Bisphosphonates (BPs) reduce bone pain and fractures by balancing the osteoblast/osteoclast ratio. The behavior of ion channels in the presence of BPs is not known. To investigate this, the effect of zoledronic acid BP (ZOL) (3 × 10⁻⁸ to 5 × 10⁻⁴ M) treatment, on ion channels, cell proliferation, and mineralization, has been investigated on preosteoclast-like cells, RAW264.7, preosteoblast-like cells MC3T3-E1, and rat/mouse native bone marrow-derived osteoblasts. In whole-cell patch clamp on cell line- and bone marrow-derived osteoblasts, ZOL potentiated outward currents. On RAW264.7, ZOL (10⁻⁴ M)-evoked current was reduced by the Kv channel blocker tetraethylammonium hydrochloride (TEA), but not by the selective TRPV1-channel antagonist capsazepine. On MC3T3-E1 cells and bone marrow-derived osteoblasts, ZOL-evoked current (5 × 10⁻⁸ to 10⁻⁴ M) was reduced by capsazepine, whereas the selective TRPV1-channel agonist capsaicin potentiated the control current. In the cell proliferation assay, 72 h incubation of RAW264.7 and MC3T3-E1 cells with ZOL reduced proliferation, with IC₅₀ values of 2.62 × 10⁻⁷ M and 2.02 × 10⁻⁵ M, respectively. Mineralization of MC3T3-E1 cells and bone marrow-derived osteoblasts was observed in the presence of capsaicin and ZOL (5 × 10⁻⁸–10⁻⁷ M); ZOL effects were antagonized by capsazepine. In summary, the ZOL-induced activation of TRPV1 channel mediates the mineralization of osteoblasts and counterbalances the antiproliferative effects, increasing the IC₅₀. This mechanism is not operative in osteoclasts lacking the TRPV1 channel.

The effects of Ca²⁺-activated K⁺ (BK) channel modulation by Paxilline (PAX) (10⁻⁷–10⁻⁴ M), Iberiotoxin (IbTX) (0.1–1 × 10⁻⁶ M) and Resveratrol (RESV) (1–2 × 10⁻⁴ M) on cell cycle and proliferation, AKT1p^Ser473 phosphorylation, cell diameter, and BK currents were investigated in SH-SY5Y cells using Operetta-high-content-Imaging-System, ELISA-assay, impedentiometric counting method and patch-clamp technique, respectively. IbTX (4 × 10⁻⁷ M), PAX (5 × 10⁻⁵ M) and RESV (10⁻⁴ M) caused a maximal decrease of the outward K⁺ current at +30 mV (Vm) of −38.3 ± 10%, −31.9 ± 9% and −43 ± 8%, respectively, which was not reversible following washout and cell depolarization. After 6h of incubation, the drugs concentration dependently reduced proliferation. A maximal reduction of cell proliferation, respectively of −60 ± 8% for RESV (2 × 10⁻⁴ M) (IC50 = 1.50 × 10⁻⁴ M), −65 ± 6% for IbTX (10⁻⁶ M) (IC50 = 5 × 10⁻⁷ M), −97 ± 6% for PAX (1 × 10⁻⁴ M) (IC50 = 1.06 × 10⁻⁵ M) and AKT1p^ser473 dephosphorylation was observed. PAX induced a G1/G2 accumulation and contraction of the S-phase, reducing the nuclear area and cell diameter. IbTX induced G1 contraction and G2 accumulation reducing diameter. RESV induced G2 accumulation and S contraction reducing diameter. These drugs share common actions leading to a block of the surface membrane BK channels with cell depolarization and calcium influx, AKT1p^ser473 dephosphorylation by calcium-dependent phosphatase, accumulation in the G2 phase, and a reduction of diameter and proliferation. In addition, the PAX action against nuclear membrane BK channels potentiates its antiproliferative effects with early apoptosis.

(1) Background: Cantu syndrome (CS) arises from gain-of-function (GOF) mutations in the ABCC9 and KCNJ8 genes, which encode ATP-sensitive K⁺ (KATP) channel subunits SUR2 and Kir6.1, respectively. Most CS patients have mutations in SUR2, the major component of skeletal muscle KATP, but the consequences of SUR2 GOF in skeletal muscle are unknown. (2) Methods: We performed in vivo and ex vivo characterization of skeletal muscle in heterozygous SUR2[A478V] (SUR2^wt/AV) and homozygous SUR2[A478V] (SUR2^AV/AV) CS mice. (3) Results: In SUR2^wt/AV and SUR2^AV/AV mice, forelimb strength and diaphragm amplitude movement were reduced; muscle echodensity was enhanced. KATP channel currents recorded in Flexor digitorum brevis fibers showed reduced MgATP-sensitivity in SUR2^wt/AV, dramatically so in SUR2^AV/AV mice; IC₅₀ for MgATP inhibition of KATP currents were 1.9 ± 0.5 × 10⁻⁵ M in SUR2^wt/AV and 8.6 ± 0.4 × 10⁻⁶ M in WT mice and was not measurable in SUR2^AV/AV. A slight rightward shift of sensitivity to inhibition by glibenclamide was detected in SUR2^AV/AV mice. Histopathological and qPCR analysis revealed atrophy of soleus and tibialis anterior muscles and up-regulation of atrogin-1 and MuRF1 mRNA in CS mice. (4) Conclusions: SUR2[A478V] “knock-in” mutation in mice impairs KATP channel modulation by MgATP, markedly so in SUR2^AV/AV, with atrophy and non-inflammatory edema in different skeletal muscle phenotypes.

Zoledronic acid (ZOL) is used as a bone-specific antiresorptive drug with antimyeloma effects. Adverse drug reactions (A.D.R.) are associated with ZOL-therapy, whose mechanics are unknown. ZOL is a nitrogen-containing molecule whose structure shows similarities with nucleotides, ligands of ATP-sensitive K⁺ (KATP) channels. We investigated the action of ZOL by performing in vitro patch-clamp experiments on native KATP channels in murine skeletal muscle fibers, bone cells, and recombinant subunits in cell lines, and by in silico docking the nucleotide site on KIR and SUR, as well as the glibenclamide site. ZOL fully inhibited the KATP currents recorded in excised macro-patches from Extensor digitorum longus (EDL) and Soleus (SOL) muscle fibers with an IC₅₀ of 1.2 ± 1.4 × 10⁻⁶ and 2.1 ± 3.7 × 10⁻¹⁰ M, respectively, and the KATP currents recorded in cell-attached patches from primary long bone cells with an IC₅₀ of 1.6 ± 2.8 × 10⁻¹⁰ M. ZOL fully inhibited a whole-cell KATP channel current of recombinant KIR6.1-SUR2B and KIR6.2-SUR2A subunits expressed in HEK293 cells with an IC₅₀ of 3.9 ± 2.7 × 10⁻¹⁰ M and 7.1 ± 3.1 × 10⁻⁶ M, respectively. The rank order of potency in inhibiting the KATP currents was: KIR6.1-SUR2B/SOL-KATP/osteoblast-KATP > KIR6.2-SUR2A/EDL-KATP >>> KIR6.2-SUR1 and KIR6.1-SUR1. Docking investigation revealed that the drug binds to the ADP/ATP sites on KIR6.1/2 and SUR2A/B and on the sulfonylureas site showing low binding energy <6 Kcal/mol for the KIR6.1/2-SUR2 subunits vs. the <4 Kcal/mol for the KIR6.2-SUR1. The IC₅₀ of ZOL to inhibit the KIR6.1/2-SUR2A/B channels were correlated with its musculoskeletal and cardiovascular risks. We first showed that ZOL blocks at subnanomolar concentration musculoskeletal KATP channels and cardiac and vascular KIR6.2/1-SUR2 channels.

Cantú syndrome (CS) is caused by the gain of function mutations in the ABCC9 and KCNJ8 genes encoding, respectively, for the sulfonylureas receptor type 2 (SUR2) and the inwardly rectifier potassium channel 6.1 (Kir6.1) of the ATP-sensitive potassium (KATP) channels. CS is a multi-organ condition with a cardiovascular phenotype, neuromuscular symptoms, and skeletal malformations. Glibenclamide has been proposed for use in CS, but even in animals, the drug is incompletely effective against severe mutations, including the Kir6.1^wt/V65M. Patch-clamp experiments showed that zoledronic acid (ZOL) fully reduced the whole-cell KATP currents in bone calvaria cells from wild type (WT/WT) and heterozygous Kir6.1^wt/V65MCS mice, with IC₅₀ for ZOL block < 1 nM in each case. ZOL fully reduced KATP current in excised patches in skeletal muscle fibers in WT/WT and CS mice, with IC₅₀ of 100 nM in each case. Interestingly, KATP currents in the bone of heterozygous SUR2^wt/A478V mice were less sensitive to ZOL inhibition, showing an IC₅₀ of ~500 nM and a slope of ~0.3. In homozygous SUR2^A478V/A478V cells, ZOL failed to fully inhibit the KATP currents, causing only ~35% inhibition at 100 μM, but was responsive to glibenclamide. ZOL reduced the KATP currents in Kir6.1^wt/VMCS mice in both skeletal muscle and bone cells but was not effective in the SUR2^[A478V] mice fibers. These data indicate a subunit specificity of ZOL action that is important for appropriate CS therapies.