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Jean-françois Liégeois - One of the best experts on this subject based on the ideXlab platform.
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Chemical modifications of the N-methyl-laudanosine scaffold point to new directions for SK Channels exploration.
Bioorganic & Medicinal Chemistry Letters, 2014Co-Authors: Eduard Badarau, Vincent Seutin, Sébastien Dilly, Johan Wouters, Jean-françois LiégeoisAbstract:An asparagine or a histidine are present in a similar position in the outer pore region of SK2 and SK3 Channels, respectively. Therefore, this structural difference was targeted in order to develop selective blockers of SK Channel subtypes. Following docking investigations, based on theoretical models of truncated SK2 and SK3 Channels, the benzyl side chain of N-methyl-laudanosine (NML) was functionalized in order to target this specific amino-acid residues. Chiral butanamide and benzyloxy analogues were prepared, resolved and tested for their affinity for SK2 and SK3 Channels. Isoquinolinium (NMIQ) derivatives have a higher affinity for both SK Channel subtypes than the corresponding derivative with no functionalized side chain. This trend was observed also for the 1,2,3,4-tetrahydroisoquinoline (THIQ) analogues. A benzyloxy functionalized NML enantiomer has a higher affinity than NML stereoisomers. Otherwise, the conserved affinity of these analogues led to the opportunity to further investigate in terms of possible labeling for in vivo investigations of the role of SK Channels.
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synthesis and radioligand binding studies of bis 8 isopropyl isoquinolinium derivatives as ligands for apamin sensitive sites on cloned SK2 and SK3 Channels
Bioorganic & Medicinal Chemistry Letters, 2011Co-Authors: Eduard Badarau, Vincent Seutin, Sébastien Dilly, Fabien Dufour, Sylvie Poncin, Jean-françois LiégeoisAbstract:Abstract A structure–activity relationship study of N-methyl-laudanosine, a SK Channel blocker, has indicated that the 6,7-dimethoxy group could be successfully replaced by a hydrophobic moiety such as an isopropyl substituent in position 8 of the isoquinoline ring. In the present study, bis-(8-isopropyl-isoquinolinium) derivatives (2a–e) were synthesized and tested for their affinity for cloned SK2 and SK3 Channels in comparison with their 6,7-dimethoxy analogues (4a–f). Several ligands were investigated, both in flexible (propyl, butyl and pentyl) and rigid (m- or p-xylyl) series, the m-xylyl derivative (2d) having the best profile in terms of affinity and selectivity for SK3/SK2 Channels. Molecular studies showed that the optimal conformation of compound 2d fits well with our SK pharmacophore model.
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crucial role of a shared extracellular loop in apamin sensitivity and maintenance of pore shape of small conductance calcium activated potassium SK Channels
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Kate L Weatherall, Jean-françois Liégeois, Vincent Seutin, Neil V MarrionAbstract:Activation of small-conductance calcium (Ca2+)-dependent potassium (KCa2) Channels (herein called “SK”) produces membrane hyperpolarization to regulate membrane excitability. Three subtypes (SK1–3) have been cloned and are distributed throughout the nervous system, smooth muscle, and heart. It is difficult to discern the physiological role of individual Channel subtypes as most blockers or enhancers do not discriminate between subtypes. The archetypical blocker apamin displays some selectivity between SK Channel subtypes, with SK2 being the most sensitive, followed by SK3 and then SK1. Sensitivity of SK1 is species specific, with the human isoform being blocked by the toxin, whereas the rat is not. Mutation studies have identified residues within the outer pore that suggest apamin blocks by an allosteric mechanism. Apamin also uses a residue within the S3–S4 extracellular loop to produce a high-sensitivity block. We have identified that a 3-amino acid motif within this loop regulates the shape of the Channel pore. This motif is required for binding and block by apamin, suggesting that a change in pore shape underlies allosteric block. This motif is absent in rat SK1, explaining why it is insensitive to block by apamin. The overlapping distribution of SK Channel subtype expression suggests that native heteromeric Channels may be common. We show that the S3–S4 loop of one subunit overlaps the outer pore of the adjacent subunit, with apamin interacting with both regions. This arrangement provides a unique binding site for each combination of SK subunits within a coassembled Channel that may be targeted to produce blockers specific for heteromeric SK Channels.
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SK Channel blockade promotes burst firing in dorsal raphe serotonergic neurons
European Journal of Neuroscience, 2008Co-Authors: Nathalie Rouchet, Jean-françois Liégeois, Olivier Waroux, Cedric Lamy, Laurent Massotte, Jacqueline Scuveemoreau, Vincent SeutinAbstract:: Previous in vivo studies have shown that blockade of small-conductance Ca(2+)-activated potassium (SK) Channels enhances burst firing in dopaminergic neurons. As bursting has been found to be physiologically relevant for the synaptic release of serotonin (5-HT), we investigated the possible role of SK Channels in the control of this firing pattern in 5-HT neurons of the dorsal raphe nucleus. In these cells, bursts are usually composed of doublets consisting of action potentials separated by a small interval (< 20 ms). Both in vivo and in vitro extracellular recordings were performed, using anesthetized rats and rat brain slices, respectively. In vivo, the specific SK blocker UCL 1684 (200 microm) iontophoresed onto presumed 5-HT neurons significantly increased the production of bursts in 13 out of 25 cells. Furthermore, the effect of UCL 1684 persisted in the presence of both the GABA(A) antagonist SR 95531 (10 mm) and the GABA(B) antagonist CGP 35348 (10 mm), whereas these agents by themselves did not significantly influence the neuronal firing pattern. In vitro, bath superfusion of the SK Channel blocker apamin (300 nm) induced bursting in only three out of 18 neurons, although it increased the coefficient of variation of the interspike intervals in all the other cells. Our results suggest that SK Channel blockade promotes bursting activity in 5-HT neurons via a direct action. An input which is present only in vivo seems to be important for the induction of this firing pattern in these cells.
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metaplastic effect of apamin on ltp and paired pulse facilitation
Learning & Memory, 2007Co-Authors: Brigitte Capron, Coralie Sclavons, Jean-françois Liégeois, Vincent Seutin, Emile GodauxAbstract:In area CA1 of hippocampal slices, a single 1-sec train of 100-Hz stimulation generally triggers a short-lasting long-term potentiation (S-LTP) of 1-2 h. Here, we found that when such a train was applied 45 min after application of the small conductance Ca2+-activated K+ (SK) Channel blocker apamin, it induced a long-lasting LTP (L-LTP) of several hours, instead of an S-LTP. Apamin-induced SK Channel blockage is known to resist washing. Nevertheless, the aforementioned effect is not a mere delayed effect; it is metaplastic. Indeed, when a single train was delivered to the Schaffer's collaterals during apamin application, it induced an S-LTP, like in the control situation. At the moment of this LTP induction (15th min of apamin application), the SK Channel blockage was nevertheless complete. Indeed, at that time, under the influence of apamin, the amplitude of the series of field excitatory postsynaptic potentials (fEPSPs) triggered by a stimulation train was increased. We found that the metaplastic effect of apamin on LTP was crucially dependent on the NO-synthase pathway, whereas the efficacy of the NMDA receptors was not modified at the time of its occurrence. We also found that apamin produced an increase in paired-pulse facilitation not during, but after, the application of the drug. Finally, we found that the induction of each of these two metaplastic phenomena was mediated by NMDA receptors. A speculative unitary hypothesis to explain these phenomena is proposed.
Vincent Seutin - One of the best experts on this subject based on the ideXlab platform.
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Chemical modifications of the N-methyl-laudanosine scaffold point to new directions for SK Channels exploration.
Bioorganic & Medicinal Chemistry Letters, 2014Co-Authors: Eduard Badarau, Vincent Seutin, Sébastien Dilly, Johan Wouters, Jean-françois LiégeoisAbstract:An asparagine or a histidine are present in a similar position in the outer pore region of SK2 and SK3 Channels, respectively. Therefore, this structural difference was targeted in order to develop selective blockers of SK Channel subtypes. Following docking investigations, based on theoretical models of truncated SK2 and SK3 Channels, the benzyl side chain of N-methyl-laudanosine (NML) was functionalized in order to target this specific amino-acid residues. Chiral butanamide and benzyloxy analogues were prepared, resolved and tested for their affinity for SK2 and SK3 Channels. Isoquinolinium (NMIQ) derivatives have a higher affinity for both SK Channel subtypes than the corresponding derivative with no functionalized side chain. This trend was observed also for the 1,2,3,4-tetrahydroisoquinoline (THIQ) analogues. A benzyloxy functionalized NML enantiomer has a higher affinity than NML stereoisomers. Otherwise, the conserved affinity of these analogues led to the opportunity to further investigate in terms of possible labeling for in vivo investigations of the role of SK Channels.
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synthesis and radioligand binding studies of bis 8 isopropyl isoquinolinium derivatives as ligands for apamin sensitive sites on cloned SK2 and SK3 Channels
Bioorganic & Medicinal Chemistry Letters, 2011Co-Authors: Eduard Badarau, Vincent Seutin, Sébastien Dilly, Fabien Dufour, Sylvie Poncin, Jean-françois LiégeoisAbstract:Abstract A structure–activity relationship study of N-methyl-laudanosine, a SK Channel blocker, has indicated that the 6,7-dimethoxy group could be successfully replaced by a hydrophobic moiety such as an isopropyl substituent in position 8 of the isoquinoline ring. In the present study, bis-(8-isopropyl-isoquinolinium) derivatives (2a–e) were synthesized and tested for their affinity for cloned SK2 and SK3 Channels in comparison with their 6,7-dimethoxy analogues (4a–f). Several ligands were investigated, both in flexible (propyl, butyl and pentyl) and rigid (m- or p-xylyl) series, the m-xylyl derivative (2d) having the best profile in terms of affinity and selectivity for SK3/SK2 Channels. Molecular studies showed that the optimal conformation of compound 2d fits well with our SK pharmacophore model.
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crucial role of a shared extracellular loop in apamin sensitivity and maintenance of pore shape of small conductance calcium activated potassium SK Channels
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Kate L Weatherall, Jean-françois Liégeois, Vincent Seutin, Neil V MarrionAbstract:Activation of small-conductance calcium (Ca2+)-dependent potassium (KCa2) Channels (herein called “SK”) produces membrane hyperpolarization to regulate membrane excitability. Three subtypes (SK1–3) have been cloned and are distributed throughout the nervous system, smooth muscle, and heart. It is difficult to discern the physiological role of individual Channel subtypes as most blockers or enhancers do not discriminate between subtypes. The archetypical blocker apamin displays some selectivity between SK Channel subtypes, with SK2 being the most sensitive, followed by SK3 and then SK1. Sensitivity of SK1 is species specific, with the human isoform being blocked by the toxin, whereas the rat is not. Mutation studies have identified residues within the outer pore that suggest apamin blocks by an allosteric mechanism. Apamin also uses a residue within the S3–S4 extracellular loop to produce a high-sensitivity block. We have identified that a 3-amino acid motif within this loop regulates the shape of the Channel pore. This motif is required for binding and block by apamin, suggesting that a change in pore shape underlies allosteric block. This motif is absent in rat SK1, explaining why it is insensitive to block by apamin. The overlapping distribution of SK Channel subtype expression suggests that native heteromeric Channels may be common. We show that the S3–S4 loop of one subunit overlaps the outer pore of the adjacent subunit, with apamin interacting with both regions. This arrangement provides a unique binding site for each combination of SK subunits within a coassembled Channel that may be targeted to produce blockers specific for heteromeric SK Channels.
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SK Channel blockade promotes burst firing in dorsal raphe serotonergic neurons
European Journal of Neuroscience, 2008Co-Authors: Nathalie Rouchet, Jean-françois Liégeois, Olivier Waroux, Cedric Lamy, Laurent Massotte, Jacqueline Scuveemoreau, Vincent SeutinAbstract:: Previous in vivo studies have shown that blockade of small-conductance Ca(2+)-activated potassium (SK) Channels enhances burst firing in dopaminergic neurons. As bursting has been found to be physiologically relevant for the synaptic release of serotonin (5-HT), we investigated the possible role of SK Channels in the control of this firing pattern in 5-HT neurons of the dorsal raphe nucleus. In these cells, bursts are usually composed of doublets consisting of action potentials separated by a small interval (< 20 ms). Both in vivo and in vitro extracellular recordings were performed, using anesthetized rats and rat brain slices, respectively. In vivo, the specific SK blocker UCL 1684 (200 microm) iontophoresed onto presumed 5-HT neurons significantly increased the production of bursts in 13 out of 25 cells. Furthermore, the effect of UCL 1684 persisted in the presence of both the GABA(A) antagonist SR 95531 (10 mm) and the GABA(B) antagonist CGP 35348 (10 mm), whereas these agents by themselves did not significantly influence the neuronal firing pattern. In vitro, bath superfusion of the SK Channel blocker apamin (300 nm) induced bursting in only three out of 18 neurons, although it increased the coefficient of variation of the interspike intervals in all the other cells. Our results suggest that SK Channel blockade promotes bursting activity in 5-HT neurons via a direct action. An input which is present only in vivo seems to be important for the induction of this firing pattern in these cells.
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metaplastic effect of apamin on ltp and paired pulse facilitation
Learning & Memory, 2007Co-Authors: Brigitte Capron, Coralie Sclavons, Jean-françois Liégeois, Vincent Seutin, Emile GodauxAbstract:In area CA1 of hippocampal slices, a single 1-sec train of 100-Hz stimulation generally triggers a short-lasting long-term potentiation (S-LTP) of 1-2 h. Here, we found that when such a train was applied 45 min after application of the small conductance Ca2+-activated K+ (SK) Channel blocker apamin, it induced a long-lasting LTP (L-LTP) of several hours, instead of an S-LTP. Apamin-induced SK Channel blockage is known to resist washing. Nevertheless, the aforementioned effect is not a mere delayed effect; it is metaplastic. Indeed, when a single train was delivered to the Schaffer's collaterals during apamin application, it induced an S-LTP, like in the control situation. At the moment of this LTP induction (15th min of apamin application), the SK Channel blockage was nevertheless complete. Indeed, at that time, under the influence of apamin, the amplitude of the series of field excitatory postsynaptic potentials (fEPSPs) triggered by a stimulation train was increased. We found that the metaplastic effect of apamin on LTP was crucially dependent on the NO-synthase pathway, whereas the efficacy of the NMDA receptors was not modified at the time of its occurrence. We also found that apamin produced an increase in paired-pulse facilitation not during, but after, the application of the drug. Finally, we found that the induction of each of these two metaplastic phenomena was mediated by NMDA receptors. A speculative unitary hypothesis to explain these phenomena is proposed.
George Lykotrafitis - One of the best experts on this subject based on the ideXlab platform.
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tonic pka activity regulates SK Channel nanoclustering and somatodendritic distribution
Journal of Molecular Biology, 2016Co-Authors: Krithika Abiraman, Randall S Walikonis, George Lykotrafitis, Anastasios V TzingounisAbstract:Small-conductance calcium-activated potassium (SK) Channels mediate a potassium conductance in the brain and are involved in synaptic plasticity, learning, and memory. SK Channels show a distinct subcellular localization that is crucial for their neuronal functions. However, the mechanisms that control this spatial distribution are unknown. We imaged SK Channels labeled with fluorophore-tagged apamin and monitored SK Channel nanoclustering at the single molecule level by combining atomic force microscopy and toxin (i.e., apamin) pharmacology. Using these two complementary approaches, we found that native SK Channel distribution in pyramidal neurons, across the somatodendritic domain, depends on ongoing cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) levels, strongly limiting SK Channel expression at the pyramidal neuron soma. Furthermore, tonic cAMP-PKA levels also controlled whether SK Channels were expressed in nanodomains as single entities or as a group of multiple Channels. Our study reveals a new level of regulation of SK Channels by cAMP-PKA and suggests that ion Channel topography and nanoclustering might be under the control of second messenger cascades.
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SK Channel distribution on neuronal axons revealed by single molecule atomic force microscopy
Biophysical Journal, 2015Co-Authors: Krithika Abiraman, Anastasios V Tzingounis, George LykotrafitisAbstract:Introduction: The axon initial segment (AIS) is a specialized cellular compartment where action potentials (APs) initiate. Calcium-activated small conductance potassium (SK) Channels reduce excitability by mediating the afterhyperpolarizing (AHP) current and thus modulate the intervals between spikes during a burst of action potentials. SK Channels have a polarized distribution in neurons with high density in dendrites and lower density in the soma. However, it is not clear if and which SK Channels are expressed on the axon and their spatial distribution. Here, we employ single molecule atomic force microscopy (AFM) combined with natural toxins to determine the localization of SK Channels on neuronal axons.Methods and Results: We have previously demonstrated that integration of single molecule AFM and toxin pharmacology allows for high resolution mapping of native SK2 Channels on soma and dendrites of living neurons. AFM tip functionalized with SK Channel blocker-apamin, was used to detect the distribution of SK Channels by measuring the unbinding forces between apamin and SK Channels. The axon was identified by transfecting rat pyramidal neurons with tau-gfp which localizes at the axon. Apamin-functionalized AFM tips were used to probe 1µm∧2 scan area on the axon of transfected pyramidal neurons. We detected unbinding forces with a mean of µ=20± 9pN and a surface density of 5.9% (n=8). To determine the specificity of the detected unbinding forces, the experiments were repeated with neurons pretreated with apamin. Along with a lower frequency of unbinding events, we found that in neurons pretreated with apamin, there was a decrease in the unbinding forces. The results above indicate that apamin sensitive SK Channels reside on the axon of pyramidal neurons.Conclusion: Employing single molecule AFM, we have revealed that SK Channels reside on axonal surfaces of neurons.
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regulation of SK Channel spatial distribution by tonic pka
Biophysical Journal, 2014Co-Authors: Krithika Abiraman, Anastasios V Tzingounis, George LykotrafitisAbstract:Introduction: Small-conductance Ca2+-activated K+ (SK) Channels mediate a ubiquitous expressed potassium conductance in the brain involved in synaptic plasticity and learning and memory. SK Channel spatial distribution shows a polarized topographic expression being highly enriched in neuronal dendrites with limited expression on somatic membranes. However, the mechanism that controls this spatial distribution is unknown. To investigate the mechanisms that control the spatial distribution of SK Channels at the single molecule level we combine single molecule atomic force microscopy and toxin pharmacology.Methods and results: AFM tip functionalized with apamin, a SK Channel blocker, is used to detect the distribution of SK Channels in living neurons by measuring the binding forces between apamin and SK Channels. Employing the above technique, we test the effect of PKA on the clustering and dendritic localization of SK Channels. Here, we show that SK2 Channel nanoclustering is under the control of cAMP and PKA activity and demonstrate that SK2 Channel polarized distribution in neurons is dictated by basal PKA activity.Conclusion: Our work reveals a new level of regulation of SK2 Channels by PKA and also demonstrates for the first time that SK2 spatial distribution is plastic.
Anastasios V Tzingounis - One of the best experts on this subject based on the ideXlab platform.
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tonic pka activity regulates SK Channel nanoclustering and somatodendritic distribution
Journal of Molecular Biology, 2016Co-Authors: Krithika Abiraman, Randall S Walikonis, George Lykotrafitis, Anastasios V TzingounisAbstract:Small-conductance calcium-activated potassium (SK) Channels mediate a potassium conductance in the brain and are involved in synaptic plasticity, learning, and memory. SK Channels show a distinct subcellular localization that is crucial for their neuronal functions. However, the mechanisms that control this spatial distribution are unknown. We imaged SK Channels labeled with fluorophore-tagged apamin and monitored SK Channel nanoclustering at the single molecule level by combining atomic force microscopy and toxin (i.e., apamin) pharmacology. Using these two complementary approaches, we found that native SK Channel distribution in pyramidal neurons, across the somatodendritic domain, depends on ongoing cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) levels, strongly limiting SK Channel expression at the pyramidal neuron soma. Furthermore, tonic cAMP-PKA levels also controlled whether SK Channels were expressed in nanodomains as single entities or as a group of multiple Channels. Our study reveals a new level of regulation of SK Channels by cAMP-PKA and suggests that ion Channel topography and nanoclustering might be under the control of second messenger cascades.
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SK Channel distribution on neuronal axons revealed by single molecule atomic force microscopy
Biophysical Journal, 2015Co-Authors: Krithika Abiraman, Anastasios V Tzingounis, George LykotrafitisAbstract:Introduction: The axon initial segment (AIS) is a specialized cellular compartment where action potentials (APs) initiate. Calcium-activated small conductance potassium (SK) Channels reduce excitability by mediating the afterhyperpolarizing (AHP) current and thus modulate the intervals between spikes during a burst of action potentials. SK Channels have a polarized distribution in neurons with high density in dendrites and lower density in the soma. However, it is not clear if and which SK Channels are expressed on the axon and their spatial distribution. Here, we employ single molecule atomic force microscopy (AFM) combined with natural toxins to determine the localization of SK Channels on neuronal axons.Methods and Results: We have previously demonstrated that integration of single molecule AFM and toxin pharmacology allows for high resolution mapping of native SK2 Channels on soma and dendrites of living neurons. AFM tip functionalized with SK Channel blocker-apamin, was used to detect the distribution of SK Channels by measuring the unbinding forces between apamin and SK Channels. The axon was identified by transfecting rat pyramidal neurons with tau-gfp which localizes at the axon. Apamin-functionalized AFM tips were used to probe 1µm∧2 scan area on the axon of transfected pyramidal neurons. We detected unbinding forces with a mean of µ=20± 9pN and a surface density of 5.9% (n=8). To determine the specificity of the detected unbinding forces, the experiments were repeated with neurons pretreated with apamin. Along with a lower frequency of unbinding events, we found that in neurons pretreated with apamin, there was a decrease in the unbinding forces. The results above indicate that apamin sensitive SK Channels reside on the axon of pyramidal neurons.Conclusion: Employing single molecule AFM, we have revealed that SK Channels reside on axonal surfaces of neurons.
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regulation of SK Channel spatial distribution by tonic pka
Biophysical Journal, 2014Co-Authors: Krithika Abiraman, Anastasios V Tzingounis, George LykotrafitisAbstract:Introduction: Small-conductance Ca2+-activated K+ (SK) Channels mediate a ubiquitous expressed potassium conductance in the brain involved in synaptic plasticity and learning and memory. SK Channel spatial distribution shows a polarized topographic expression being highly enriched in neuronal dendrites with limited expression on somatic membranes. However, the mechanism that controls this spatial distribution is unknown. To investigate the mechanisms that control the spatial distribution of SK Channels at the single molecule level we combine single molecule atomic force microscopy and toxin pharmacology.Methods and results: AFM tip functionalized with apamin, a SK Channel blocker, is used to detect the distribution of SK Channels in living neurons by measuring the binding forces between apamin and SK Channels. Employing the above technique, we test the effect of PKA on the clustering and dendritic localization of SK Channels. Here, we show that SK2 Channel nanoclustering is under the control of cAMP and PKA activity and demonstrate that SK2 Channel polarized distribution in neurons is dictated by basal PKA activity.Conclusion: Our work reveals a new level of regulation of SK2 Channels by PKA and also demonstrates for the first time that SK2 spatial distribution is plastic.
Krithika Abiraman - One of the best experts on this subject based on the ideXlab platform.
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tonic pka activity regulates SK Channel nanoclustering and somatodendritic distribution
Journal of Molecular Biology, 2016Co-Authors: Krithika Abiraman, Randall S Walikonis, George Lykotrafitis, Anastasios V TzingounisAbstract:Small-conductance calcium-activated potassium (SK) Channels mediate a potassium conductance in the brain and are involved in synaptic plasticity, learning, and memory. SK Channels show a distinct subcellular localization that is crucial for their neuronal functions. However, the mechanisms that control this spatial distribution are unknown. We imaged SK Channels labeled with fluorophore-tagged apamin and monitored SK Channel nanoclustering at the single molecule level by combining atomic force microscopy and toxin (i.e., apamin) pharmacology. Using these two complementary approaches, we found that native SK Channel distribution in pyramidal neurons, across the somatodendritic domain, depends on ongoing cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) levels, strongly limiting SK Channel expression at the pyramidal neuron soma. Furthermore, tonic cAMP-PKA levels also controlled whether SK Channels were expressed in nanodomains as single entities or as a group of multiple Channels. Our study reveals a new level of regulation of SK Channels by cAMP-PKA and suggests that ion Channel topography and nanoclustering might be under the control of second messenger cascades.
-
SK Channel distribution on neuronal axons revealed by single molecule atomic force microscopy
Biophysical Journal, 2015Co-Authors: Krithika Abiraman, Anastasios V Tzingounis, George LykotrafitisAbstract:Introduction: The axon initial segment (AIS) is a specialized cellular compartment where action potentials (APs) initiate. Calcium-activated small conductance potassium (SK) Channels reduce excitability by mediating the afterhyperpolarizing (AHP) current and thus modulate the intervals between spikes during a burst of action potentials. SK Channels have a polarized distribution in neurons with high density in dendrites and lower density in the soma. However, it is not clear if and which SK Channels are expressed on the axon and their spatial distribution. Here, we employ single molecule atomic force microscopy (AFM) combined with natural toxins to determine the localization of SK Channels on neuronal axons.Methods and Results: We have previously demonstrated that integration of single molecule AFM and toxin pharmacology allows for high resolution mapping of native SK2 Channels on soma and dendrites of living neurons. AFM tip functionalized with SK Channel blocker-apamin, was used to detect the distribution of SK Channels by measuring the unbinding forces between apamin and SK Channels. The axon was identified by transfecting rat pyramidal neurons with tau-gfp which localizes at the axon. Apamin-functionalized AFM tips were used to probe 1µm∧2 scan area on the axon of transfected pyramidal neurons. We detected unbinding forces with a mean of µ=20± 9pN and a surface density of 5.9% (n=8). To determine the specificity of the detected unbinding forces, the experiments were repeated with neurons pretreated with apamin. Along with a lower frequency of unbinding events, we found that in neurons pretreated with apamin, there was a decrease in the unbinding forces. The results above indicate that apamin sensitive SK Channels reside on the axon of pyramidal neurons.Conclusion: Employing single molecule AFM, we have revealed that SK Channels reside on axonal surfaces of neurons.
-
regulation of SK Channel spatial distribution by tonic pka
Biophysical Journal, 2014Co-Authors: Krithika Abiraman, Anastasios V Tzingounis, George LykotrafitisAbstract:Introduction: Small-conductance Ca2+-activated K+ (SK) Channels mediate a ubiquitous expressed potassium conductance in the brain involved in synaptic plasticity and learning and memory. SK Channel spatial distribution shows a polarized topographic expression being highly enriched in neuronal dendrites with limited expression on somatic membranes. However, the mechanism that controls this spatial distribution is unknown. To investigate the mechanisms that control the spatial distribution of SK Channels at the single molecule level we combine single molecule atomic force microscopy and toxin pharmacology.Methods and results: AFM tip functionalized with apamin, a SK Channel blocker, is used to detect the distribution of SK Channels in living neurons by measuring the binding forces between apamin and SK Channels. Employing the above technique, we test the effect of PKA on the clustering and dendritic localization of SK Channels. Here, we show that SK2 Channel nanoclustering is under the control of cAMP and PKA activity and demonstrate that SK2 Channel polarized distribution in neurons is dictated by basal PKA activity.Conclusion: Our work reveals a new level of regulation of SK2 Channels by PKA and also demonstrates for the first time that SK2 spatial distribution is plastic.