AsKC11

AsKC11 (also known as U-actitoxin-Avd3n or U-AITX-Avd3n) is a toxin found in the venom of the sea anemone, Anemonia sulcata. This toxin is part of the Kunitz peptide family and has been shown to be an activator of G protein-coupled inwardly-rectifying potassium (GIRK) channels 1/2, involved in the regulation of cellular excitability. 

AsKC11
3D structure AsKC11[1]
Identifiers
OrganismAnemonia sulcata
SymbolN/A
UniProtP0DN15
Search for
StructuresSwiss-model
DomainsInterPro

Etymology

The name AsKC11 is an acronym for Anemonia sulcata kalicludine, the species it originates from and its predicted mechanism of action.[2]

Sources

AsKC11 is a peptide that can be purified from the venom of the Mediterranean snakelocks sea anemone, Anemonia sulcata.[3] A. sulcata is sometimes also referred to as Anemonia viridis. 

Chemistry

Structure

AsKC11 is characterized by a single polypeptide sequence of 59 amino acid residues, in its mature form, containing six cysteine residues. The peptide is stabilized by three disulfide bridges.[1][4]

The amino acid sequence of the Kunitz peptide AsKC11:

INKDCLLPKVVGFCRARFPRYYYNSSSRRCEKFIYGGCGGNANNFSSYYECHIKCFGPR [3]

Homology

AsKC11 exhibits sequence homology with other venom Kunitz-type peptides from various species, such as DTX-1 (extracted from black mamba venom) and APHC1 (extracted from leathery sea anemone venom).[3] In Basic Local Alignment Search Tool (BLAST), twenty proteins showed more than 70% similarity with AsKC11. All originate from the Anemonia genus and the majority originate from A. viridis.[3]

Family

AsKC11 belongs to the toxin Kunitz-type peptide family and is considered part of the sea anemone type 2 potassium channel toxin subfamily.[3][1] Kunitz-type peptides are found in several species such as scorpions, snakes, spiders, and sea anemones.[5] All Kunitz peptides share the Kunitz domain, which is characterized by a peptide chain of approximately 60 amino acid residues, rich in cysteine. The domain is stabilized by three disulfide bonds and contains an alpha and beta fold. Kunitz peptides modulate the activity of different ion channels and inhibit proteases (Kunitz inhibitors).[4] Unlike AsKC11, AsKC1-3, which are also Kunitz peptides, block voltage-gated potassium channels. Additionally, they show protease inhibition.[2] AsKC11 is expected to execute protease inhibition as well and it is the first Kunitz peptide to be an activator of GIRK1/2 channels.[3] Besides affecting potassium channels, other Kunitz peptides are known to inhibit voltage-gated sodium and voltage-gated calcium channels.[2][6][7]

Target

AsKC11 targets inward-rectifier potassium channels (Kir). More specifically, it activates GIRKs 1/2 channels (heterotetrameric protein), predominantly in cardiac cells and in the brain.[3][8] This peptidic activation is direct, the Gi protein alpha subunit is not involved. Additionally, AsKC11 has the ability to activate the homotetrameric GIRK2 channel.[3] AsKC11 has no effect on Kir2.1 or the voltage-gated potassium channels, Kv1.1-1.4 channels, and showed a small inhibition of Kv1.6. channels.[3]

AsKC11 binding is reversible. For GIRK1/2 channel activation induced by AsKC11, at a concentration of 48 μM of AsKC11, the first order-association rate constant (Kon) is 5.71 x 10−3 s−1T and the second-order association rate constant (α) is 7.82 x 10−5 μM−1s−1. The first-order dissociation rate constant (Koff = β) is 6.33 x 10−3 s−1. The calculated equilibrium dissociation constant to represent the affinity of AsKC11 to GIRK1/2 channels has been found to be 80.9 μM. Under the assumption of a linear relationship between the channel occupancy and the response, EC50 = 80.9 μM.[3]

Mode of action

AsKC11 has shown to be an activator of GIRK1/2 channels.[3] These channels are involved in the regulation of cellular excitability.[8] GIRK channels become activated following G protein-coupled receptor (GPCR) activation. Heterotrimeric G protein (Gβγ) subunits dissociate from the Gβγ and activate GIRK channels via binding of this subunit to the GIRK channel.[9][10] AsKC11 can activate GIRK1/2 channels without an activated Gi/o GPCR.[3] GIRK1/2 activation increases inward rectification of potassium, which aids in resting membrane maintenance and regulation of the length and shape of action potentials.[8] The membrane potential of neurons becomes hyperpolarized, causing a reduction in action potential firing.[11] The inward potassium current is dependent on the concentration of AsKC11. The reversal potential (close to 0 mV) is not affected by AsKC11-induced currents through GIRK1/2 channels. The current amplitude increases, which is presumed to be due to the higher chord conductance of GIRK1/2 channels.[3]

References
  1. "UniProt". www.uniprot.org. Retrieved 2022-10-23.
  2. Schweitz H, Bruhn T, Guillemare E, Moinier D, Lancelin JM, Béress L, Lazdunski M (October 1995). "Kalicludines and kaliseptine. Two different classes of sea anemone toxins for voltage sensitive K+ channels". The Journal of Biological Chemistry. 270 (42): 25121–25126. doi:10.1074/jbc.270.42.25121. PMID 7559645.
  3. An D, Pinheiro-Junior EL, Béress L, Gladkikh I, Leychenko E, Undheim EA, et al. (February 2022). "AsKC11, a Kunitz Peptide from Anemonia sulcata, Is a Novel Activator of G Protein-Coupled Inward-Rectifier Potassium Channels". Marine Drugs. 20 (2): 140. doi:10.3390/md20020140. PMC 8876855. PMID 35200669.
  4. Mishra M (September 2020). "Evolutionary Aspects of the Structural Convergence and Functional Diversification of Kunitz-Domain Inhibitors". Journal of Molecular Evolution. 88 (7): 537–548. Bibcode:2020JMolE..88..537M. doi:10.1007/s00239-020-09959-9. PMID 32696206. S2CID 220656912.
  5. Sintsova O, Gladkikh I, Monastyrnaya M, Tabakmakher V, Yurchenko E, Menchinskaya E, et al. (March 2021). "Sea Anemone Kunitz-Type Peptides Demonstrate Neuroprotective Activity in the 6-Hydroxydopamine Induced Neurotoxicity Model". Biomedicines. 9 (3): 283. doi:10.3390/biomedicines9030283. PMC 8001995. PMID 33802055.
  6. You D, Hong J, Rong M, Yu H, Liang S, Ma Y, et al. (August 2009). "The first gene-encoded amphibian neurotoxin". The Journal of Biological Chemistry. 284 (33): 22079–22086. doi:10.1074/jbc.M109.013276. PMC 2755932. PMID 19535333.
  7. Schweitz H, Heurteaux C, Bois P, Moinier D, Romey G, Lazdunski M (February 1994). "Calcicludine, a venom peptide of the Kunitz-type protease inhibitor family, is a potent blocker of high-threshold Ca2+ channels with a high affinity for L-type channels in cerebellar granule neurons". Proceedings of the National Academy of Sciences of the United States of America. 91 (3): 878–882. Bibcode:1994PNAS...91..878S. doi:10.1073/pnas.91.3.878. PMC 521415. PMID 8302860.
  8. Zhao Y, Gameiro-Ros I, Glaaser IW, Slesinger PA (March 2021). "Advances in Targeting GIRK Channels in Disease". Trends in Pharmacological Sciences. 42 (3): 203–215. doi:10.1016/j.tips.2020.12.002. PMC 7990506. PMID 33468322.
  9. Reuveny E, Slesinger PA, Inglese J, Morales JM, Iñiguez-Lluhi JA, Lefkowitz RJ, et al. (July 1994). "Activation of the cloned muscarinic potassium channel by G protein beta gamma subunits". Nature. 370 (6485): 143–146. Bibcode:1994Natur.370..143R. doi:10.1038/370143a0. PMID 8022483. S2CID 4345632.
  10. Logothetis DE, Kurachi Y, Galper J, Neer EJ, Clapham DE (1987-01-22). "The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart". Nature. 325 (6102): 321–326. Bibcode:1987Natur.325..321L. doi:10.1038/325321a0. PMID 2433589. S2CID 4338529.
  11. Glaaser IW, Slesinger PA (July 2017). "Dual activation of neuronal G protein-gated inwardly rectifying potassium (GIRK) channels by cholesterol and alcohol". Scientific Reports. 7 (1): 4592. Bibcode:2017NatSR...7.4592G. doi:10.1038/s41598-017-04681-x. PMC 5496853. PMID 28676630.
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