Direct Evidence for a Similar Molecular Mechanism Underlying Shaker Kv Channel Fast Inactivation and Clustering

Lewin, Limor; Nirenberg, Valerie Abigail; Yehezkel, Rinat; Naim, Shany; Orr, Irit; Yifrach, Ofer

doi:10.1016/j.jmb.2018.12.002

Cited by 4 publications

(30 citation statements)

References 33 publications

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“…To test this assertion, we used a series of Kv channel variants presenting different C-terminal 'chain' lengths, ranging from 9 to 144 amino acids, all presenting the same terminal PDZ-binding 'ball' motif, and examined their effects on PSD-95-mediated clustering metrics under identical conditions. These 'chain' length variants were generated by artificially shortening the C-terminal 'chain' of the B variant by 40, 83, 120 or 135 amino acids 17 , or by replacing the original native clustering (C) 'chains' with short (S) or long (L) native versions of the N-terminal inactivation (I) 'chain' , known to be entropic random 'chains' and previously shown as being able to replace the original native C-terminal chains with respect to PSD-95 binding and PSD-95-mediated channel expression and clustering 20 . Together with the native A and B channel variants, the channel series thus presented C-terminal 'chain' lengths of 9, 24, 39, 50, 61, 88, 104, 106 and 144 amino acids.…”

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confidence: 99%

Molecular and cellular correlates in Kv channel clustering: entropy-based regulation of cluster ion channel density

Lewin

Nsasra

Golbary

et al. 2020

Sci Rep

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Scaffold protein-mediated ion channel clustering at unique membrane sites is important for electrical signaling. Yet, the mechanism(s) by which scaffold protein-ion channel interactions lead to channel clustering or how cluster ion channel density is regulated is mostly not known. the voltage-activated potassium channel (Kv) represents an excellent model to address these questions as the mechanism underlying its interaction with the post-synaptic density 95 (PSD-95) scaffold protein is known to be controlled by the length of the extended 'ball and chain' sequence comprising the C-terminal channel region. Here, using sub-diffraction high-resolution imaging microscopy, we show that Kv channel 'chain' length regulates Kv channel density with a 'bell'-shaped dependence, reflecting a balance between thermodynamic considerations controlling 'chain' recruitment by PSD-95 and steric hindrance due to the spatial proximity of multiple channel molecules. our results thus reveal an entropy-based mode of channel cluster density regulation that mirrors the entropy-based regulation of the Kv channel-PSD-95 interaction. The implications of these findings for electrical signaling are discussed. Action potential generation, propagation and the evoked synaptic potential all rely on precisely timed events associated with activation and inactivation gating transitions of voltage-dependent Na + and K + channels, clustered in multiple copies at unique membrane sites, such as the initial segment of an axon, nodes of Ranvier, pre-synaptic terminals or at the post-synaptic density (PSD) 1-3. Changes in either ionic current shape or density, reflecting changes in temporal and spatial dimensions, respectively, affect action potential shape and frequency and may lead a neuron to change its mode of firing 4-9. Despite emerging evidence attesting to the importance of ion channel density for efficient electrical signaling 7 and information encoding 7-9 , little is currently known of the clustering process itself or its regulation. It is, however, clear that ion channel clustering is an active process, involving the interaction of a channel with a specific member of one of several scaffold protein families. For example, Nav channel clustering at nodes of Ranvier is mediated by interaction of the channel with the ankyrin G scaffold protein 10,11. Kv channel clustering at the PSD of excitatory synapses (of Drosophila melanogaster but not of mammalians), on the other hand, is mediated by binding to the PSD-95 synapse-associated scaffold protein 12-15. Yet, the mechanism by which these elementary binding events lead to the clustering of ion channel molecules in a restricted area of the membrane remains unclear. Furthermore, it is also generally not known if and how ion channel membrane density is regulated in the spatial and/or temporal dimensions. In the absence of a molecular mechanism describing the channel protein-scaffold protein interaction, bridging this molecularcellular gap to understand ion channel clustering has proven challenging. The pr...

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confidence: 99%

Molecular and cellular correlates in Kv channel clustering: entropy-based regulation of cluster ion channel density

Lewin

Nsasra

Golbary

et al. 2020

Sci Rep

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“…The same variants further exhibited differences in cluster area size, with the short "chain" variant presenting clusters covering larger areas ( Figure 3A) (Zandany et al, 2015a). Moreover, confocal imaging analyses of embryonic Drosophila Schneider cells transfected to express PSD-95, together with a series of Kv channel native and artificial "chain"-length variants, revealed "chain length"-dependence of Kv channel cluster area size (Lewin et al, 2019). Specifically, the shorter the Kv channel C-terminal "chain", the larger was the cluster area size observed ( Figure 3B) (Lewin et al, 2019).…”

Section: Bridging the Molecular-cellular Gap In Understanding Kv Chanmentioning

confidence: 90%

“…Direct support for the analogy between the fast inactivation and channel binding to PSD-95 "ball and chain mechanisms" was recently obtained using a "chain"-level chimeric channel approach, where it was shown that different alternatively spliced and intrinsically-disordered N-and C-terminal "chain" variants were able to replace one another in the corresponding fast inactivation or PSD-95 binding processes (Lewin et al, 2019). Furthermore, the swapped "chains" affected the relevant fast inactivation or PSD-95 binding processes in a length-dependent manner, as predicted by the random flight "chain" theory underlying the "ball and chain" mechanism (Lewin et al, 2019).…”

Section: Bridging the Molecular-cellular Gap In Understanding Kv Chanmentioning

confidence: 98%

“…Moreover, confocal imaging analyses of embryonic Drosophila Schneider cells transfected to express PSD-95, together with a series of Kv channel native and artificial "chain"-length variants, revealed "chain length"-dependence of Kv channel cluster area size (Lewin et al, 2019). Specifically, the shorter the Kv channel C-terminal "chain", the larger was the cluster area size observed ( Figure 3B) (Lewin et al, 2019). Furthermore, the cluster area size of any Kv channel-PSD-95 protein pair was found to linearly correlate with the observed affinity between the two proteins ( Figure 3C).…”

Section: Bridging the Molecular-cellular Gap In Understanding Kv Chanmentioning

confidence: 99%

“…In the past, ion channel clustering was usually studied using conventional confocal imaging light microscopy that allows for resolutions up to 250 nm in the lateral direction. At such low resolution, quantitative assessment of channel clustering yields only limited basic information on surface expression and megacluster area size (Zandany et al, 2015a;Lewin et al, 2019). Recent advances in imaging techniques, in particular, in superresolution imaging methodologies, offer improved spatial resolution reaching 30 nm, a value that may enable the further studying of clustering at the single-molecule level (Sieben et al, 2018;Schermelleh et al, 2019).…”

Section: Future Challenges and Directionsmentioning

confidence: 99%

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Bridging the Molecular-Cellular Gap in Understanding Ion Channel Clustering

Nirenberg

Yifrach

2020

Front. Pharmacol.

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The clustering of many voltage-dependent ion channel molecules at unique neuronal membrane sites such as axon initial segments, nodes of Ranvier, or the post-synaptic density, is an active process mediated by the interaction of ion channels with scaffold proteins and is of immense importance for electrical signaling. Growing evidence indicates that the density of ion channels at such membrane sites may affect action potential conduction properties and synaptic transmission. However, despite the emerging importance of ion channel density for electrical signaling, how ion channel-scaffold protein molecular interactions lead to cellular ion channel clustering, and how this process is regulated are largely unknown. In this review, we emphasize that voltagedependent ion channel density at native clustering sites not only affects the density of ionic current fluxes but may also affect the conduction properties of the channel and/or the physical properties of the membrane at such locations, all changes that are expected to affect action potential conduction properties. Using the concrete example of the prototypical Shaker voltage-activated potassium channel (Kv) protein, we demonstrate how insight into the regulation of cellular ion channel clustering can be obtained when the molecular mechanism of ion channel-scaffold protein interaction is known. Our review emphasizes that such mechanistic knowledge is essential, and when combined with super-resolution imaging microscopy, can serve to bridge the molecular-cellular gap in understanding the regulation of ion channel clustering. Pressing questions, challenges and future directions in addressing ion channel clustering and its regulation are discussed.

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Regulating Shaker Kv channel clustering by hetero-oligomerization

Nsasra

Peretz²,

Orr³

et al. 2023

Front. Mol. Biosci.

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Scaffold protein-mediated voltage-dependent ion channel clustering at unique membrane sites, such as nodes of Ranvier or the post-synaptic density plays an important role in determining action potential properties and information coding. Yet, the mechanism(s) by which scaffold protein-ion channel interactions lead to channel clustering and how cluster ion channel density is regulated are mostly unknown. This molecular-cellular gap in understanding channel clustering can be bridged in the case of the prototypical Shaker voltage-activated potassium channel (Kv), as the mechanism underlying the interaction of this channel with its PSD-95 scaffold protein partner is known. According to this mechanism, changes in the length of the intrinsically disordered channel C-terminal chain, brought about by alternative splicing to yield the short A and long B chain subunit variants, dictate affinity to PSD-95 and further controls cluster homo-tetrameric Kv channel density. These results raise the hypothesis that heteromeric subunit assembly serves as a means to regulate Kv channel clustering. Since both clustering variants are expressed in similar fly tissues, it is reasonable to assume that hetero-tetrameric channels carrying different numbers of high- (A) and low-affinity (B) subunits could assemble, thereby giving rise to distinct cluster Kv channel densities. Here, we tested this hypothesis using high-resolution microscopy, combined with quantitative clustering analysis. Our results reveal that the A and B clustering variants can indeed assemble to form heteromeric channels and that controlling the number of the high-affinity A subunits within the hetero-oligomer modulates cluster Kv channel density. The implications of these findings for electrical signaling are discussed.

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Direct Evidence for a Similar Molecular Mechanism Underlying Shaker Kv Channel Fast Inactivation and Clustering

Cited by 4 publications

References 33 publications

Molecular and cellular correlates in Kv channel clustering: entropy-based regulation of cluster ion channel density

Molecular and cellular correlates in Kv channel clustering: entropy-based regulation of cluster ion channel density

Bridging the Molecular-Cellular Gap in Understanding Ion Channel Clustering

Regulating Shaker Kv channel clustering by hetero-oligomerization

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