The VRAC blocker DCPIB directly gates the BK channels and increases intracellular Ca²⁺ in melanoma and pancreatic duct adenocarcinoma cell lines

Abstract

Background and purpose The volume regulated anion channel (VRAC) is known to be involved in different aspects of cancer cell behaviour and response to therapies. For this reason, we investigated the effect of DCPIB, a presumably specific blocker of VRAC, in two types of cancer: pancreatic duct adenocarcinoma (PDAC) and melanoma. Experimental approach We used patch‐clamp electrophysiology, supported by Ca2+ imaging, gene expression analysis, docking simulation and mutagenesis. We employed two PDAC lines (Panc‐1 and MiaPaCa‐2), as well as a primary (IGR39) and a metastatic (IGR37) melanoma line. Key results DCPIB markedly increased whole‐cell currents in Panc‐1, MiaPaca2 and IGR39, but not in IGR37 cells. The currents were mostly mediated by KCa1.1 channels, commonly known as BK channels. We confirmed DCPIB activation of BK channels also in HEK293 cells transfected with α subunits of this channel. Further experiments showed that in IGR39, and to a smaller degree also in Panc‐1 cells, DCPIB induced a rapid Ca2+ influx. This, in turn, indirectly potentiated BK channels and, in IGR39 cells, additionally activated other Ca2+‐dependent channels. However, Ca2+ influx was not required for activation of BK channels by DCPIB, as such activation involved the extracellular part of the protein and we have identified a residue crucial for binding. Conclusion and implications DCPIB directly targeted BK channels and, also, acutely increased intracellular Ca2+. Our findings extend the list of DCPIB effects that should be taken into consideration for future development of DCPIB‐based modulators of ion channels and other membrane proteins.

BK Channel in the Physiology and in the Cancer of Pancreatic Duct: Impact and Reliability of BK Openers

“…NS11021 decreased cell viability within 72 h. However, several lines of evidence raised serious doubts that this effect is dependent on BK activation. First, another cancer cell line (melanoma IGR37) displayed a similar viability drop after a 72 hours-long treatment with NS11021 even though the expression of the channel is ∼10 4 times lower in IGR37 cells than in Panc-1 cells ( Ferrera et al, 2021 ; Zuccolini et al, 2022 ), and no BK like currents were activated by NS11021 in IGR37 cells ( Remigante et al, 2021 ). Moreover, the other tested BK activator, NS19504, did not have any impact on Panc-1 viability in the same experimental conditions.…”

“…This splice variant is characterized by a normal BK unitary conductance of ∼250 pS but displays slower activation and higher sensitivity to [Ca 2+ ] i than its closest homolog hbr5 ( Liu et al, 2002 ). The endogenous currents displayed by Panc-1 cells at low [Ca 2+ ] i (∼20 nM) are characterized by a deviation from linearity and a sudden increase of current density at voltages ≥ +80 mV, which are likely due to the activation of BK channels ( Zuccolini et al, 2022 ). No clear evidence of LRRC26-mediated strong left shift of BK voltage-dependence can be observed in this cell line.…”

“…PDAC is one of the deadliest types of cancer, given the lack of specific symptoms and resistance to chemotherapy ( Fan et al, 2020 ; Zhang et al, 2020 ). BK has been found to be expressed both in the pancreatic duct epithelia cells (PDECs) ( Gray et al, 1990 ; Hede et al, 2005 ; Venglovecz et al, 2011 ) and in PDAC cell lines ( Zuccolini et al, 2022 ). Many studies in the literature report that sustained opening of the BK channel with activators reduces the ability to proliferate and migrate of different cancer cell lines.…”

Nicotinic Acid Adenine Dinucleotide Phosphate Induces Intracellular Ca2+ Signalling and Stimulates Proliferation in Human Cardiac Mesenchymal Stromal Cells

“…All reactions were performed in a 96-well format with the 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, MA, United States). The relative quantities of specific mRNA were obtained with the use of the comparative Ct method and were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ( Maione et al, 2021 ; Zuccolini et al, 2022 ). The expression of each target gene was assessed in triplicate ( Ferrera et al, 2021 ; Maione et al, 2021 ).…”

Recent Advances in the Structural Biology of the Volume-Regulated Anion Channel LRRC8

“…Moreover, DCPIB is not just a specific VRAC inhibitor. At concentrations used to inhibit VRAC, various studies suggested that it regulates other membrane proteins including glutamate transporter GLT-1 ( Bowens et al, 2013 ), Connexin 43 (Cx43) ( Bowens et al, 2013 ), gastric H + ,K + -ATPase ( Fujii et al, 2015 ), components of mitochondrial electron transport chains ( Afzal et al, 2019 ), K2P K + channels ( Minieri et al, 2013 ; Lv et al, 2019 ), inward rectifying K + (Kir) channels ( Deng et al, 2016 ), and BK K + channel ( Zuccolini et al, 2022 ). Nevertheless, this successful result of the structure-based approach to design DCPIB analogs suggests that further potent inhibitors targeting the native VRAC pore can be designed using the SN-407- and DCPIB-bound MmLRRC8A homo-hexamer structures in the future.…”