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Toxins (Basel)
2023 Oct 13;1510:. doi: 10.3390/toxins15100612.
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Molecular Basis for Mambalgin-2 Interaction with Heterotrimeric α-ENaC/ASIC1a/γ-ENaC Channels in Cancer Cells.
Lyukmanova EN
,
Zaigraev MM
,
Kulbatskii DS
,
Isaev AB
,
Kukushkin ID
,
Bychkov ML
,
Shulepko MA
,
Chugunov AO
,
Kirpichnikov MP
.
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Cancer progression is characterized by microenvironmental acidification. Tumor cells adapt to low environmental pH by activating acid-sensing trimeric ion channels of the DEG/ENaC family. The α-ENaC/ASIC1a/γ-ENaC heterotrimeric channel is a tumor-specific acid-sensing channel, and its targeting can be considered a new strategy for cancer therapy. Mambalgin-2 from the Dendroaspis polylepis venom inhibits the α-ENaC/ASIC1a/γ-ENaC heterotrimer more effectively than the homotrimeric ASIC1a channel, initially proposed as the target of mambalgin-2. Although the molecular basis of such mambalgin selectivity remained unclear. Here, we built the models of the complexes of mambalgin-2 with the α-ENaC/ASIC1a/γ-ENaC and ASIC1a channels, performed MD and predicted the difference in the binding modes. The importance of the 'head' loop region of mambalgin-2 for the interaction with the hetero-, but not with the homotrimeric channel was confirmed by site-directed mutagenesis and electrophysiology. A new mode of allosteric regulation of the ENaC channels by linking the thumb domain of the ASIC1a subunit with the palm domain of the γ-ENaC subunit was proposed. The data obtained provide new insights into the regulation of various types of acid-sensing ion channels and the development of new strategies for cancer treatment.
Figure 1. Models of the complexes of Mamb-2 with ASIC1 homotrimer (a,b) and α-ENaC/ASIC1/γ-ENaC heterotrimer (c,d): side (a,c) and top (b,d) views are shown. Both models are immersed in the phospholipid bilayer and solvated to perform MD calculations; the presented structures result from 500 ns MD.
Figure 2. Contact maps of the Mamb-2 interaction with the primary ASIC1a(+) subunit in heterotrimeric and homotrimeric complexes. Map cells are colored according to the contact intensity, which corresponds to the summarized lifetime of the contacts of all types between each pair of residues under consideration during the MD simulation of 500 ns. The following types of contacts were considered: ionic bridges, hydrogen bonds, π-cation, stacking, and hydrophobic interactions. ASIC1a residues outside the 280–370 range did not give a significant contribution to the interaction and therefore were not included in the map. The position of the loops’ and ‘head’ regions of Mamb-2 is shown.
Figure 3. Amino-acid residues implicated in Mamb-2 interaction with the complementary (−) subunits in the α-ENaC/ASIC1a/γ-ENaC hetero- (a) and ASIC1a homotrimer (b). Note that interactions with (−)-subunits determine the difference between these two complexes, while the constant part of the Mamb-2/ASIC1a(+) binding interface is not shown for clarity (see also Figure 2). The binding sites Mamb-2/(−)-subunit are shown by dotted lines. The contacting residues from the (−)-subunits are shown in italics. (c). Mamb-2 model: loops and ‘head’ are subscribed, along with receptor-interacting residues in the latter.
Figure 4. Mamb-2 inhibitory activity at the α-ENaC/ASIC1a/γ-ENaC channel with mutated γ-ENaC(−) subunit (F89A, R90A and K91A). (a) Representative current traces recorded in X. laevis the wild-type (WT) α-ENaC/ASIC1a/γ-ENaC channel or its mutants with substitutions F89A, R90A, and K91A in the γ-ENaC subunit after pH decrease without (black lines) and with (red lines) application of 370 nM of Mamb-2. Oocytes were pre-incubated with Mamb-2 for 15 s (red bar above the trace, the beginning of pre-incubation is off the time scale), and the stimulation phase (pH 5.0) was 7 s. Time and current scale are shown by bars near the traces. (b) Dose–response curves for the Mamb-2 inhibitory effect at WT and mutated α-ENaC/ASIC1a/γ-ENaC channels. The response current was normalized to the control experiment for each Mamb-2 concentration. Each data point represents an average from independent experiments in different oocytes ± SEM (n = 4–7). The fitted curves are described by Hill’s equation. Parameters describing the curves’ fit are presented in Table 2. (c) Protocol of experiments within the two-electrode patch-clamp technique.
Figure 5. Inhibitory activity of Mamb-2 and its mutants H13A, R14A, D15A, and R14A/D15A at the α-ENaC/ASIC1a/γ-ENaC channel. (a) Representative traces of currents recorded in X. laevis oocytes, which express WT α-ENaC/ASIC1a/γ-ENaC channel after pH decrease without (black lines) and with (red lines) application of 370 nM of Mamb-2 and its mutants. Oocytes were pre-incubated with Mamb-2 for 15 s (red bar above the trace, the beginning of pre-incubation is off the time scale), and the pH drop phase (pH 5.0) was 7 s. Time and current scale are shown by bars near the traces. (b) Dose–response curves for the Mamb-2 and its mutants at the α-ENaC/ASIC1a/γ-ENaC channel. The response current was normalized to the control experiment for each Mamb-2 concentration. Each data point represents an average from independent experiments in different oocytes ±SEM (n = 4–7). The fitted curves are described by Hill’s equation. Parameters describing the curves’ fit are presented in Table 3.
Figure 6. Inhibitory activity of Mamb-2 and its mutants H13A, R14A, D15A, and R14A/D15A at the ASIC1a channel. (a) Representative traces of currents recorded in X. laevis oocytes, which express WT ASIC1a channel after pH decrease without (black lines) and with (red lines) application of 1.1 µM of Mamb-2 and its mutants. Oocytes were pre-incubated with Mamb-2 for 15 s (red bar above the trace, the beginning of pre-incubation is off the time scale), and the pH drop phase (pH 5.0) was 7 s. Time and current scale are shown by bars near the traces. (b) Dose–response curves for the Mamb-2 and its mutants at the ASIC1a channel. The response current was normalized to the control experiment for each Mamb-2 concentration. Each data point represents an average from independent experiments in different oocytes ±SEM (n = 4–7). The fitted curves are described by the single-component Hill’s equation. Parameters describing the curves’ fit are presented in Table 4.
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