| 1. |
- Choudhury, Koushik, et al.
(författare)
-
An alpha-pi transition in S6 shapes the conformational cycle of the bacterial sodium channel NavAb
- 2022
-
Ingår i: The Journal of General Physiology. - : Rockefeller University Press. - 0022-1295 .- 1540-7748. ; 155:2
-
Tidskriftsartikel (refereegranskat)abstract
- Voltage-gated sodium channels play an important role in electrical signaling in excitable cells. In response to changes in membrane potential, they cycle between nonconducting and conducting conformations. With recent advances in structural biology, structures of sodium channels have been captured in several distinct conformations, which are thought to represent different functional states. However, it has been difficult to capture the intrinsically transient open state. We recently showed that a proposed open state of the bacterial sodium channel NavMs was not conductive and that a conformational change involving a transition to a pi-helix in the pore-lining S6 helix converted this structure into a conducting state. However, the relevance of this structural feature in other sodium channels, and its implications for the broader gating cycle, remained unclear. Here, we propose a comparable open state of another class of bacterial channel from Aliarcobacter butzleri (NavAb) with characteristic pore hydration, ion permeation, and drug binding properties. Furthermore, we show that a pi-helix transition can lead to pore opening and that such a conformational change blocks fenestrations in the inner helix bundle. We also discover that a region in the C-terminal domain can undergo a disordering transition proposed to be important for pore opening. These results support a role for a pi-helix transition in the opening of NavAb, enabling new proposals for the structural annotation and drug modulation mechanisms in this important sodium channel model. We propose a new conformational cycle for NavAb wherein an alpha- to pi-helix transition in S6 and disordering of the neck region of the C-terminal domain is important for pore opening.
|
|
| 2. |
- Choudhury, Koushik, et al.
(författare)
-
An open state of a voltage-gated sodium channel involving a p-helix and conserved pore-facing asparagine
- 2022
-
Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 121:1, s. 11-22
-
Tidskriftsartikel (refereegranskat)abstract
- Voltage-gated sodium (Nav) channels play critical roles in propagating action potentials and otherwise manipulating ionic gradients in excitable cells. These channels open in response to membrane depolarization, selectively permeating sodium ions until rapidly inactivating. Structural characterization of the gating cycle in this channel family has proved challenging, particularly due to the transient nature of the open state. A structure from the bacterium Magnetococcus marinus Nav (NavMs) was initially proposed to be open, based on its pore diameter and voltage-sensor conformation. However, the functional annotation of this model, and the structural details of the open state, remain disputed. In this work, we used molecular modeling and simulations to test possible open-state models of NavMs. The full-length experimental structure, termed here the cc-model, was consistently dehydrated at the activation gate, indicating an inability to conduct ions. Based on a spontaneous transition observed in extended simulations, and sequence/structure comparison to other Nav channels, we built an alternative p-model featuring a helix transition and the rotation of a conserved asparagine residue into the activation gate. Pore hydration, ion permeation, and state-dependent drug binding in this model were consistent with an open functional state. This work thus offers both a functional annotation of the full-length NavMs structure and a detailed model for a stable Nav open state, with potential conservation in diverse ion-channel families.
|
|
| 3. |
- Choudhury, Koushik
(författare)
-
Gating and modulation mechanism of voltage gated sodium channels
- 2023
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Voltage-gated sodium channels (Nav channels) play an essential role in nerve impulse conduction in excitable cells. Thus, these channels are involved in several neurological and muscular disorders. Understanding their mechanism of functioning is essential for designing drugs targeting them. These are tetrameric membrane proteins that selectively transport sodium ions across the membrane. They regulate ion flow by cycling through three main functional states - resting state, open state, and inactivated state. Structural biology techniques have captured Nav channels in several functional states. However, most of the structures are captured in the inactivated state. Although it is quite challenging to capture the open state experimentally because of its transient nature, several structures of bacterial and eukaryotic Nav channels have been captured in the putative open state. However, a rigorous functional annotation of these open-state structures awaits. I performed molecular dynamics simulations to show that the experimental bacterial Nav channels captured in the putative open state, the pore was dehydrated and had a high free energy barrier for ion/drug permeation suggesting that these structures do not correspond to a functional open state. The pore-lining helices of these channels are ? helical. Sequence/structure conservation analysis showed the possibility of ?-helices in the pore-lining helices. Introducing ?-helices in the middle of these pore-lining helices hydrated the pore and removed the free energy barrier for ion/drug permeation. The ?-helices might also be relevant for pore opening as they dehydrate the peripheral cavities/reduce the interactions between the hydrophobic pore-lining residues and hence allow the opening of the hydrophobic pore. Additionally, I also determined a disordered region in the C-terminal domain which is known to be relevant to pore opening.I also studied the effect of ?-helices on drug access and binding to sodium channels. I found that ?-helices in the bacterial Nav channel blocked the fenestrations irrespective of the pore diameter thus inhibiting drug access through the fenestrations. Exploring further on drug binding, I investigated lidocaine binding to different functional states which revealed that the drug binds in different orientations and positions across the functional states. This implies that there might be a change in the lidocaine-binding affinity as the channel cycles through different functional states. I also investigated the drug binding site and access pathway of cannabidiol in sodium channels and the effect of cannabidiol on membrane properties. Our computational results were complemented by experimental results. Molecular dynamics simulations suggest that cannabidiol does not affect the membrane rigidity and causes an ordering of the membrane methylenes, which is in excellent agreement with the NMR results. Mutagenesis experiments show that cannabidiol blocks the pore by interacting with a phenylalanine residue which is in good agreement with our docking results. Adiabatic biased molecular dynamics simulations were performed to confirm the pathway for CBD to reach the pore is through the fenestrations in the ion channel. The idea of investigating the relevance of ?-helices in pore-lining helices was extended to eukaryotic Nav channels as well. Eukaryotic channels are heterotetrameric, so the pore lining helices of different subunits might contribute differently to the channel function. I concluded that increasing the number of ?-helices not only increased the pore hydration and ion conductance but also reduced the barrier for ion permeation. ?-helices in pore-lining helices of subunit-I and subunit-IV in an expanded pore are essential for a functional open state.Putting the above results together, I show that the bacterial experimental structures initially proposed to represent open states might correspond instead to inactivated states. In eukaryotes, the experimental structure initially proposed to represent the open state corresponds to a sub-conductance open state. Thus, I propose that a ? to ? helix transition and vice-versa might be relevant to the gating of Nav channels. By showing these results I would like to highlight the importance of rigorously annotating experimental structures and assigning their functional states. Finally, I would also like to highlight the power of molecular dynamics simulations to not only rigorously annotate experimental structures but also to provide atomistic details to explain experimental results.
|
|
| 4. |
- Choudhury, Koushik, et al.
(författare)
-
Modulation of pore opening of Eukaryotic sodium channels by π-helices in S6
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Voltage-gated sodium channels are heterotetrameric sodium selective ion channels that play a central role in electrical signaling in excitable cells. With recent advances in structural biology, structures of eukaryotic sodium channels have been captured in several distinct conformations corresponding to different functional states. The secondary structure of the pore lining S6 helices of subunit DI, DII, and DIV has been captured with both short π-helix stretches and in fully α-helical conformations. The relevance of these secondary structure elements for pore gating is not yet understood. Here, we propose that a π helix in at least DI-S6, DIII-S6, and DIV-S6 results in a fully conductive state. On the other hand, the absence of π-helix in either DI-S6 or DIV-S6 yields a sub-conductance state, and its absence from both DI-S6 and DIV-S6 yields a non-conducting state. This work highlights the impact of the presence of a π-helix in the different S6 helices of an expanded pore on pore conductance, thus opening new doors towards reconstructing the entire conformational landscape along the functional cycle of Nav Channels and paving the way to the design of state-dependent modulators.
|
|
| 5. |
- Choudhury, Koushik, et al.
(författare)
-
Modulation of Pore Opening of Eukaryotic Sodium Channels by π-Helices in S6
- 2023
-
Ingår i: The Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185. ; 14:25, s. 5876-5881
-
Tidskriftsartikel (refereegranskat)abstract
- Voltage-gated sodium channels are heterotetrameric sodiumselectiveion channels that play a central role in electrical signaling in excitablecells. With recent advances in structural biology, structures of eukaryoticsodium channels have been captured in several distinct conformationscorresponding to different functional states. The secondary structureof the pore lining S6 helices of subunits DI, DII, and DIV has beencaptured with both short & pi;-helix stretches and in fully & alpha;-helicalconformations. The relevance of these secondary structure elementsfor pore gating is not yet understood. Here, we propose that a & pi;-helixin at least DI-S6, DIII-S6, and DIV-S6 results in a fully conductivestate. On the other hand, the absence of & pi;-helix in either DI-S6or DIV-S6 yields a subconductance state, and its absence from bothDI-S6 and DIV-S6 yields a nonconducting state. This work highlightsthe impact of the presence of a & pi;-helix in the different S6helices of an expanded pore on pore conductance, thus opening newdoors toward reconstructing the entire conformational landscape alongthe functional cycle of Nav Channels and paving the way to the design of state-dependent modulators.
|
|
| 6. |
|
|
| 7. |
|
|
| 8. |
- Frampton, Damon, et al.
(författare)
-
Subtype-specific responses of hKv7.4 and hKv7.5 channels to polyunsaturated fatty acids reveal an unconventional modulatory site and mechanism
- 2022
-
Ingår i: eLIFE. - : eLife Sciences Publications, Ltd. - 2050-084X. ; 11
-
Tidskriftsartikel (refereegranskat)abstract
- The K(V)7.4 and K(V)7.5 subtypes of voltage -gated potassium channels play a role in important physiological processes such as sound amplification in the cochlea and adjusting vascular smooth muscle tone. Therefore, the mechanisms that regulate K(V)7.4 and K(V)7.5 channel function are of interest. Here, we study the effect of polyunsaturated fatty acids (PUFAs) on human K(V)7.4 and KV7.5 channels expressed in Xenopus oocytes. We report that PUFAs facilitate activation of hK(V)7.5 by shifting the V50 of the conductance versus voltage (G(V)) curve toward more negative voltages. This response depends on the head group charge, as an uncharged PUFA analogue has no effect and a positively charged PUFA analogue induces positive V-50 shifts. In contrast, PUFAs inhibit activation of hK(V)7.4 by shifting V-50 toward more positive voltages. No effect on V-50 of hK(V)7.4 is observed by an uncharged or a positively charged PUFA analogue. Thus, the hK(V)7.5 channel's response to PUFAs is analogous to the one previously observed in hK(V)7.1-7.3 channels, whereas the hK(V)7.4 channel response is opposite, revealing subtype-specific responses to PUFAs. We identify a unique inner PUFA interaction site in the voltage-sensing domain of hKV7.4 underlying the PUFA response, revealing an unconventional mechanism of modulation of hK(V)7.4 by PUFAs.
|
|
| 9. |
- Galleano, Iacopo, et al.
(författare)
-
Functional cross-talk between phosphorylation and disease-causing mutations in the cardiac sodium channel Na(v)1.5
- 2021
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 118:33
-
Tidskriftsartikel (refereegranskat)abstract
- The voltage-gated sodium channel Nav1.5 initiates the cardiac action potential. Alterations of its activation and inactivation properties due to mutations can cause severe, life-threatening arrhythmias. Yet despite intensive research efforts, many functional aspects of this cardiac channel remain poorly understood. For instance, Nav1.5 undergoes extensive posttranslational modification in vivo, but the functional significance of these modifications is largely unexplored, especially under pathological conditions. This is because most conventional approaches are unable to insert metabolically stable posttranslational modification mimics, thus preventing a precise elucidation of the contribution by these modifications to channel function. Here, we overcome this limitation by using protein semisynthesis of Nav1.5 in live cells and carry out complementary molecular dynamics simulations. We introduce metabolically stable phosphorylation mimics on both wild-type (WT) and two pathogenic long-QT mutant channel backgrounds and decipher functional and pharmacological effects with unique precision. We elucidate the mechanism by which phosphorylation of Y1495 impairs steady-state inactivation in WT Nav1.5. Surprisingly, we find that while the Q1476R patient mutation does not affect inactivation on its own, it enhances the impairment of steady-state inactivation caused by phosphorylation of Y1495 through enhanced unbinding of the inactivation particle. We also show that both phosphorylation and patient mutations can impact Nav1.5 sensitivity toward the clinically used antiarrhythmic drugs quinidine and ranolazine, but not flecainide. The data highlight that functional effects of Nav1.5 phosphorylation can be dramatically amplified by patient mutations. Our work is thus likely to have implications for the interpretation of mutational phenotypes and the design of future drug regimens.
|
|
| 10. |
|
|
