Electrophysiological properties of two axonal sodium channels, Nav1.2 and Nav1.6, expressed in mouse spinal sensory neurones

Abstract

Sodium channels Na(v)1.2 and Na(v)1.6 are both normally expressed along premyelinated and myelinated axons at different stages of maturation and are also expressed in a subset of demyelinated axons, where coexpression of Na(v)1.6 together with the Na(+)/Ca(2+) exchanger is associated with axonal injury. It has been difficult to distinguish the currents produced by Na(v)1.2 and Na(v)1.6 in native neurones, and previous studies have not compared these channels within neuronal expression systems. In this study, we have characterized and directly compared Na(v)1.2 and Na(v)1.6 in a mammalian neuronal cell background and demonstrate differences in their properties that may affect neuronal behaviour. The Na(v)1.2 channel displays more depolarized activation and availability properties that may permit conduction of action potentials, even with depolarization. However, Na(v)1.2 channels show a greater accumulation of inactivation at higher frequencies of stimulation (20-100 Hz) than Na(v)1.6 and thus are likely to generate lower frequencies of firing. Na(v)1.6 channels produce a larger persistent current that may play a role in triggering reverse Na(+)/Ca(2+) exchange, which can injure demyelinated axons where Na(v)1.6 and the Na(+)/Ca(2+) exchanger are colocalized, while selective expression of Na(v)1.2 may support action potential electrogenesis, at least at lower frequencies, while producing a smaller persistent current.

Figure 1. Comparison of Na_v1.2_R and Na_v1.6_R current properties

Representative families of currents recorded from Na_v1.8-null DRG neurones expressing Na_v1.2_R channels (A) or Na_v1.6_R channels (B) are shown. Currents were elicited by 40 ms depolarizations from a holding potential of −100 mV to a range of potentials between −65 mV and +60 mV (inset). C, average absolute current–voltage relationship for the two channels (Na_v1.2, n = 25; Na_v1.6, n = 27). D, activation and availability curves for Na_v1.2_R (n = 23/10) and Na_v1.6_R (n = 24/11). Activation was calculated from current–voltage experiments, as detailed in Methods. Availability of channels was estimated by measuring the peak current amplitude elicited by 40 ms test pulses to −10 mV following 500 ms prepulses to a range of voltages from −110 mV to 0 mV. Values were normalized to peak and plotted versus voltage. E and F, Inactivation time constants (E, fast; F, slow) for double exponential fits of the decay phase of currents elicited at the potentials shown for 40 ms. Relative amplitudes of the fits were 60–88% for τ_fast and 12–40% for τ_slow. C–F show mean ± s.e.m.

Figure 2. Na_v1.6_R displays a larger persistent current than Na_v1.2_R in Na_v1.8-null DRG neurones

Representative currents, on an expanded scale, demonstrating the larger persistent current, in response to a −10 mV stimulus from a holding potential of −100 mV displayed by Na_v1.6r (B) (n = 12), compared to Na_v1.2r (A) (n = 14). Currents were elicited from a holding potential of −100 mV to the voltages between −65 mV and +20 mV for 40 ms. The amplitude of the current was measured 30 ms into the voltage step, as the arrows indicate, and plotted for a range of voltages (C). *P < 0.05. Scale in B refers to panels A and B. C show mean ± s.e.m.

Figure 3. Repriming (recovery from inactivation) from a single depolarizing stimulus is faster for Na_v1.2_R than Na_v1.6_R channels, expressed in DRG neurones

Families of currents of DRG neurones expressing Na_v1.2_R (A) and Na_v1.6_R (B) elicited using a recovery from inactivation protocol (inset), using −80 mV as the recovery voltage (V_rec) voltage in these cases. For this set of experiments, cells were held at −100 mV, depolarized to −10 mV for 40 ms and then allowed to recover for increasing amounts of time, at a variety of voltages, before the test potential to −10 mV for 40 ms to measure the extent of recovery. Averaged data at a V_rec of −80 mV are plotted in C, demonstrating single exponential fits to the results. The fitted time constants at the range of voltages tested (−100 to −70 mV) are plotted in D, showing fast repriming for both channels and faster recovery from inactivation for Na_v1.2_R*P < 0.05, under these conditions (n = 6–8). C, D show mean ± s.e.m.

Figure 4. Development of inactivation is more rapid for Na_v1.6_R than Na_v1.2_R channels

Experiments designed to examine onset of inactivation were carried out using the protocol shown in the inset, and representative records are shown for Na_v1.2_R (A) and Na_v1.6_R (B). Cells were held at −100 mV, depolarized to a particular potential (V_dev) for increasing amounts of time, until a test depolarization to −10 mV for 40 ms was given to measure the extent of inactivation. Averaged data are presented at V_dev potentials of −80 mV (C) and −60 mV (D). Double exponential decay fits were used to compare the development of inactivation and the fast time constants are shown in E and the slow in F (n = 5–6; *P < 0.02). Relative amplitudes of the fits were 38–94% for τ_fast and 6–62% for τ_slow. Development of inactivation was rapid for both channels but was faster for Na_v1.6_R than Na_v1.2_R. C–F show mean ± s.e.m.

Figure 5. Na_v1.2_R and Na_v1.6_R can produce resurgent current in Na_v1.8-null DRG neurones

Representative resurgent currents are shown for Na_v1.2_R (A) and Na_v1.6_R (B) after activation by a protocol (inset) where cells were held at −100 mV, depolarized to +30 mV for 20 ms, followed by repolarizations to a range of voltages (−80 to +20 mV) to elicit resurgent current. In 8% (2 out of 25) of the cells transfected with Na_v1.2_R, a resurgent current could be clearly observed. Resurgent current could be found in 22% (6 out of 27) of cells where Na_v1.6_R was expressed. Averaged data from only those cells producing the current are shown in C. C show mean ± s.e.m.

Figure 6. Na_v1.6_R currents can follow high-frequency stimulation more faithfully than Na_v1.2_R currents

Experiments were performed to examine the behaviour of the two currents with high-frequency stimulation using two protocols (both insets). Cells were held at −80 mV and depolarized to −10 mV for 40 ms for 20 episodes at a variety of frequencies. (A) Representative first and last currents from such a train are shown at 0.5 Hz (top panels) or 20 Hz (bottom panels). Averaged data are summarized in C. B, representative first and last currents from a train using 5 ms episodes are shown at 0.5 Hz (top panels) or 100 Hz (bottom panels). Averaged data are summarized in D. These experiments were performed at a range of frequencies (0.5–20/100 Hz) and the fraction of current remaining over the 20 episodes is plotted versus frequency for 40 ms episodes (E) and for 5 ms episodes (F) (n = 6–11). Na_v1.6_R currents were able to maintain their amplitude more than Na_v1.2_R currents (*P < 0.05), suggesting that cells that express Na_v1.6 may be better high-frequency followers than cells that express Na_v1.2. C–F show mean ± s.e.m.