The author | Liu Runan
The pain is very uncomfortable, it is an experience that almost everyone has experienced, and sometimes the pain is really "terrible".
Pain, which seems to occur in a momentary sensation, actually contains a series of signal transmissions. To block the pain sensation, we must first clarify its sensing pathway.
On July 7, the latest results of Princeton University Professor Yan Ning's team were published online in Nature. The study is the first to analyze the high-definition three-dimensional structure of the N-type voltage-gated calcium ion channel of human nerve tissue at 3.1 angstrom resolution, providing clues for understanding its function. The study also analyzed the structure of the 3.0 angstrom painkiller Ziconotide (Ziconotide) blocking the channel, providing ideas for the development of specific painkillers for this channel.
<h1 class="pgc-h-arrow-right" data-track="29" > cuts off the pathway and cannot perceive pain</h1>
When the tip of the needle is inserted into the human body, when the oil is splashed on the skin, the nerve endings throughout the body are the first to feel, it converts the stimulation signal into an electrical signal, which is transmitted to the central nervous system located in the spinal cord, which in turn converts the electrical signal into a chemical signal - a neurotransmitter, and finally transmits it to the brain.
In the process of conversion from electrical signal to chemical signal, Cav2.2, that is, the participation of N-type voltage gated calcium ion channels in neural tissues, is inseparable.
Voltage-gated ion channels are an important class of proteins that cells control ion transport, including the transport of calcium ions, potassium ions, sodium ions, etc. They play an important role in many physiological processes, such as gene expression, nerve signaling, muscle contraction, neurodegenerative diseases, heart disease, mental illness, etc. Ion channel proteins are currently the second largest drug therapeutic target after GPCRs (G protein-coupled receptors).
Calcium ion (Ca2+), as an important second messenger in the organism, is involved in life processes such as muscle contraction, nerve signaling, glandular secretion, gene transcription regulation, and apoptosis. The voltage-gated calcium ion channel (Cav) protein family includes 10 members, which can be divided into three subfamilys according to their sequence differences, namely Cav1.1-1.4, Cav2.1-2.3, and Cav3.1-3.3.
In recent years, Yan Ning's team has been working on analyzing the three-dimensional structure of different subtypes of Cav, and has successfully analyzed the complex structure of Cav1.1 and Cav3.1.
In the study, they focused on family member Cav2.2. When the cell membrane potential changes, Cav2.2 is immediately activated, opening the channel gate so that the messenger Ca2+ can smoothly enter the membrane from outside the cell membrane, thereby promoting the release of neurotransmitters and triggering a series of downstream signaling pathways. Therefore, if Cav2.2 is artificially cut off, nerve signals are difficult to conduct, and the brain cannot perceive pain.
<h1 class="pgc-h-arrow-right" data-track="28" > Cav2.2 resembles a jumping "horse."</h1>
It's not easy to cut off Cav2.2. It looks very similar to "family members" and can regulate Cav2.2 molecules, often other members, causing abnormal physiological responses. This has become a bottleneck in the development of related painkillers.
At present, there is only one painkiller on the market that relies on blocking Cav2.2 to play a role - ziconotide. Compared with opioid painkillers, it is not addictive, the effect does not deteriorate with the increase in dosage, it can relieve almost all pain, and it has a strong painkiller effect, which is 1000 times that of opioid painkillers.
However, the testonotide can only be administered by intrathecal injection, that is, the drug is injected directly into the subarachnoid cavity by lumbar puncture, so that the drug is dispersed in the cerebrospinal fluid. "Everyone hopes to use siconolide as a template to develop a more convenient, safer and more effective painkiller." The first author of the paper, Gao Shuai, a postdoctoral fellow in the Department of Molecular Biology at Princeton University, said.
"From a structural biology perspective, we wanted to know what the structure of Cav2.2 was, how siconolide was combined with it. Only by understanding its structure and mechanism can we design targeted drugs for the corresponding proteins. Yao Xia, co-first author of the paper and a postdoctoral fellow in Princeton University's Department of Molecular Biology, said.
Using the single-particle cryo-EM method, the researchers reconstructed the three-dimensional structure of the human Cav2.2 protein complex before and after binding to ziconolotide with a resolution of 3.0 angstroms.
The three-dimensional reconstruction diagram shows that the three subunits of Cav2.2 overlap each other, resembling a "horse" leaping up in the air, the "horse head and neck" composed of α2δ-1 extending outside the cell membrane, and the α1 constituting the "horse body and front hoof" across the entire transmembrane region, leaving the "hind legs" β3 pedaling inside the cell membrane as a support.
Gao Shuai introduced, "Zicophenolate is bound at the entrance of calcium ion transport. Relative to the pre-binding, the extramembrane membrane annular structure of α2δ-1 and α1 is synergistically displaced upwards, vacating the position and 'giving way' to ziconolotide to facilitate its binding to the protein. ”
<h1 class="pgc-h-arrow-right" data-track="20" provides ideas for the development of pain drugs ></h1>
After in-depth analytical calculations of its three-dimensional structure, the researchers resolved the interaction of β3 with the α1 subunit.
"We also found that unlike Cav1.1 and Cav3.1 in previous studies, Cav2.2 has two more structural fragments, CH1 and CH2." Yao Xia said that at the same time, Cav2.2's four voltage receptors (VSDs) did not rise at the same time, of which VSD2 showed a "down" conformational state, which has never been observed in the VSD conformation of other family members.
After further analysis, the researchers also found the phospholipid molecule PIP2 in its VSD2, which contributes to the stabilization of its "down" conformation.
"It was a pleasant surprise. At present, we cannot accurately determine what functional state the entire protein is in and why it is presented in this way, which is also a problem that needs to be studied in the future. She said.
Gao Shuai said, "Our structural analysis of Cav2.2, a representative member of the family, has laid a foundation for future research on the structure and mechanism of Cav channel regulation, and also provided ideas for the design and development of painkillers for Cav2.2. ”
Related thesis information: https://doi.org/10.1038/s41586-021-03699-6
Yan Ning re-issued Nature to crack the structural secret of "ziconotide" pain relief
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