NAD⁺ Activates K_Na Channels in Dorsal Root Ganglion Neurons

K_Na channels recorded from adult small DRG neurons. A, Large conductance “K_Na-like” channels recorded in cell-attached patches on small DRG neurons bathed in physiological saline (140 mm Na⁺ and 3 mm K⁺). Pipette solution contained 130 mm K⁺ and 1 mm TEA. Voltage indicated is the applied pipette potential. Excision into high external Na⁺ confirmed K_Na channels. Patches were subsequently perfused with varying concentrations of Na⁺ to confirm Na⁺ dependence. Channels here were recorded at holding potential of −80 mV. B, Current–voltage relationship in symmetrical K⁺ revealed a large conductance channel. C, Na⁺ dose–response in small neurons (n = 6). EC₅₀, 53 mm; Hill coefficient, 2.4.

Rundown of K_Na channels after patch excision. A, Representative traces of high basal K_Na channel activity. Rundown occurred in three of six patches from small DRG neurons. B, NP(o) values were calculated for three patches every 2 s after patch excision in inside-out configuration. Channels were recorded at −80 mV. The nP(o)s were averaged after every 10 s, normalized to highest nP(o) in each neuron, and the averages for each time point were taken. Arrows indicate approximate commencement of perfusion of patches at each concentration of Na⁺ (all values in millimolars). A Savitzky–Golay smoothing curve was used to filter data.

Slack and Slick contain putative NAD⁺-binding sites. NAD⁺-binding sequence in Slack, Slick, and known NAD⁺-dependent K⁺ transporters from Methanocaldococcus jannaschii (Mja), Bacillus subtilis (Bsu), Vibro alginolyticus (Val), and Bacteroides fragilis (BacF). Yellow highlights indicate β sheets, whereas red highlights indicate α helices. The fingerprint region consists of a βαβαβ structure. The first green highlights indicated the conserved positive charge in beginning of the first β sheet in the fingerprint region. The second green highlights indicate the conserved negatively charged residue at the end of the second β sheet in the fingerprint region. The blue highlights are the phosphate-binding consensus sequence “GXGXXG” connecting the first β sheet to the first α helix. The purple highlights a consensus protein kinase C phosphorylation site present in Slack but not in Slick.

NAD⁺ modulates K_Na channels in small DRG neurons. A, Representative trace of channels recorded in an inside-out-excised patch from a small DRG neuron with X mm Na⁺ (control) and with X mm Na⁺ and 1 mm NAD⁺. Voltage held at −80 mV. B, Na⁺ dose–response relationship of control and NAD⁺ perfusions in small adult rat DRG neurons. NP(o) values were calculated from 30 sweeps of 2400 ms time samples at each dose of Na⁺ and Na⁺/NAD⁺ solution. Each point represents an n = 6. Error bars represent SEM. EC₅₀ was determined to be 50 mm for Na⁺ and 17 mm for Na⁺/NAD⁺ solutions in small adult DRG neurons with a Hill coefficient of 2.4. C, NADH does not modulate K_Na channel activity in DRG neurons. Na⁺ (30 mm; control) was perfused onto inside-out-excised patches and compared with 30 mm Na⁺ + 1 mm NADH and 30 mm + 1 mm NAD⁺. Voltage was held at −80 mV. NP(o) values were calculated from 30 sweeps of 2400 ms time samples (n = 5; *p < 0.01). Error bars represent SEM.