Kuzmenkov AI et al. (2022), Apamin structure and pharmacology revisited.

XB-ART-59367

Front Pharmacol 2022 Jan 01;13:977440. doi: 10.3389/fphar.2022.977440.

Show Gene links Show Anatomy links

Apamin structure and pharmacology revisited.

Kuzmenkov AI , Peigneur S , Nasburg JA , Mineev KS , Nikolaev MV , Pinheiro-Junior EL , Arseniev AS , Wulff H , Tytgat J , Vassilevski AA .

???displayArticle.abstract???
Apamin is often cited as one of the few substances selectively acting on small-conductance Ca2+-activated potassium channels (KCa2). 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 (KCa, KV, NaV, nAChR, ASIC, and others) and confirmed its unique selectivity to KCa2 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 KCa2 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 KCa2-selective pharmacological tool and as a template for drug design.

???displayArticle.pubmedLink??? 36188602
???displayArticle.pmcLink??? PMC9523135
???displayArticle.link??? Front Pharmacol
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: asic1 trpv1
GO keywords: calcium channel activity [+]

???attribute.lit??? ???displayArticles.show???

	FIGURE 1. Analytical RP-HPLC of apamin and its structure in solution. (A) 10 nmol of peptide was injected onto a Vydac C18 column (4.6 × 250 mm). Reflector-mode MALDI mass spectrum with an isotopic resolution of the purified peptide is shown in the inset. (B) Amino acid sequence of apamin. The numbering is shown above. Amino acid residues are colored with respect to their chemical properties: positively charged residues are shown in blue, negatively charged in red, hydrophobic in green, hydrophilic uncharged in magenta, and cysteines in yellow. Disulfide bridges are shown with lines. (C) A set of 10 NMR structures with the fewest restraint violations (PDB ID: 7OXF). Disulfide bridges are colored in yellow and labeled. (D) The spatial structure of apamin is shown in a ribbon representation. The ɑ-helix is colored pink, and the rest of the main chain is gray. Side chains of the peptide are labeled and colored, the coloring is as in panel (B). Disulfide bridges are shown as yellow sticks.
	FIGURE 2. Apamin effects on KCa channels expressed in HEK293 or COS7 cells (in the case of KCa2.3). (A) Effect of increasing concentrations of apamin (10 nM and 500 nM in red and blue, respectively) on KCa2.1 currents. Note that the currents visible above 0 mV are carried by endogenous KV channels in HEK cells. (B) Effect of increasing concentrations of apamin (75 pM and 100 nM in red and blue, respectively) on KCa2.2 currents. (C) Effect of increasing concentrations of apamin (20 nM and 500 nM in red and blue, respectively) on KCa2.3 currents. (D) Concentration-response curves for KCa2.1 (in dark purple), KCa2.2 (teal), and KCa2.3 (red). Data points are mean ± SD from five independent measurements per concentration. (E) KCa3.1 currents (in black) are insensitive to 5 µM apamin (red) but are potently blocked by 1 µM TRAM-34 (blue). (F) KCa1.1 currents evoked by depolarization steps to +80 mV (in black) are insensitive to 5 µM of apamin (red).
	FIGURE 3. Electrophysiological profiling of apamin. Shown are representative traces of currents through the corresponding ion channels in control (gray) and after application of 5 μM toxin (blue). In the case of nAChR, TRPV1, and GIRK1/2, blue bars indicate apamin application. In nAChR, arrows indicate agonist (ACh) application. In TRPV1, the open bar shows agonist (capsaicin, CAP) application, and gray bar, antagonist (capsazepine, CZP) application. In GIRK1/2, presentation of different bath solutions is shown as line segments. In ASIC traces, application of the activating pH is shown with gray bars. And in GluR traces, gray bars depict the application of agonists. CaImp, Ca2+-impermeable; CaP, Ca2+-permeable.
	Figure S1. Experimental data used for the structure determination of apamin. The following parameters are listed top down: protein sequence with S-S-bonds, NOE connectivities (dij), and 3JHNHA couplings. Widths of the bars represent the relative intensity of cross-peaks in NOESY spectra. Squares have three colors according to the value of J-coupling: black (>8 Hz), gray (6–8 Hz), and white (<6 Hz).
	Figure S2. Absolute NMDA current amplitudes in control and in the present of 5μM apamin.

References [+] :

Alexander, THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: Ion channels. 2021, Pubmed