, at different stages of development, each of the three known TTX-R Na 1 channels is expressed within these cells.

Acknowledgements We thank Dr. Joel A. Black and William N. Hormuzdiar for providing tissues and cultures, and Dr. Ted Cummins for helpful discussions. This work was supported in part by grants from the National Multiple Sclerosis Society and the Rehabilitation Research and Development Service and

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Medical Research Services, Department of Veterans Affairs, and by gifts from the Paralyzed Veterans of America and Eastern Paralyzed Veterans Association.

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