Regulation of a sodium-activated potassium channel by protein kinase A

Inflammatory pain occurs as a result of tissue injury. During inflammation, a complex neuron-immune interaction arises, characterized by nociceptive sensitization and hyperalgesia. Nociceptor sensitization is the direct result of hyperexcitability of the pain-sensing neurons located within the dorsal root ganglion (DRG). DRG neuronal hyperexcitability is caused by activation of second messenger pathways by immune factors, which alter ion channel conductances in the neuron. Previous studies have shown that the protein kinase A (PKA) pathway is critical for DRG hyperexcitability and inflammatory pain. However, the mechanisms by which PKA activation leads to hyperexcitability largely remain unresolved. Here, I examined the effect of PKA on the Na+-activated K + conductance, (KNa), that is highly expressed in DRG neurons. The goal of my work was to determine if and how PKA modulates KNa. Specifically, I looked at (1) if PKA affects the KNa channel subunit Slack expressed heterologously in HEK-293 and CHO cells and (2) if PKA affects KNa in DRG neurons. My results showed that although recombinant Slack channels are unaffected by PKA, PKA activation does cause a decrease in KNa in DRG neurons. I then showed that the decrease is due to the internal trafficking of Slack channels from the DRG membrane, and not due to an inhibition in channel gating. (1) The C-terminal of the KNa channel subunit, Slack contains several putative PKA phosphorylation consensus sites. While previous studies have shown modulation of recombinant Slack channels by protein kinase C, the effect of PKA phosphorylation on these channels has not been examined. In this study, I investigated if Slack serves as a substrate for phosphorylation by PKA and what effect this phosphorylation has on Slack channel gating in heterologous expression systems. I found that the Slack protein is phosphorylated by PKA. Using electrophysiological techniques to examine Slack channel activity however, I found that in HEK and CHO cells, there is no effect of PKA activation on Slack channel activity. These results suggested that additional factors, not present in heterologous expression systems, may be needed for PKA to exert its effect on Slack.(2) PKA is known to play an important role in nociceptive sensitization characterized by DRG hyperexcitability. Although Slack is highly expressed in DRG neurons, its role in sensitization and hyperexcitability has not been previously described. In this study, I investigated the effect of PKA activation on KNa in DRG neurons. In DRG neurons, I found that PKA activation led to a loss of firing accommodation of the neurons, resulting in hyperexcitability. I also found that KNa is significantly decreased in response to PKA. While KNa channel activity is unaffected by PKA, I found there was a significant reduction in the membrane expression levels of the K Na channel subunit Slack after PKA activation. The decrease in Slack membrane expression may be responsible for the loss of firing accommodation observed in these neurons after PKA activation. These results suggest that KNa may be involved in nociceptive sensitization and could be a novel target for the development of new analgesics.