Periodic Reporting for period 2 - CRYO-EM TRPV5 (Structure-function analysis of the calcium channel TRPV5)
Reporting period: 2018-09-01 to 2019-08-31
Ion channels are proteins composed of a hydrophillic pore that facilitate ion flow across a plasma membrane. This ionic permeability is controlled by a set of essential properties affecting the channel activation and inactivation in response to voltage, ligands, or intracellular second messengers. The focus of the present project proposal, the transient receptor potential vanilloid channel (TRPV5), forms a specific category within the large TRP familiy of ion channels as it comprises a unique high selectivity for calcium ions together with a calcium-dependent inactivation mechanism that is incompletely understood. Detailed analysis of the TRPV5 channel will provide new structural insights into channel gating that can be extrapolated to other TRP channels, as the current knowledge on the TRP protein structure and its impact on the regulation of the channel function was still limited.
The key objective of my project was to deliver the first detailed mechanistic view of TRPV5 by connecting Prof. Cheng’s expertise in structural biology with my biophysical background on TRP channel functioning. The following work packages were addressed:
1) Channel activation mechanism of TRPV5
Elucidation of the 3D structure of integral TRPV5 by single-particle cryo-EM will provide critical structural and mechanistic insight into calcium-dependent regulation of channel function.
2) Intramolecular regulation of TRPV5
Reconstitution of TRPV5 into lipid nanodiscs and liposomes allows detailed study on lipid regulation and the mechanism of channel inactivation
In this project, we delivered several high resolution structural maps of TRPV5: i) carboxy-terminally truncated TRPV5 in nanodisc, ii) full length TRPV5 in nanodisc, iii) TRPV5 mutant (W583A) in nanodisc, and iv) TRPV5-calmodulin complex in detergent. This provided critical structural and mechanistic insight into channel function and forms the basis to further investigate the calmodulin-dependent mechanism of channel inactivation by advanced fluorescence microscopy. Ultimately, this can be extrapolated to other ion channels to advance studies on channel-related diseases (channelopathies) and guide effective treatment.
The key objective of my project was to deliver the first detailed mechanistic view of TRPV5 by connecting Prof. Cheng’s expertise in structural biology with my biophysical background on TRP channel functioning. The following work packages were addressed:
1) Channel activation mechanism of TRPV5
Elucidation of the 3D structure of integral TRPV5 by single-particle cryo-EM will provide critical structural and mechanistic insight into calcium-dependent regulation of channel function.
2) Intramolecular regulation of TRPV5
Reconstitution of TRPV5 into lipid nanodiscs and liposomes allows detailed study on lipid regulation and the mechanism of channel inactivation
In this project, we delivered several high resolution structural maps of TRPV5: i) carboxy-terminally truncated TRPV5 in nanodisc, ii) full length TRPV5 in nanodisc, iii) TRPV5 mutant (W583A) in nanodisc, and iv) TRPV5-calmodulin complex in detergent. This provided critical structural and mechanistic insight into channel function and forms the basis to further investigate the calmodulin-dependent mechanism of channel inactivation by advanced fluorescence microscopy. Ultimately, this can be extrapolated to other ion channels to advance studies on channel-related diseases (channelopathies) and guide effective treatment.
Within physiology, TRPV5 and TRPV6 serve as apical entryways into epithelial cells that line parts of the gut and nephron, initiating transcellular calcium transport pathways that help fine-tune serum calcium levels.
To understand how the structure of the pore conveys the extraordinary calcium selectivity and the calcium-dependent channel inactivation, we set out to determine the structure of the epithelial calcium channel TRPV5, using cryo-electron microscopy. To this end, TRPV5 was purified from mammalian cells and reconstituted in lipid nanodiscs, small disc-like lipid bilayers surrounded by membrane scaffolding protein. Following cryo-EM analysis, we can now report the structures of a truncated version and full length rabbit TRPV5. In addition, we resolved the structure of TRPV5 in complex with its accessory protein calmodulin and provide a structural explanation for the calcium-dependent channel inactivation. And finally, we resolved the TRPV5 W583A mutant as an open structure giving insight into the channel gating mechanism.
Our work has been finalized by a publication in PNAS in 2019, which has led to media attention, several prizes, and follow-up grants to perform further studies on this topic. Moreover, the work was presented at European and national meetings, led to an invited review, and is also incorporated in teaching activities at the university.
To understand how the structure of the pore conveys the extraordinary calcium selectivity and the calcium-dependent channel inactivation, we set out to determine the structure of the epithelial calcium channel TRPV5, using cryo-electron microscopy. To this end, TRPV5 was purified from mammalian cells and reconstituted in lipid nanodiscs, small disc-like lipid bilayers surrounded by membrane scaffolding protein. Following cryo-EM analysis, we can now report the structures of a truncated version and full length rabbit TRPV5. In addition, we resolved the structure of TRPV5 in complex with its accessory protein calmodulin and provide a structural explanation for the calcium-dependent channel inactivation. And finally, we resolved the TRPV5 W583A mutant as an open structure giving insight into the channel gating mechanism.
Our work has been finalized by a publication in PNAS in 2019, which has led to media attention, several prizes, and follow-up grants to perform further studies on this topic. Moreover, the work was presented at European and national meetings, led to an invited review, and is also incorporated in teaching activities at the university.
The calcium-selective transient receptor potential (TRP) channel, TRPV5 and TRPV6, are essential for calcium homeostasis. Unlike the other TRPV channels, they do not exhibit thermosensitivity or a ligand-activated permeation mechanism. Instead, they are constitutively opened at physiological membrane potentials. Despite recent TRPV structures, structural details underscoring the distinction between the TRPV family members are limited. Here, we report the electron cryo-microscopy (cryo-EM) structures of nanodisc-reconstituted truncated and full-length TRPV5 in closed conformation, as well as a TRPV5 W583A mutant in open confirmation, with nominal resolutions of 2.85 - 3.0 Å.
Follow-up studies are focused at comparing TRPV5 with the other TRPV channel members and undertake studies towards unraveling their structure-function relationships as well as delineate important regulation mechanisms of these ion channels.
These high resolution structures of ion channels such as TRPV5 can have a number of potential future outcomes and impacts:
-Given increasing amount of evidence that links several TRP channels to pathologic conditions, a full understanding of how molecular components, like natural or non-natural substances and lipids, activate or inhibit the channel is necessary. A high-resolution structure can provide detailed insight into the actions of pharmacological compounds, to ultimately support drug development programs.
-Structure-function outline and established experimental setups will be applicable to other TRP channels and is important for unravelling functional diversities within the TRP family.
-Knowledge from high-resolution structures can translate into understanding the pathophysiology of various human diseases. How a mutation leads to impaired function can be studied in detail by structural analysis.
Taken together, the combined knowledge obtained in this project will support a full understanding of the role of the protein in a physiological and pathophysiological process, and should help in development of ion channel (and transporter) therapeutics.
Follow-up studies are focused at comparing TRPV5 with the other TRPV channel members and undertake studies towards unraveling their structure-function relationships as well as delineate important regulation mechanisms of these ion channels.
These high resolution structures of ion channels such as TRPV5 can have a number of potential future outcomes and impacts:
-Given increasing amount of evidence that links several TRP channels to pathologic conditions, a full understanding of how molecular components, like natural or non-natural substances and lipids, activate or inhibit the channel is necessary. A high-resolution structure can provide detailed insight into the actions of pharmacological compounds, to ultimately support drug development programs.
-Structure-function outline and established experimental setups will be applicable to other TRP channels and is important for unravelling functional diversities within the TRP family.
-Knowledge from high-resolution structures can translate into understanding the pathophysiology of various human diseases. How a mutation leads to impaired function can be studied in detail by structural analysis.
Taken together, the combined knowledge obtained in this project will support a full understanding of the role of the protein in a physiological and pathophysiological process, and should help in development of ion channel (and transporter) therapeutics.