44 research outputs found
RNA editing regulates insect gamma-aminobutyric acid receptor function and insecticide sensitivity
A-to-I pre-mRNA editing by adenosine deaminase enzymes has been reported to enhance protein diversity in the nervous system. In Drosophila, the resistance to dieldrin (RDL) gamma-aminobutyric acid (GABA) receptor subunit displays an editing site (R122) that is close to the putative GABA-binding site. We assessed the functional effects of editing at this site by expressing homomeric RDL receptors in Xenopus oocytes. After replacement of arginine 122 with a glycine, both agonist and fipronil potencies were shifted to the right in either fipronil-sensitive receptors or mutated resistant receptors (A301G/T350M). These data provide the first insight on the influence of RNA editing on GABA receptor function
Inhibition of protein kinase C decreases sensitivity of GABA receptor subtype to fipronil insecticide in insect neurosecretory cells.
Phosphorylation by serine/threonine kinases has been described as a new mechanism for regulating the effects of insecticides on insect neuronal receptors and channels. Although insect GABA receptors are commercially important targets for insecticides (e.g. fipronil), their modulation by kinases is poorly understood and the influence of phosphorylation on insecticide sensitivity is unknown. Using the whole-cell patch-clamp technique, we investigated the modulatory effect of PKC and CaMKinase II on GABA receptor subtypes (GABAR1 and GABAR2) in DUM neurons isolated from the terminal abdominal ganglion (TAG) of Periplaneta americana. Chloride currents through GABAR2 were selectively abolished by PMA and PDBu (the PKC activators) and potentiated by Gö6983, an inhibitor of PKC. Furthermore, using KN-62, a specific CaMKinase II inhibitor, we demonstrated that CaMKinase II activation was also involved in the regulation of GABAR2 function. In addition, using CdCl(2) (the calcium channel blocker) and LOE-908, a blocker of TRPγ, we revealed that calcium influx through TRPγ played an important role in kinase activations. Comparative studies performed with CACA, a selective agonist of GABAR1 in DUM neurons confirmed the involvement of these kinases in the specific regulation of GABAR2. Furthermore, our study reported that GABAR1 was less sensitive than GABAR2 to fipronil. This was demonstrated by the biphasic concentration-response curve and the current-voltage relationship established with both GABA and CACA. Finally, we demonstrated that GABAR2 was 10-fold less sensitive to fipronil following inhibition of PKC, whereas inhibition of CaMKinase II did not alter the effect of fipronil
Functional analysis of missense variants in the TRESK (KCNK18) K+ channel
A loss of function mutation in the TRESK K2P potassium channel (KCNK18), has recently been linked with typical familial migraine with aura. We now report the functional characterisation of additional TRESK channel missense variants identified in unrelated patients. Several variants either had no apparent functional effect, or they caused a reduction in channel activity. However, the C110R variant was found to cause a complete loss of TRESK function, yet is present in both sporadic migraine and control cohorts, and no variation in KCNK18 copy number was found. Thus despite the previously identified association between loss of TRESK channel activity and migraine in a large multigenerational pedigree, this finding indicates that a single non-functional TRESK variant is not alone sufficient to cause typical migraine and highlights the genetic complexity of this disorder
Fluorescence-Tracking of Activation Gating in Human ERG Channels Reveals Rapid S4 Movement and Slow Pore Opening
Background: hERG channels are physiologically important ion channels which mediate cardiac repolarization as a result of their unusual gating properties. These are very slow activation compared with other mammalian voltage-gated potassium channels, and extremely rapid inactivation. The mechanism of slow activation is not well understood and is investigated here using fluorescence as a direct measure of S4 movement and pore opening. Methods and Findings: Tetramethylrhodamine-5-maleimide (TMRM) fluorescence at E519 has been used to track S4 voltage sensor movement, and channel opening and closing in hERG channels. Endogenous cysteines (C445 and C449) in the S1–S2 linker bound TMRM, which caused a 10 mV hyperpolarization of the VK of activation to 227.562.0 mV, and showed voltage-dependent fluorescence signals. Substitution of S1–S2 linker cysteines with valines allowed unobstructed recording of S3–S4 linker E519C and L520C emission signals. Depolarization of E519C channels caused rapid initial fluorescence quenching, fit with a double Boltzmann relationship, F-VON, with VK,1 = 237.861.7 mV, and VK,2 = 43.567.9 mV. The first phase, VK,1, was,20 mV negative to the conductance-voltage relationship measured from ionic tail currents (G-VK = 218.361.2 mV), and relatively unchanged in a non-inactivating E519C:S620T mutant (V K = 234.461.5 mV), suggesting the fast initial fluorescence quenching tracked S4 voltage sensor movement. The second phase of rapid quenching was absent in the S620T mutant. The E519C fluorescence upon repolarizatio
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SUMOylation and calcium signalling: potential roles in the brain and beyond
Small ubiquitin-like modi er (SUMO) conjugation (or SUMOylation) is a post-translational protein modi cation implicated in alterations to protein expression, localization and func- tion. Despite a number of nuclear roles for SUMO being well characterized, this process has only started to be explored in relation to membrane proteins, such as ion channels. Cal- cium ion (Ca2+) signalling is crucial for the normal functioning of cells and is also involved in the pathophysiological mechanisms underlying relevant neurological and cardiovascu- lar diseases. Intracellular Ca2+ levels are tightly regulated; at rest, most Ca2+ is retained in organelles, such as the sarcoplasmic reticulum, or in the extracellular space, whereas depolarization triggers a series of events leading to Ca2+ entry, followed by extrusion and reuptake. The mechanisms that maintain Ca2+ homoeostasis are candidates for modulation at the post-translational level. Here, we review the effects of protein SUMOylation, including Ca2+ channels, their proteome and other proteins associated with Ca2+ signalling, on vital cellular functions, such as neurotransmission within the central nervous system (CNS) and in additional systems, most prominently here, in the cardiac system
hERG S4-S5 linker acts as a voltage-dependent ligand that binds to the activation gate and locks it in a closed state
International audienceDelayed-rectifier potassium channels (hERG and KCNQ1) play a major role in cardiac repolarization. These channels are formed by a tetrameric pore (S5-S6) surrounded by four voltage sensor domains (S1-S4). Coupling between voltage sensor domains and the pore activation gate is critical for channel voltage-dependence. However, molecular mechanisms remain elusive. Herein, we demonstrate that covalently binding, through a disulfide bridge, a peptide mimicking the S4-S5 linker (S4-S5L) to the channel S6 C-terminus (S6T) completely inhibits hERG. This shows that channel S4-S5L is sufficient to stabilize the pore activation gate in its closed state. Conversely, covalently binding a peptide mimicking S6T to the channel S4-S5L prevents its inhibiting effect and renders the channel almost completely voltage-independent. This shows that the channel S4-S5L is necessary to stabilize the activation gate in its closed state. Altogether, our results provide chemical evidence that S4-S5L acts as a voltage-controlled ligand that binds S6T to lock the channel in a closed state, elucidating the coupling between voltage sensors and the gate in delayed rectifier potassium channels and potentially other voltage-gated channels