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Anatrace社 Glyco-Diosgenin and Ion Channels: A Great Match!

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Glyco-Diosgenin and Ion Channels:

 A Great Match

 

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The 2021 Nobel Prize in Physiology or Medicine awarded to Drs. Julius and Patapoutian “for their discoveries of receptors for temperature and touch” highlighted the recent successes in ion channel research. These important drug targets are present in the membranes of all cell types and are famously difficult (or even impossible) to crystallize. Add to that the ongoing ‘resolution revolution’ in cryo-EM and we have a winning case for this protein class!

 

Indeed, the PDB database has contained a consistently high number of ion channel structures over the years, with 738 in 2019 and 744 in 2020. Efforts to combat COVID-19 slowed down the research, reducing the count to 614 in 2021, but current trending makes us expect 2020’s record will be easily surpassed in 2022!

 

TRPV3.jpgThe Anatrace surfactant glyco-diosgenin (GDN), a drop-in substitute for the plant-derived digitonin, has been a helpful tool for stabilizing complex and fragile ion channels in this research. GDN is widely used for solubilization, purification, and stabilization of membrane proteins, and has significant advantages over digitonin: no batch-to-batch variability, none of the harmful toxic byproducts commonly found in digitonin, and a lower cost!

 

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We are excited to share a list of recent, high-impact publications we’ve read that make great use of the GDN/cryo-EM combo in tackling ion channels:

 

  • TRPV3 is a cation-permeable TRP channel found in skin keratinocytes: it is responsible for cutaneous sensation and is linked to several skin diseases. This study describes TRPV3 inhibition using a local anesthetic dyclonine, which is an ingredient in some anti-itch ointments. The protein was purified using 0.01% (w/v) GDN.1
  • TMEM16A is a calcium-activated chloride channel linked to diseases such as hypertension, asthma, and cystic fibrosis. This study describes a cryo-EM structure of TMEM16A in complex with the channel blocker 1PBC and provides a detailed functional analysis of its inhibition mechanism. The channel was solubilized and purified in the presence of GDN and cryo-EM data were collected in 0.01% GDN. 2
  • KV4 is a voltage-gated potassium channel expressed in the brain and heart: it is involved in neuronal and muscular excitability regulation, as well as orchestrating firing rhythms. This study investigates the details of gating, activation, and inactivation of the human KV2 channel expressed in mammalian HEK293 cells. The complex with 2 auxiliary subunits was solubilized in LMNG with CHS, purified in DDM with CHS, and stabilized in 0.005% GDN. 3
  • NaV are voltage-gated sodium channels that initiate action potentials: proper inactivation is crucial to avoid hyperexcitability. This study describes NaVEh from the coccolithophore Emiliania huxleyi that has a molecular gating mechanism different from that of other NaV. The protein was solubilized in DDM with CHS and subsequently exchanged into GDN; the final purification buffer contained 0.007% GDN. 4
  • NALCN is a channel that mediates voltage-modulated sodium leak currents and is important for maintaining proper resting membrane potential. Mutations in the channel are linked to disorders such as hypotonia, congenital contractures, and developmental delay. Here, we present the cryo-EM structure of the mammalian NALCNFAM155A-UNC79-UNC80 quaternary complex. The protein was solubilized, purified, and stabilized in GDN (final concentration 0.006% w/v). 5

 

1 Neuberger, A., Nadezhdin, K. D. & Sobolevsky, A. I. Structural mechanism of TRPV3 channel inhibition by the anesthetic dyclonine. Nat Commun 13, 2795 (2022).

2 Lam, A. K. M., Rutz, S. & Dutzler, R. Inhibition mechanism of the chloride channel TMEM16A by the pore blocker 1PBC. Nat Commun 13, 2798 (2022).

3 Ye, W. et al. Activation and closed-state inactivation mechanisms of the human voltage-gated KV4 channel complexes. Mol Cell (2022) doi:10.1016/j.molcel.2022.04.032.

4 Zhang, J. et al. N-type fast inactivation of a eukaryotic voltage-gated sodium channel. Nat Commun 13, 2713 (2022).

5 Kang, Y. & Chen, L. Structure and mechanism of NALCN-FAM155A-UNC79-UNC80 channel complex. Nat Commun 13, 2639 (2022).

  

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