Specific sites in the Beta Interaction Domain of a schistosome Ca²⁺ channel β subunit are key to its role in sensitivity to the anti-schistosomal drug praziquantel

Abstract

Praziquantel, the drug of choice against schistosomiasis, disrupts calcium (Ca2+) homeostasis in schistosomes via an unknown mechanism. Voltage-gated Ca2+ channels are heteromultimeric transmembrane protein complexes that contribute to impulse propagation and also regulate intracellular Ca2+ levels. Beta subunits modulate the properties of the pore-forming alpha1 subunit of high voltage-activated Ca2+ channels. Unlike other Ca2+ channel beta subunits, which have current stimulatory effects, a beta subunit subtype found in S. mansoni (SmbetaA) and S. japonicum (Sjbeta) dramatically reduces current levels when co-expressed with Ca2+ channel alpha1 subunits in Xenopus oocytes. It also confers praziquantel sensitivity to the mammalian Cav2.3 alpha1 subunit. The Beta Interaction Domains (BIDs) of SmbetaA and Sjbeta lack 2 conserved serines that each constitute a consensus site for protein kinase C (PKC) phosphorylation. Here, we use site-directed mutagenesis of schistosome beta subunits to show that these unique functional properties are correlated with the absence of these consensus PKC sites in the BID. Furthermore, a second schistosome beta subunit subtype contains both serines in the BID, enhances currents through alpha1 subunits, and does not confer praziquantel sensitivity. Thus, phosphorylation sites in the BID may play important roles in defining the modulatory properties and pharmacological sensitivities of schistosome Ca2+ channel beta subunits.

Praziquantel for Schistosomiasis: Single-Drug Metabolism Revisited, Mode of Action, and Resistance

“…Guglielmo et al (120) developed a series of PZQ NO-donors furoxans that are worthy of investigation in view of their potential activity against PZQ-resistant schistosomes. Involvement of Ca 2ϩ channel changes in resistance to PZQ has been widely described (121). Nonetheless, whether these phenomena are responsible for drug action or represent downstream consequences has not been established (83,122).…”

Schistosomiasis—a century searching for chemotherapeutic drugs

“…It is known that praziquantel (Table 1, entry 1) interferes with Ca 2+ homeostasis (Greenberg 2005), promoting sustained muscular contraction (Fetterer et al 1980) and tegument disruption Mehlhorn et al 1981), which leads to antigen exposure on the worm surface (Harnett and Kusel 1986). A possible target that triggers those Ca 2+ -dependent events in the presence of praziquantel was recently described by Kohn and collaborators (Kohn et al 2001(Kohn et al , 2003. They cloned from S. mansoni and S. japonicum cDNA libraries the α1 and β subunits of voltage-gated Ca 2+ channels.…”

“…This unusual effect was reverted in the presence of praziquantel. Thus, it seems that the absence of the phosphorylation site in the parasite's β subunit is directly linked to praziquantel sensitivity of this, otherwise insensitive, α1 subunit (Kohn et al 2001(Kohn et al , 2003.…”

“…Cloning and functional expression of α1 and β subunits of voltage-gated Ca 2+ channels provided direct evidences that sensitivity to praziquantel is linked to the absence of fosforilation sites in those S. mansoni and S. japonicum particular β subunits (Kohn et al 2003) It positively interferes with the immunological response S. mansoni, S. japonicum, S. haematobium, S. intercalatum, S. mekongi. Cercariae, very young schistosomula, and adult forms (Ross et al 2002) 2.…”

The N Terminus of a Schistosome β Subunit Regulates Inactivation and Current Density of a Cav2 Channel

“…Indeed, a single amino acid is responsible for the sensitivity of insect Na v channels to pyrethroid insecticides (12,13). Similarly, the capability of the other schistosome (variant) ␤ subunit to confer praziquantel sensitivity to an otherwise non-susceptible ␣1 subunit can be abolished by mutating a single amino acid residue (14). In another case the sensitivity of the GABA receptor to ethanol requires a string of 8 amino acids in one of the ␥ subunits (15).…”