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Role of tetrodotoxin-resistant Na+ current slow inactivation in adaptation of action potential firing in small-diameter dorsal root ganglion neurons - PubMed

️Wed Jan 01 2003

Role of tetrodotoxin-resistant Na+ current slow inactivation in adaptation of action potential firing in small-diameter dorsal root ganglion neurons

Nathaniel T Blair et al. J Neurosci. 2003.

Abstract

When acutely dissociated small-diameter dorsal root ganglion (DRG) neurons were stimulated with repeated current injections or prolonged application of capsaicin, their action potential firing quickly adapted. Because TTX-resistant (TTX-R) sodium current in these presumptive nociceptors generates a large fraction of depolarizing current during the action potential, we examined the possible role of inactivation of TTX-R sodium channels in producing adaptation. Under voltage clamp, TTX-R current elicited by short depolarizations showed strong use dependence at frequencies as low as 1 Hz, although recovery from fast inactivation was complete in approximately 10-30 msec. This use-dependent reduction was the result of the entry of TTX-R sodium channels into slow inactivated states. Slow inactivation was more effectively produced by steady depolarization than by cycling channels through open states. Slow inactivation was steeply voltage dependent, with a Boltzmann slope factor of 5 mV, a midpoint near -45 mV (5 sec conditioning pulses), and completeness of approximately 93% positive to -20 mV. The time constant for entry (approximately 200 msec) was independent of voltage from -20 mV to +60 mV, whereas recovery kinetics were moderately voltage dependent (time constant, approximately 1.5 sec at -60 mV and approximately 0.5 sec at -100 mV). Using a prerecorded current-clamp response to capsaicin as a voltage-clamp command waveform, we found that adaptation of firing occurred with a time course similar to that of development of slow inactivation. Thus, slow inactivation of the TTX-R sodium current limits the duration of small DRG cell firing in response to maintained stimuli and may contribute to cross desensitization between chemical and electrical stimuli.

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Figures

**Figure 1.**
Current-clamp responses of DRG cells to capsaicin application and to repeated current ramp injections. A, Top, Application of 500 n
m
capsaicin (bar) elicited a brief period of action potential firing, after which the cell remained depolarized at -27 mV. A, Bottom, 800 msec of the same capsaicin response on an expanded time base. The internal solution contained the following (in m
m
): 140 K-methanesulfonate, 13.5 NaCl, 1.6 MgCl₂, 0.09 EGTA, 9 HEPES, 4 MgATP, 14 Tris-creatine PO₄, and 0.3 Tris-GTP, pH 7.4 (with KOH). The external solution was Tyrode's solution. B, Ten 1 sec current ramps to 1 nA were injected at 0.5 Hz. The internal solution contained the following (in m
m
): 140 K-aspartate, 13.5 NaCl, 1.6 MgCl₂, 0.09 EGTA, 9 HEPES, 4 MgATP, 14 Tris-creatine PO₄, and 0.3 Tris-GTP, pH 7.4 (with KOH). The external solution was Tyrode's solution. C, Comparison of action potential firing elicited during first current ramp (open bars) and 10th current ramp (gray bars) applied at 0.5 Hz. The total number of action potentials (peaks >0 mV), the maximal upstroke of the first action potential, and the latency from the current ramp onset to the first action potential are shown. Error bars indicate SD.

**Figure 8.**
TTX-S and TTX-R sodium currents flowing during the voltage waveform elicited by capsaicin application. Top, Voltage command taken from current-clamp response to 500 n
m
capsaicin (Fig. 1 A). Middle, TTX-R sodium currents flowing in response to capsaicin response waveform. To improve voltage control, currents were recorded in an external solution with reduced sodium containing the following (in m
m
): 50 NaCl, 100 TEA-Cl, 4 CsCl, 2 CaCl₂, 2 MgCl₂, 0.03 CdCl₂, 10 glucose, and 10 HEPES, pH 7.4 (with TEA-OH). Outward currents occur when spike height exceeds sodium reversal potential in reduced sodium (∼31 mV). Bottom, TTX-S sodium current flowing during the capsaicin response waveform, determined as the current sensitive to block by 300 n
m
TTX in an external solution containing the following (in m
m
): 50 NaCl, 100 TEA-Cl, 2 BaCl₂, 0.3 CdCl₂, 10 glucose, and 10 HEPES, pH 7.4 (with TEA-OH) (different cell from top). The internal solution for both recordings contained the following (in m
m
): 126 NMDG, 120 aspartate, 15 NaCl, 1.8 MgCl₂, 9 EGTA, and 9 HEPES, pH 7.4 (with 7 m
m
CsOH).

**Figure 9.**
Inactivation of TTX-R sodium current during capsaicin response. A, Voltage protocol and TTX-R sodium currents evoked during a single sweep to measure slow inactivation elicited by an ∼490 msec segment of the capsaicin response. TTX-R sodium currents during 2.5 msec test steps to 0 mV (arrows) are shown on an expanded time scale. A 30 msec step to -100 mV preceded the second test pulse to remove fast inactivation. Large inward tail currents flowing during repolarizations to -100 mV have been truncated. Persistent sodium current between spikes is compressed because of the vertical scale. The internal solution contained the following (in m
m
): 130 NMDG, 120 aspartate, 15 NaCl, 1.8 MgCl₂, 9 EGTA, 9 HEPES, 4 MgATP, 14 Tris-creatine PO₄, and 0.3 Tris-GTP, pH 7.4 (with 7 m
m
CsOH). The external solution contained the following (in m
m
): 50 NaCl, 100 TEA-Cl, 4 CsCl, 2 CaCl₂, 2 MgCl₂, 0.03 CdCl₂, 10 glucose, and 10 HEPES, pH 7.4 (with TEA-OH). B, Fraction of TTX-R sodium current available (second test step current normalized to the first) plotted as a function of time during the capsaicin response. Total inactivation (open circles) was measured with test steps directly to 0 mV; slow inactivation (crosses) was measured with test steps preceded by hyperpolarizing pulses to remove fast inactivation. Error bars indicate SD.

**Figure 10.**
Recovery from fast inactivation during interspike interval in the capsaicin response waveform. A, Top, Voltage protocol used to measure TTX-R sodium channel recovery from fast inactivation in the interval between the third and fourth action potentials (outlined in B). For comparison, the TTX-R sodium current available after removal of all fast inactivation by a 30 msec step to -100 mV applied near the peak of the AHP is shown at right (69% of initial current). Dotted lines show zero current level. Large inward tail currents flowing during repolarizations to -100 mV have been truncated, as have outward currents during the action potential upstroke and the test step onset. The internal solution contained the following (in m
m
): 130 NMDG, 120 aspartate, 15 NaCl, 1.8 MgCl₂, 9 EGTA, 9 HEPES, 4 MgATP, 14 Tris-creatine PO₄, and 0.3 Tris-GTP, pH 7.4 (with 7 m
m
CsOH). The external solution contained the following (in m
m
): 50 NaCl, 100 TEA-Cl, 4 CsCl, 2 CaCl₂, 2 MgCl₂, 0.03 CdCl₂, 10 glucose, and 10 HEPES, pH 7.4 (with TEA-OH). B, Average fraction available TTX-R sodium current is shown throughout the capsaicin response. Available current ∼5 msec after a spike (open squares) and later in interspike interval (filled circles). Error bars indicate SD. Lines connect values during the same interspike interval. Reduction in TTX-R current availability caused by slow inactivation is shown in crosses (error bars omitted).

**Figure 2.**
Use dependence of TTX-R sodium current. A, TTX-R sodium current elicited during 30 msec steps to 0 mV applied at 5 Hz, from a holding potential of -80 mV. For clarity, only the 1st, 5th, 10th, and 15th sweeps are shown. Dotted line indicates zero current level. At the onset of the voltage step, 200 μsec were blanked to remove residual uncompensated capacity current. The internal solution contained the following (in m
m
): 130 NMDG, 120 aspartate, 15 NaCl, 1.8 MgCl₂, 9 EGTA, 9 HEPES, 4 MgATP, 14 Tris-creatine PO₄, and 0.3 Tris-GTP, pH 7.4 (with 7 m
m
CsOH). The external solution contained the following (in m
m
): 50 NaCl, 100 TEA-Cl, 4 CsCl, 2 CaCl₂, 2 MgCl₂, 0.03 CdCl₂, 10 glucose, and 10 HEPES, pH 7.4 (with TEA-OH). B, Peak TTX-R sodium currents from the cell in A were normalized to the initial value and plotted against the step number. Results for holding potentials of -60 and -80 mV and stimulation rates of 1 and 5 Hz are also shown for this cell. C, Average decline in TTX-R sodium current amplitude over 15 steps for holding potentials of -60 and -80 mV and stimulation rates of 1 and 5 Hz. Error bars indicate SD.

**Figure 11.**
TTX-R sodium current use dependence elicited by action potential waveforms. A, top, Single action potentials recorded from two DRG neurons applied as voltage commands at 1 Hz, from a holding potential of -60 or -70 mV. Solid line, Short-duration action potential recorded with internal solution containing the following (in m
m
): 140 K-methanesulfonate, 13.5 NaCl, 1.6 MgCl₂, 0.09 EGTA, 9 HEPES, 0.9 glucose, 14 Tris-creatine PO₄, 4 MgATP, and 0.3 Tris-GTP, pH 7.4. Dashed line, Long-duration action potential recorded with the same internal solution as above, with 140 m
m
K-aspartate replacing K-methanesulfonate. In both cases, the external solution was Tyrode's solution. A, Bottom, Peak inward current elicited during action potential waveforms (applied in the same cell), was normalized to the initial value and plotted against sweep number. Filled triangles, The degree of TTX-R sodium current use dependence in this cell during 30 msec steps to 0 mV, applied at 1 Hz from a holding potential of -60 mV. The internal solution contained the following (in m
m
): 140 K-aspartate, 13.5 NaCl, 1.6 MgCl₂, 0.09 EGTA, 9 HEPES, 14 Tris-creatine PO₄, 4 MgATP, and 0.3 Tris-GTP, pH 7.4. The external solution contained the following (in m
m
): 150 NaCl, 4 KCl, 2 CaCl₂, 2 MgCl₂, 10 HEPES, 10 glucose, and 5 TEA-Cl, pH 7.4 with 300 n
m
TTX.

**Figure 4.**
Rate of TTX-R sodium channel entry into slow inactivated states. A, Protocol for measuring the rate of entry into slow inactivated states. Test steps to 0 mV were separated by conditioning pulses of 10-1500 msec at potentials from -40 to +60 mV. To isolate the contribution of slow inactivation, fast inactivation was removed during a 12 msec step to -100 mV preceding the second test step. B, TTX-R sodium currents during measurement of slow inactivation elicited by 10 msec (gray) and 100 msec (black) conditioning pulses at 0 mV. Approximately 5% of TTX-R channels were slow inactivated after the 10 msec pulse to 0 mV, whereas ∼50% were slow inactivated after the 100 msec pulse. (Conditioning pulse durations do not include the initial 4 msec step to 0 mV.) The fast inward tail current during the repolarization to -100 mV is probably carried by a mixture of sodium ions flowing through TTX-R channels and calcium ions flowing through calcium channels; the block of calcium channels by 30 μ
m
is partially relieved at strongly hyperpolarized potentials. The internal solution contained the following (in m
m
): 130 NMDG, 120 aspartate, 15 NaCl, 1.8 MgCl₂, 9 EGTA, 9 HEPES, 4 MgATP, 14 Tris-creatine PO₄, and 0.3 Tris-GTP, pH 7.4 (with 7 m
m
CsOH). The external solution contained the following (in m
m
): 50 NaCl, 100 TEA-Cl, 4 CsCl, 2 CaCl₂, 2 MgCl₂, 0.03 CdCl₂, 10 glucose, and 10 HEPES, pH 7.4 (with TEA-OH). C, Fraction of available TTX-R sodium current (second test step current normalized to the first) plotted against the duration of the conditioning pulse at 0 and +40 mV (same cell). The dashed line shows the monoexponential fit to the data for 0 mV conditioning potential, with a time constant of 111 msec. D, Average time constants for TTX-R sodium channel entry to slow inactivation at conditioning potentials between -20 and +60 mV. At most, two conditioning potentials were tested in a single cell. Error bars indicate SD.

**Figure 3.**
TTX-R sodium channels recover rapidly from fast inactivation. A, Top, Voltage protocol to measure recovery from fast inactivation. A 30 msec step to 0 mV is followed by varying durations at the recovery potential (here, -60 mV), before a second test step to 0 mV to assay available TTX-R sodium current. A, Bottom, TTX-R sodium currents recorded during the above protocol with recovery durations of 1, 3, 10, 25, and 50 msec. The internal solution contained the following (in m
m
): 130 NMDG, 120 aspartate, 15 NaCl, 1.8 MgCl₂, 9 EGTA, 9 HEPES, 4 MgATP, 14 Tris-creatine PO₄, and 0.3 Tris-GTP, pH 7.4 (with 7 m
m
CsOH). The external solution contained the following (in m
m
): 50 NaCl, 100 TEA-Cl, 4 CsCl, 2 CaCl₂, 2 MgCl₂, 0.03 CdCl₂, 10 glucose, and 10 HEPES, pH 7.4 (with TEA-OH). B, TTX-R sodium current (cell in A), during the second test step normalized to the current during the initial step, is plotted against the recovery period duration (symbols). Dashed lines show monoexponential fits to the data, with indicated time constants. C, Average TTX-R sodium current recovery rates plotted against voltage. Error bars indicate SD; absent error bars are smaller than symbols.

**Figure 5.**
TTX-R sodium channel slow inactivation elicited by steps of varying duration. A, Schematic of voltage protocol: a train of 1, 2, 5, and 50 msec pulses to 0 mV was applied, with a total duration of 500 msec spent at 0 mV in each case. A test step to 0 mV after the train (preceded by 15 msec at -100 mV) assayed TTX-R sodium channel slow inactivation. The interpulse interval was 2 msec for the 1 and 2 msec pulse trains and 5 msec for the 5 and 50 msec pulse trains. B, Comparison of TTX-R sodium currents flowing during 2 and 50 msec pulses. The first seven pulses during the 2 msec train and the first 50 msec pulse are shown. Large inward tail currents during repolarizations were truncated. The dotted line indicates zero current level. Note different time scales: bar corresponds to 3 msec of 2 msec pulse train and 5 msec of 50 msec pulse train. The internal solution contained the following (in m
m
): 130 NMDG, 120 aspartate, 15 NaCl, 1.8 MgCl₂, 9 EGTA, 9 HEPES, 14 Tris-creatine PO₄, 4 MgATP, and 0.3 Tris-GTP, pH 7.4. The external solution contained the following (in m
m
): 50 NaCl, 100 TEA-Cl, 4 CsCl, 2 CaCl₂, 2 MgCl₂, 0.03 CdCl₂, 10 glucose, and 10 HEPES, pH 7.4 (with TEA-OH). C, Average available TTX-R sodium current (second test step current normalized to test step current applied before pulse train) for different pulse durations. Error bars indicate SD.

**Figure 6.**
Kinetics of recovery from slow inactivation. A, Slow inactivation was produced by a 500 msec conditioning pulse to 40 mV. Recovery was assayed by periodic 3 msec steps to 0 mV, preceded by 15 msec at -100 mV to recover fast inactivation. Current was normalized to that elicited by an initial test step to 0 mV. Dashed lines show single exponential fits to the data, with indicated time constants. B, Average time constants of recovery from slow inactivation at -60, -80, and -100 mV. Recovery duration includes the 15 msec periods at -100 mV used to remove fast inactivation. Error bars indicate SD.

**Figure 7.**
Voltage dependence of TTX-R sodium channel slow inactivation. A, Top, Protocol for measuring steady-state voltage dependence of slow inactivation. Conditioning pulses of 5 sec to potentials between -120 and +20 mV were followed by a 20 msec step to -100 mV to remove fast inactivation and then a test step to 0 mV. A, Bottom, Representative TTX-R sodium currents elicited during 0 mV test step. To remove residual uncompensated capacity current, 200 μsec of current traces at the leading and falling edges of the voltage step were blanked. The internal solution contained the following (in m
m
): 130 NMDG, 120 aspartate, 15 NaCl, 1.8 MgCl₂, 9 EGTA, 9 HEPES, 4 MgATP, 14 Tris-creatine PO₄, and 0.3 Tris-GTP, pH 7.4 (with 7 m
m
CsOH). The external solution contained the following (in m
m
): 50 NaCl, 100 TEA-Cl, 4 CsCl, 2 CaCl₂, 2 MgCl₂, 0.03 CdCl₂, 10 glucose, and 10 HEPES, pH 7.4 (with TEA-OH). B, TTX-R sodium currents during 0 mV step from cell in B were normalized to their maximum and plotted against conditioning pulse voltage. Filled circles, Test pulse current after 5 sec conditioning pulses and 20 msec at -100 mV to remove fast inactivation. Open squares, Test pulse current after 500 msec conditioning pulses, and no removal of fast inactivation. This measurement includes the effects of slow inactivation, and is therefore labeled “total inactivation.” Lines show the best fit to the Boltzmann equation, I/I_max = 1/(1 + exp[(V - V_1/2)/k]), where V is the conditioning pulse potential, V_1/2 is the half-maximal voltage in millivolts, and k is the slope factor in millivolts. Slow inactivation, V_1/2 = -43 mV, k = 4.2 mV. Total inactivation, V_1/2 = -33 mV, k = 4.4 mV.

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References

1. Akopian AN, Souslova V, England S, Okuse K, Ogata N, Ure J, Smith A, Kerr BJ, McMahon SB, Boyce S, Hill R, Stanfa LC, Dickenson AH, Wood JN (1999) The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways. Nat Neurosci 2:541-548. - PubMed
1. Andrew D, Greenspan JD (1999) Mechanical and heat sensitization of cutaneous nociceptors after peripheral inflammation in the rat. J Neurophysiol 82:2649-2656. - PubMed
1. Baker MD, Wood JN (2001) Involvement of Na+ channels in pain pathways. Trends Pharmacol Sci 22:27-31. - PubMed
1. Baumann TK, Burchiel KJ, Ingram SL, Martenson ME (1996) Responses of adult human dorsal root ganglion neurons in culture to capsaicin and low pH. Pain 65:31-38. - PubMed
1. Blair NT, Bean BP (2002) Roles of tetrodotoxin (TTX)-sensitive Na+ current, TTX-resistant Na+ current, and Ca2+ current in the action potentials of nociceptive sensory neurons. J Neurosci 22:10277-10290. - PMC - PubMed

Role of tetrodotoxin-resistant Na+ current slow inactivation in adaptation of action potential firing in small-diameter dorsal root ganglion neurons - PubMed