Apamin structure and pharmacology revisited

Abstract

Apamin is often cited as one of the few substances selectively acting on smallconductance Ca 2+ -activated potassium channels (K Ca 2). However, published pharmacological and structural data remain controversial. Here, we investigated the molecular pharmacology of apamin by two-electrode voltage-clamp in Xenopus laevis oocytes and patch-clamp in HEK293, COS7, and CHO cells expressing the studied ion channels, as well as in isolated rat brain neurons. The microtitre broth dilution method was used for antimicrobial activity screening. The spatial structure of apamin in aqueous solution was determined by NMR spectroscopy. We tested apamin against 42 ion channels (K Ca , K V , Na V , nAChR, ASIC, and others) and confirmed its unique selectivity to K Ca 2 channels. No antimicrobial activity was detected for apamin against Gram-positive or Gram-negative bacteria. The NMR solution structure of apamin was deposited in the Protein Data Bank. The results presented here demonstrate that apamin is a selective nanomolar or even subnanomolar-affinity K Ca 2 inhibitor with no significant effects on other molecular targets. The spatial structure as well as ample functional data provided here support the use of apamin as a K Ca 2-selective pharmacological tool and as a template for drug design.

The Bis(1,2,3,4-tetrahydroisoquinoline) Alkaloids Cepharanthine and Berbamine Are Ligands of SK Channels

“…Finally, additional testing showed that the reinforcement of the positive charges on both nitrogens, obtained by Nmethylation yielding quaternary analogues, resulted in an increased affinity for SK targets (data not shown). This is in accordance with the pharmacophore 15,16 and with previous investigations of related molecules, such as tetrandrine and its stronger N-methylated analogue, and the AG525 isomers, which were also N-methylated, yielding stronger derivatives. 22 Such quaternary compounds are, however, less interesting in therapeutic contexts, as their biological availability is strongly reduced due to cell-membrane impermeability, especially when considering the central nervous system distribution.…”

“…Apamin is a specific ligand of the SK family and has an extremely high affinity for all three subtypes, in the nanomolar to sub-nanomolar range, with no effective subtype selectivity. More precisely, its blocking activity, in order, is stronger on SK2 with an IC 50 of 87.7 pM, than on SK3 with an IC 50 of 2.3 nM, and finally on SK1 channels with an IC 50 of 4.1 nM. − …”

“…Although apamin was the first ligand to be identified for SK channels and has been known for a long time, 13,14 it still interests the scientific community to this day. 15 In this pharmacological context, new molecules are investigated for their ability to block SK channels, notably by using a pharmacophore model 16,17 built on the basis of the similarities between apamin's spatial disposition and that of UCL1684, 18 one of the strongest non-peptidic blockers (Figure 2). Indeed, potent SK blockers typically bear two positively charged nitrogens separated by a comparable distance.…”

Role of Calcium-Activated Potassium Channels in Proliferation, Migration and Invasion of Human Myeloid Leukemia Cells

“…Particularly, SK2 channels are the most sensitive to apamin with a half maximal blocking concentration (IC50: 27-140 pM, SK1 are less sensitive (IC50: 0.7-12 nM), and SK3 show an intermediate sensitivity to apamin (IC50: 0.6-4 nM). Importantly, apamin has no inhibitory effects on other types of K + channels as well as on ion channels from other families as it was confirmed recently on a broad repertoire of channel proteins [6]. For functional identification of IK activity, TRAM-34, the analog of clotrimazole, a potent and selective blocker of IK (KCa3.1), is widely used [7,8].…”

Kalium 3.0 is a comprehensive depository of natural, artificial, and labeled polypeptides acting on potassium channels

“…Differentiation into “apamin‐sensitive” and “apamin‐insensitive” K Ca has been common since 1980s to indicate the small‐conductance K Ca channels (K Ca 2 or SK Ca ) and intermediate‐conductance (K Ca 3.1 or IK Ca ) or large‐conductance K Ca channels (K Ca 1.1 or BK Ca ; Burgess et al, 1981; Pennefather et al, 1985; Romey & Lazdunski, 1984). The cloning and expression of mammalian K Ca 2 channels in Xenopus laevis oocytes was then used to demonstrate apamin activity on the molecular level (Grunnet et al, 2001; Köhler et al, 1996), whereas large‐scale pharmacological profiling revealed a unique selectivity of apamin (Kuzmenkov et al, 2022). Human β‐defensin 2, plectasin from the fungus Pseudoplectania nigrella , and peptide AcK1 from the parasitic worm Ancylostoma ceylanicum serve as examples of non‐venom‐derived ligands of K + channels (Chhabra et al, 2014; Xiang et al, 2015; Yang et al, 2015).…”