Dissertations / Theses on the topic 'Tandem Pore Domain Potassium Channels'

Endocytic trafficking dynamically regulates neuronal plasma membrane protein presentation and activity, and plays a central role in excitability and plasticity. Over the course of my dissertation research I investigated endocytic mechanisms regulating two neuronal membrane proteins: the anesthetic-activated potassium leak channel, KCNK3, as well as the psychostimulant-sensitive dopamine transporter (DAT). My results indicate that KCNK3 internalizes in response to Protein Kinase C (PKC) activation, using a novel pathway that requires the phosphoserine binding protein, 14-3-3β, and demonstrates for the first time regulated KCNK3 channel trafficking in neurons. Additionally, PKC-mediated KCNK3 trafficking requires a non-canonical endocytic motif, which is shared exclusively between KCNK3 and sodium-dependent neurotransmitter transporters, such as DAT. DAT trafficking studies in intact ex vivo adult striatal slices indicate that DAT endocytic trafficking has both dynamin-dependent and –independent components. Moreover, DAT segregates into two populations at the neuronal plasma membrane: trafficking-competent and -incompetent. Taken together, these results demonstrate that novel, non-classical endocytic mechanisms dynamically control the plasma membrane presentation of these two important neuronal proteins.

The membrane potential of arterial smooth cells, and consequently arterial tone, is. regulated by the activity of plasmalemmal K+ selective ion channels. K2P channels are responsible for background K+ (KB) conductances which contribute to generation of the resting membrane potential. They have been identified in arterial smooth muscle cells but their significance is poorly understood. A Ks conductance is expressed in rat femoral arterial smooth muscle cells (rFASMCs), and the aim of this project was to (i) determine the expression profile of K2P subunits in rat femoral artery and (ii) identify the molecular correlate of the KB conductance in rFASMCs. Using RT-PCR, we identified transcripts encoding TWIK-2, TASK-I, TASK-2, TREK-I, TREK-2 and THIK-I in total rat femoral artery RNA. TWIK-2, TASK-I, TASK-2 and TREK-I were further identified at the protein level in isolated FASMCs by antibody staining. Only TWIK-2 was detected at the cell surface, while TASK-I, TASK-2 and TREK-I had a predominantly intracellular localisation. Immunohistochemistry of femoral artery sections may also support the expression of TWIK-2, TASK-I and TASK-2 (but not TREK-I) in the endothelium. To allow comparison between cloned and native conductances, the open reading frames of rTWIK-2, rTASK-2 and rTREK-I were cloned by PCR amplification. The sequence encoding rTASK-2 was cloned de novo and submitted to the EBI gene data base (Accession no: AM229406). Functional expression ofrTWIK-2 and rTASK-2 was investigated in Xenopus 'laevis oocytes. rTWIK-2 did not exhibit strong expression in the Xenopus laevis expression system. Conversely, recombinant rTASK-2 channels formed K+ selective, extracellular pH sensitive ion channels. The basic pharmacological properties of cloned rTASK-2 channels were also investigated. Currents were insensitive to extracellular TEA, 4-AP and Cs+, but were sensitive to inhibition by Ba2+, Zn2+and quinidine. Inhibition by extracellular Ba2+was strongly time and voltage-dependent. Ba2+ inhibition of rTASK-2 was characterised by steady-state analysis and voltage pulses. Although we were unable to describe the functional correlate of the KB conductance in rFASMCs, this thesis has established the groundwork for further correlative studies. The information provided will assist in the identification of native TASK-2 conductances.

Introduction: Two pore domain potassium (K2P) channels are responsible for background currents that regulate membrane potential and neuronal excitability. Compounds which alter the activity of these channels are predicted to have therapeutic potential in treating CNS disorders. Members of the TREK family of K2P channels (TREK1 and TREK2) have been shown to play an active role in neuroprotection, depression and pain, whilst TRESK, with high expression in sensory neurons, has a role in nociception. Sipatrigine, a neuroprotective agent and a derivative of the anticonvulsant lamotrigine, is a known antagonist of TREK channels whilst lamotrigine is thought to primarily inhibit TRESK channels. A new compound, Cen-092-C, has also been developed which is structurally similar to lamotrigine. However, its effects on K2P channels are unknown. To understand the mechanism of channel inhibition by drugs, the structure of TREK2 was solved and was co-crystallised with norfluoxetine. This showed that fenestration sites were important in channel and current inhibition. Furthermore, TRESK docking studies showed that F145 and F352 function in a similar way to TREK2 fenestration site, as the bulky phenylalanine faces into the pore, and are thought to be important for compound binding. The aim of this study is to clarify to differences in the inhibitory effect of these compounds on the selected K2P channels and to investigate the mechanism by which these compounds inhibit the channels current. Methods: Wild-type (WT) and mutated human K2P channels were transiently expressed in tsA-201 cells. The currents were measured using whole-cell patch-clamp electrophysiology. Results: Sipatrigine was shown to inhibit both TREK1 and TREK2 current. Lamotrigine was also found to inhibit TREK1 and to a lesser extent TREK2. Cen-092-C was found to be less effective on TREK1 and TRESK current compared to sipatrigine, but similar to lamotrigine results. The sipatrigine inhibitory effect, but not lamotrigine, was reduced by mutations on the M4 region at the fenestration site of TREK1 and TREK2 (L286 and L320). This sensitivity is selective at this site as other mutations in the central cavity showed no change in sipatrigine inhibition. Interestingly, the gain-of-function mutation (TREK1 E306A) on the C terminus showed a reduced sipatrigine inhibition. The effect of sipatrigine on TREK2 showed an over-recovery of current following wash-off of the compound. The wash-off current increase was not seen if the N-terminus length is forced into intermediate and short isoform. Sipatrigine inhibition was significantly decreased when the N-terminus was truncated. Sipatrigine has been shown to strongly inhibit TRESK. Lamotrigine was seen to inhibit TRESK current, however significantly less effective compared to sipatrigine. Furthermore, lamotrigine did show state dependent inhibition when TRESK is in the fixed activated state. Cen-092-C was also found to inhibit TRESK to a similar degree to lamotrigine, however there was no state dependent inhibition on TRESK current. The effects of these antagonists on TRESK has been shown to be abolished by mutations on two sites at the central cavity (F145 and F352). Conclusion: Lamotrigine was found not to be TRESK selective, contrary to other studies. Sipatrigine and lamotrigine inhibition works through binding to the channel. The fenestration site in both TREK1 and TREK2 has been found to be an important binding site of sipatrigine, differing from lamotrigine. This suggests that the structurally similar compounds bind to different regions of the TREK channels. Furthermore, the over recovery of TREK2 current after sipatrigine wash off is believed to show the compound's biphasic effect, where the underlying enhancement of current is hidden by the action of inhibition. The N-terminus is therefore believed to be important in regulating sipatrigine action on TREK2. It remains unclear whether the TRESK potential binding sites (F1452 and F352) are important in compound binding as the inserted mutation is believed to shift the channel to constant active state. The newly developed compound Cen-092-C shows a significantly greater degree of inhibition of TRESK when compared to TREK1. Cen-092-C and lamotrigine inhibition of TRESK is not significantly different. Lamotrigine inhibition of TRESK current is state dependent whereas sipatrigine and Cen-092-C inhibition of TRESK current is shown as state independent. All of this together could lead to a better understanding of how neuroprotective agents effect TREK and TRESK channels and could contribute to the design of more efficient ligands.

TASK channels, members of the two-pore-domain potassium channel family, contribute towards the resting membrane potential and have been implicated in the mechanism of general anaesthesia. Previous work from our group with a TASK-3 channel knockout (T3KO) mouse showed a reduction in halothane sensitivity using a loss of righting reflex (LORR) assay, and absence of the theta brain oscillation induced in wild type (WT) mice by halothane anaesthesia. Two further strains of knockout mice, the TASK-1 knockout (T1KO) and the double knockout (DKO: TASK-1 and -3 channels), were compared with WT using the LORR assay, cortical electroencephalogram recording in response to halothane and during sleep. The mechanistic basis for the diminished theta oscillation in T3KO mice was investigated by recording in CA1 pyramidal cells of the hippocampus. The LORR assay revealed a decrease in halothane sensitivity in T1KO but not DKO compared with WT. The T1KO strain had a theta oscillation induced by exposure to halothane similar to that of WT mice, whereas that observed in the DKO was intermediate between WT and T3KO. T1KO differed from other strains in that the distribution of sleep and wake periods was uniform across the diurnal cycle. The resting membrane potential did not differ between strains during control or halothane exposure. During control there was no strain difference in action potential (AP). Halothane altered AP shape in WT but not the T3KO strain. WT had a greater ability to sustain AP firing than T3KO during halothane. These data show that T1KO mice have decreased anaesthetic sensitivity and altered sleep structure compared with WT, indicating a role for this channel in anaesthetic sensitivity and sleep. The similar resting membrane potential and lack of response to halothane in the T3KO makes pyramidal cells an unlikely source of the theta ablation observed.

Background: Kv2.1 and TASK-1 channels are two main contributors of K+ currents in pulmonary artery smooth muscle cells (PASMC). Dysregulation of these channels has been implicated in the pathogenesis of pulmonary hypertension (PH). This thesis aims to delve deeper into the implications of the regulation of Kv2.1 by Kv9.3 in PH. Another subject of interest would be whether NADPH oxidase type 4 (Nox4), one of the major reactive oxygen species (ROS) producers in the PASMC, modulates Kv2.1, Kv9.3, and TASK-1 channels. The effects of several redox agents are also investigated as potential modulators of Kv2.1, Kv9.3, and TASK-1. In addition, this thesis also examined the effect of a Kv2-channel blocker, stromatoxin, on Kv2.1 and Kv9.3. Finally, since amphoterin-induced gene and open reading frame (AMIGO) proteins have recently been shown as novel Kv2.1-interacting partners, their effects on Kv2.1 and/or Kv9.3 are also explored in this study. Experimental approach: Whole-cell patch clamp electrophysiology was used to measure currents of the ion channels expressed in modified tsA-201 cells, in the absence and presence of Nox4 AMIGO and other regulatory molecules. Immunohistochemistry was deployed to visualize the distribution of Kv2.1 and Kv9.3 proteins in the rat lungs and hearts. Key results and Conclusions: This study supports the findings that Kv9.3 regulates Kv2.1 by increasing the current amplitude, shifting the activation threshold to a more negative voltage range, and prolonging the slow component of time constant of deactivation. These effects could be beneficial in PH as this would mean cells could be brought back to its resting membrane potential faster and the transduction of the next action potential can be delayed. Kv2.1 and Kv9.3 have also been detected at the endothelium and PASMC in rat lungs and hearts, further substantiating the claim that these channels are potential players in regulating PH. AMIGO1 and AMIGO2 proteins are confirmed as regulators of Kv2.1 and Kv9.3 proteins. Nox4 does not regulate Kv2.1, Kv9.3, and TASK-1 channels expressed in tsA-201 cells. While hydrogen peroxide (H2O2) does not have any effect on Kv2.1 and Kv9.3, it abolished the current reduction effect of AMIGO2 on Kv2.1/Kv9.3. Other redox agents used in this study such as dithiothreitol (DTT), 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and chloramine T (Ch-T) are not modulators of these channels expressed in tsA-201 cells. The lack of effect from Nox4 and these redox agents could suggest that the redox regulation of different Nox subunit/Kv channels combination varies for different cell types due to the different regulatory proteins present in different heterologous expression systems. As with the case of H2O2 and AMIGO2, it is likely that the regulatory proteins, which could facilitate the hypoxia-sensing properties of Nox4 and the effects of the redox agents on the ion channels, are missing in our heterologous expression system, compared with other host cells.

In mammals light is the principal timing cue for alignment of physiology to the external environment. Illumination from the unrelenting 24-hour day-night cycle enters the biological system and is communicated to the master pacemaker, the suprachiasmatic nucleus (SCN) to drive circadian entrainment. The decoding of light by the retina and the signalling pathways to and from the SCN rely on neural excitation mechanisms, achieved through changes in membrane potential from a resting state stabilised by K2P channels. With TASK-3 being the most abundant K2P channel in the rodent SCN it is feasible this channel has a crucial role in regulating SCN neural transmission for effective circadian entrainment. This study investigates this role through the use of transgenic TASK-3 KO mice. In the first experimental chapter I demonstrate the presence of TASK-3 mRNA in the SCN and retina of wild type mice. Further, I reveal a circadian pattern in TASK-3 mRNA expression with significant midday nadir which feasibly influences resting membrane potential (RMP) supporting increased neuronal excitation reported at this time. The following three chapters explore TASK-3 conductance in behavioural output rhythms via locomotor activity studies under light-dark cycles and in constant darkness. This series of experiments highlights how TASK-3 is essential for effective adjustment to changing light and how loss of this channel reduces light-driven and endogenous activity intensity and rhythm amplitude. With light entering the circadian system exclusively via the eyes, the role of TASK-3 at the level of the retina is of upmost importance to entrainment. This is investigated in chapter 6 using pupillary light reflex as a measure of retinal sensitivity and decoding capacity. Through manipulation of intensity and wavelength specific classes of photoreceptor are studied for their contribution to this non-image forming response. These experiments show TASK-3 ablation significantly attenuates retinal sensitivity to sub-saturating light in a mechanism likely to be melanopsin-independent. Finally examination of mRNA expression of core clock genes reveals the role of TASK-3 at the level of the SCN. Here, loss of TASK-3 conductance is shown to alter daily rhythms in several key genes thereby linking the properties of this background leakage channel to the molecular clockwork. Overall these experiments demonstrate some of the roles TASK-3 conductance plays within the SCN and in output rhythms; and the requirement of this channel within the retina for effective retinal decoding across the visible spectrum over a range of light intensities.

Background potassium currents play an important role in the regulation of the resting membrane potential and excitability of mammalian neurons. Recently cloned two- pore domain potassium channels (K2p) are believed to underlie these currents. The roles of K2P channels in general anesthesia and neuroprotection have been proposed recently. In view of this, we investigated the ability of trichloroethanol (an active metabolite of the non-volatile general anesthetic cldoral hydrate, widely used as a pediatric sedative) to modulate the activity of human TREK-1 and TRAAK channels. We found that trichloroethanol potently activates both hTREK-1 and hTRAAK channels at pharmacologically relevant concentrations. The parent compound chloral hydrate was also found to augtnent the activity of both the channels reversibly. Studies with carboxy- terminal deletion mutants (hTREK-1A89, hTREK-1 A100 and hTREK-1 A1 19), suggested that C-terminal tail is not essential for the activation of TREK-1 by trichloroethanol. Our findings identify TREK-1 and TRCL4K channels as molecular targets for trichloroethanol and we propose that activation of both these channels might contribute to the CNS depressant effects of chloral hydrate. Another channel TASK-2, which is essentially absent in the human brain was also found to be potently activated by both trichloroethanol and chloral hydrate. In another series of experiments, we studied the effects of methyl xanthines caffeine and theophylline on hTREK-1 channels. Caffeine and theophylline are used for therapeutic purposes and frequently cause life-threatening convulsive seizures due to systemic toxicity. The mechanisms for the epileptogenicity of caffeine and theophylline are not clear. Recent experiments using knockout mice provided direct evidence for a role for TREK-1 in the control of epileptogenesis. We hypothesized that the epileptogenicity of caffeine and theophylline may be related to the inhibition of TREK-1 channels. We investigated this possibility and observed massive inhibition of TREK-1 channels at toxicologically relevant concentrations. Experiments with the mutant TREK-1 channel (S348A mutant) suggested the involvement of cANP/PKA pathway in the inhibition of TREK-1 channels by caffeine and theophylline. We suggest that inhibition of TREK-1 channels may contribute to the convulsive seizures induced by toxic levels of caffeine and theophylline. Local anesthetics exhibit their clinical effects not only by binding to voltage-gated sodium channels, but also by interacting with other ion channels such as potassium channels. Because of the physiological significance of TREK-1 channels and their abundant expression in peripheral sensory neurons, we investigated the effects of lidocaine to see whether its interaction with 'REK-1 channels contribute to the conduction blockade. Lidocaine caused dose-dependent inhibition of TREK-1channels and the inhibition was voltage-independent. Cytoplasmic C-terminal tail is critically required for lidocaine action. Inhibition of TREK-1 channels is achieved at concentrations for iiz vivo action and this effect may have implications for the clinically observed drug action of lidocaine.