Retinoic acid inhibition impairs planarian eye regeneration

Retinoic acid is a known morphogen in regulating animal growth and development. Planaria are a key model system for regeneration and their eyes are a morphological marker of anterior differentiation. We explored the requirement for retinoic acid signaling in the regeneration of body parts in the planaria S. mediterranea using an inhibitor of retinoic acid synthesis, diethylaminobenzaldehyde (DEAB). Whole planaria, soaked in DEAB for three days prior to and five days following amputation, produced trunk and tail fragments with defective anterior regeneration. Following regeneration, up to 80% of posterior fragments developed abnormal eyes. The abnormalities included animals without eyes, with only a single eye, with one enlarged eye, or two eyes of different sizes. Eyes were considered to be functional because animals responded to blue laser light with turning behavior. No abnormalities in eye regeneration were observed in side by side vehicle controls. These results suggest that retinoic acid is necessary for normal eye regeneration following injury and supports a previously undocumented signaling role in planaria eye development

A Xenopus oocyte model system to study action potentials

Action potentials (APs) are the functional units of fast electrical signaling in excitable cells. The upstroke and downstroke of an AP is generated by the competing and asynchronous action of Na+- and K+-selective voltage-gated conductances. Although a mixture of voltage-gated channels has been long recognized to contribute to the generation and temporal characteristics of the AP, understanding how each of these proteins function and are regulated during electrical signaling remains the subject of intense research. AP properties vary among different cellular types because of the expression diversity, subcellular location, and modulation of ion channels. These complexities, in addition to the functional coupling of these proteins by membrane potential, make it challenging to understand the roles of different channels in initiating and temporally shaping the AP. Here, to address this problem, we focus our efforts on finding conditions that allow reliable AP recordings from Xenopus laevis oocytes coexpressing Na+ and K+ channels. As a proof of principle, we show how the expression of a variety of K+ channel subtypes can modulate excitability in this minimal model system. This approach raises the prospect of studies on the modulation of APs by pharmacological or biological means with a controlled background of Na+ and K+ channel expression

Retigabine holds KV7 channels open and stabilizes the resting potential

The anticonvulsant Retigabine is a KV7 channel agonist used to treat hyperexcitability disorders in humans. Retigabine shifts the voltage dependence for activation of the heteromeric KV7.2/KV7.3 channel to more negative potentials, thus facilitating activation. Although the molecular mechanism underlying Retigabine\u27s action remains unknown, previous studies have identified the pore region of KV7 channels as the drug\u27s target. This suggested that the Retigabine-induced shift in voltage dependence likely derives from the stabilization of the pore domain in an open (conducting) conformation. Testing this idea, we show that the heteromeric KV7.2/KV7.3 channel has at least two open states, which we named O1 and O2, with O2 being more stable. The O1 state was reached after short membrane depolarizations, whereas O2 was reached after prolonged depolarization or during steady state at the typical neuronal resting potentials. We also found that activation and deactivation seem to follow distinct pathways, suggesting that the KV7.2/KV7.3 channel activity displays hysteresis. As for the action of Retigabine, we discovered that this agonist discriminates between open states, preferentially acting on the O2 state and further stabilizing it. Based on these findings, we proposed a novel mechanism for the therapeutic effect of Retigabine whereby this drug reduces excitability by enhancing the resting potential open state stability of KV7.2/KV7.3 channels. To address this hypothesis, we used a model for action potential (AP) in Xenopus laevis oocytes and found that the resting membrane potential became more negative as a function of Retigabine concentration, whereas the threshold potential for AP firing remained unaltered

The action of Retigabine on KV7 channels activity requires PI(4,5)P2. Society of General Physiologist

Retigabine is a KV7 channel agonist used to treat hyperexcitability disorders in humans (Rudzinski et al., 2016). Although the mechanisms of action remains unclear, it is thought that Retigabine facilitates M-current activation by shifting the voltage dependence of the heteromeric KV7.2/KV7.3 channel to more negative potentials (Schenzer et al., 2005; Wuttke et al., 2005; Gunthorpe et al., 2012; Kim et al., 2015). This notion, however, has been recently challenged as the pore domain was identified as the drug’s target (Schenzer et al., 2005; Wuttke et al., 2005; Lange et al., 2009; Kim et al., 2015), suggesting that Retigabine could, instead, stabilize already-activated channels without affecting activation. Supporting this latter idea, we have recently shown that the heteromeric KV7.2/KV7.3 channel has at least two open modes, (OPEN1 and OPEN2), that OPEN2 is more stable than OPEN1, and that Retigabine further stabilizes OPEN2 (Corbin-Leftwich et al., 2016). To further understand this process, we evaluated the role of PI(4,5)P2 in KV7 channels modal behavior. KV7 channels are subject to muscarinic regulation since these channels require PI(4,5)P2 to be active. Thus, pinpointing a potential functional link between Retigabine and PI(4,5)P2 would be essential to comprehend the action of anticonvulsants in patients. Using pharmacological and enzymatic approaches, we have found that the stability of the KV7.2/KV7.3 OPEN2 mode exhibits a higher dependence on PI(4,5)P2 than the OPEN1 mode, suggesting that the affinity for phosphoinositides of this channel changes during activation. Per these findings, we hypothesized that the action of Retigabine would depend on the concentration of PI(4,5)P2 since Retigabine targets the OPEN2mode. Indeed, we found that decreasing the PI(4,5)P2 concentration impairs the ability of Retigabine to further stabilize the OPEN2 mode. Therefore, we conclude that PI(4,5)P2 is required for the action of Retigabine on KV7 channels and can be subject to muscarinic regulation

The action of Retigabine on KV7 channels activity requires PI(4,5)P2. Society of General Physiologist

Retigabine is a KV7 channel agonist used to treat hyperexcitability disorders in humans (Rudzinski et al., 2016). Although the mechanisms of action remains unclear, it is thought that Retigabine facilitates M-current activation by shifting the voltage dependence of the heteromeric KV7.2/KV7.3 channel to more negative potentials (Schenzer et al., 2005; Wuttke et al., 2005; Gunthorpe et al., 2012; Kim et al., 2015). This notion, however, has been recently challenged as the pore domain was identified as the drug’s target (Schenzer et al., 2005; Wuttke et al., 2005; Lange et al., 2009; Kim et al., 2015), suggesting that Retigabine could, instead, stabilize already-activated channels without affecting activation. Supporting this latter idea, we have recently shown that the heteromeric KV7.2/KV7.3 channel has at least two open modes, (OPEN1 and OPEN2), that OPEN2 is more stable than OPEN1, and that Retigabine further stabilizes OPEN2 (Corbin-Leftwich et al., 2016). To further understand this process, we evaluated the role of PI(4,5)P2 in KV7 channels modal behavior. KV7 channels are subject to muscarinic regulation since these channels require PI(4,5)P2 to be active. Thus, pinpointing a potential functional link between Retigabine and PI(4,5)P2 would be essential to comprehend the action of anticonvulsants in patients. Using pharmacological and enzymatic approaches, we have found that the stability of the KV7.2/KV7.3 OPEN2 mode exhibits a higher dependence on PI(4,5)P2 than the OPEN1 mode, suggesting that the affinity for phosphoinositides of this channel changes during activation. Per these findings, we hypothesized that the action of Retigabine would depend on the concentration of PI(4,5)P2 since Retigabine targets the OPEN2mode. Indeed, we found that decreasing the PI(4,5)P2 concentration impairs the ability of Retigabine to further stabilize the OPEN2 mode. Therefore, we conclude that PI(4,5)P2 is required for the action of Retigabine on KV7 channels and can be subject to muscarinic regulation

Retigabine holds KV7 channels open and stabilizes the resting potential

The anticonvulsant Retigabine is a KV7 channel agonist used to treat hyperexcitability disorders in humans. Retigabine shifts the voltage dependence for activation of the heteromeric KV7.2/KV7.3 channel to more negative potentials, thus facilitating activation. Although the molecular mechanism underlying Retigabine\u27s action remains unknown, previous studies have identified the pore region of KV7 channels as the drug\u27s target. This suggested that the Retigabine-induced shift in voltage dependence likely derives from the stabilization of the pore domain in an open (conducting) conformation. Testing this idea, we show that the heteromeric KV7.2/KV7.3 channel has at least two open states, which we named O1 and O2, with O2 being more stable. The O1 state was reached after short membrane depolarizations, whereas O2 was reached after prolonged depolarization or during steady state at the typical neuronal resting potentials. We also found that activation and deactivation seem to follow distinct pathways, suggesting that the KV7.2/KV7.3 channel activity displays hysteresis. As for the action of Retigabine, we discovered that this agonist discriminates between open states, preferentially acting on the O2 state and further stabilizing it. Based on these findings, we proposed a novel mechanism for the therapeutic effect of Retigabine whereby this drug reduces excitability by enhancing the resting potential open state stability of KV7.2/KV7.3 channels. To address this hypothesis, we used a model for action potential (AP) in Xenopus laevis oocytes and found that the resting membrane potential became more negative as a function of Retigabine concentration, whereas the threshold potential for AP firing remained unaltered