Slack sodium-activated potassium channel membrane expression requires p38 mitogen-activated protein kinase phosphorylation

p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The K(Na) channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that K(Na) currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack K(Na) channels as p38 MAPK substrates

NAD⁺Activates K_NaChannels in Dorsal Root Ganglion Neurons

Although sodium-activated potassium channels (KNa) have been suggested to shape various firing patterns in neurons, including action potential repolarization, their requirement for high concentrations of Na+to gate conflicts with this view. We characterized KNachannels in adult rat dorsal root ganglion (DRG) neurons. Using immunohistochemistry, we found ubiquitous expression of the Slack KNachannel subunit in small-, medium-, and large-diameter DRG neurons. Basal KNachannel activity could be recorded from cell-attached patches of acutely dissociated neurons bathed in physiological saline, and yet in excised inside-out membrane patches, the Na+EC50for KNachannels was typically high, ∼50 mm. In some cases, however, KNachannel activity remained considerable after initial patch excision but decreased rapidly over time. Channel activity was restored in patches with high Na+. The channel rundown after initial excision suggested that modulation of channels might be occurring through a diffusible cytoplasmic factor. Sequence analysis indicated that the Slack channel contains a putative nicotinamide adenine dinucleotide (NAD+)-binding site; accordingly, we examined the modulation of native KNaand Slack channels by NAD+. In inside-out-excised neuronal patch recordings, we found a decrease in the Na+EC50for KNachannels from ∼50 to ∼20 mmwhen NAD+was included in the perfusate. NAD+also potentiated recombinant Slack channel activity. NAD+modulation may allow KNachannels to operate under physiologically relevant levels of intracellular Na+and hence provides an explanation as to how KNachannel can control normal neuronal excitability.</jats:p

Abnormally Expressed Low-Voltage-Activated Calcium Channels in β-Cells From NOD Mice and a Related Clonal Cell Line

A macroscopic low-voltage-activated (LVA) inward current was found in pancreatic β-cells isolated from NOD mice. However, this current was not present in nondiabetic prone mouse (e.g., Swiss-Webster) pancreatic β-cells. We performed pharmacological analyses on this current in NOD insulinoma tumor cells (NIT-1). This cell line was developed from pancreatic β-cells of a transgenic NOD mouse. The sodium-channel blocker, tetrodotoxin (TTX; 2 micromol/l) had no effect on this LVA current. The amplitudes of currents elicited by a –20 mV test pulse retained similarity when the extracellular sodium concentration was increased from 0 to 115 mmol/l; when the extracellular calcium concentration was decreased from 10 to 2 mmol/l, there was an approximate 50% reduction of this current elicited by a –30 mV test pulse. Neither the L-type calcium-channel blocker, nifedipine (3 μmol/l), nor the N-type calcium-channel blocker, ω-CgTx-GVIA (1 μmol/l), at –30 mV produced an appreciable effect. The T-type calcium-channel blockers, nickel (3 μmol/l) and amiloride (250 μmol/l), effectively reduced the peak of this current. In 2 mmol/l calcium external solution, the threshold of voltage-dependent activation of this calcium current was approximately –65 mV, and the peak current occurred at –20 mV. Half-maximum steady-state inactivation was around –43 mV. The mean time constant of slow deactivating tail currents generated by a preceding 20 mV pulse was 2.53 ms. The intracellular free calcium concentration was two- to threefold higher in NOD mouse pancreatic β-cells compared with Swiss-Webster pancreatic β-cells. We concluded that there are LVA calcium channels abnormally expressed in NOD mouse β-cells. This LVA calcium channel may be factorial to the high cytosolic free calcium concentration observed in these cells, and thereby may contribute to the pathogenesis of NOD mouse β-cells.</jats:p