A schistosome [beta] subunit remodels inactivation of a calcium channel _via_ an N-terminal polyacidic motif

The beta subunit of high voltage-activated Ca (Cav) channels targets the pore forming [alpha]~1~ subunit to the plasma membrane and defines the biophysical phenotype of the Cav channel complex. Cav channel inactivation following activation and opening is tightly regulated and is an essential property that not only prevents excessive entry of Ca^2+^ into the cell but may also have functions in signal transduction. The [beta] subunit modulates Ca^2+^-dependent and voltage-dependent components of Cav channel inactivation via its interaction with the I-II linker of the [alpha]~1~ subunit. Here, using Cav2.3 and whole-cell patch-clamp, we show that a [beta] subunit from the human parasite _Schistosoma mansoni_ ([beta]~Sm~) accelerates inactivation via a unique, long N-terminal polyacidic motif (NPAM). The accelerating effect of NPAM-containing subunits, both native ([beta]~Sm~)and chimeric mammalian [beta]~1b~, [beta]~2a~ and [beta]~3~ subunits to which NPAM had been attached, was only apparent when Ca^2+^ was internally buffered with BAPTA (5 mM) or when Ba^2+^ was used as the charge carrier, two commonly used strategies to eliminate Ca^2+^/calmodulin dependent inactivation. These results indicate that calmodulin is not involved. In addition to accelerating inactivation, NPAM-containing [beta] subunits significantly reduced current density with respect to their non NPAM-bearing counterparts. Interestingly, when the amino acids N terminal to NPAM were deleted, inactivation of Cav2.3 currents was faster than in the presence of the entire N-terminal portion of the [beta]~Sm~ subunit, as if the pre-NPAM region counteracts the effect of NPAM. Presence of NPAM also resulted in currents that activated faster, suggesting that NPAM increases open channel probability. However, NPAM does not modulate inactivation gating. In summary, this study identifies a structural determinant of Cav channel inactivation that is entirely unlike those previously known

Atypical properties of a conventional calcium channel β subunit from the platyhelminth Schistosoma mansoni

© 2008 Salvador-Recatalà et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. The definitive version was published in BMC Physiology 8 (2008): 6, doi:10.1186/1472-6793-8-6.The function of voltage-gated calcium (Cav) channels greatly depends on coupling to cytoplasmic accessory β subunits, which not only promote surface expression, but also modulate gating and kinetic properties of the α1 subunit. Schistosomes, parasitic platyhelminths that cause schistosomiasis, express two β subunit subtypes: a structurally conventional β subunit and a variant β subunit with unusual functional properties. We have previously characterized the functional properties of the variant Cavβ subunit. Here, we focus on the modulatory phenotype of the conventional Cavβ subunit (SmCavβ) using the human Cav2.3 channel as the substrate for SmCavβ and the whole-cell patch-clamp technique. The conventional Schistosoma mansoni Cavβ subunit markedly increases Cav2.3 currents, slows macroscopic inactivation and shifts steady state inactivation in the hyperpolarizing direction. However, currents produced by Cav2.3 in the presence of SmCavβ run-down to approximately 75% of their initial amplitudes within two minutes of establishing the whole-cell configuration. This suppressive effect was independent of Ca2+, but dependent on intracellular Mg2+-ATP. Additional experiments revealed that SmCavβ lends the Cav2.3/SmCavβ complex sensitivity to Na+ ions. A mutant version of the Cavβ subunit lacking the first forty-six amino acids, including a string of twenty-two acidic residues, no longer conferred sensitivity to intracellular Mg2+-ATP and Na+ ions, while continuing to show wild type modulation of current amplitude and inactivation of Cav2.3. The data presented in this article provide insights into novel mechanisms employed by platyhelminth Cavβ subunits to modulate voltage-gated Ca2+ currents that indicate interactions between the Ca2+ channel complex and chelated forms of ATP as well as Na+ ions. These results have potentially important implications for understanding previously unknown mechanisms by which platyhelminths and perhaps other organisms modulate Ca2+ currents in excitable cells.This work was supported by NIH grant #s R01 AI-40522 and R01 AI-73660 to RMG and by NIH-NCRR grant # P41 RR001395 to the Biocurrents Research Center (BRC) at MBL

Attenuation of apoptosis in enterocytes by blockade of potassium channels

Apoptosis plays an important role in maintaining the balance between proliferation and cell loss in the intestinal epithelium. Apoptosis rates may increase in intestinal pathologies such as inflammatory bowel disease and necrotizing enterocolitis, suggesting pharmacological prevention of apoptosis as a therapy for these conditions. Here, we explore the feasibility of this approach using the rat epithelial cell line IEC-6 as a model. On the basis of the known role of K+ efflux in apoptosis in various cell types, we hypothesized that K+ efflux is essential for apoptosis in enterocytes and that pharmacological blockade of this efflux would inhibit apoptosis. By probing intracellular [K+] with the K+-sensitive fluorescent dye and measuring the efflux of 86Rb+, we found that apoptosis-inducing treatment with the proteasome inhibitor MG-132 leads to a twofold increase in K+ efflux from IEC-6 cells. Blockade of K+ efflux with tetraethylammonium, 4-aminopyridine, stromatoxin, chromanol 293B, and the recently described K+ channel inhibitor 48F10 prevents DNA fragmentation, caspase activation, release of cytochrome c from mitochondria, and loss of mitochondrial membrane potential. Thus K+ efflux occurs early in the apoptotic program and is required for the execution of later events. Apoptotic K+ efflux critically depends on activation of p38 MAPK. These results demonstrate for the first time the requirement of K+ channel-mediated K+ efflux for progression of apoptosis in enterocytes and suggest the use of K+ channel blockers to prevent apoptotic cell loss occurring in intestinal pathologies

Specific and Slow Inhibition of the Kir2.1 K+ Channel by Gambogic Acid*

Although Kir2.1 channels are important in the heart and other excitable cells, there are virtually no specific drugs for this K+ channel. In search of Kir2.1 modulators, we screened a library of 720 naturally occurring compounds using a yeast strain in which mammalian Kir2.1 enables growth at low [K+]. One of the identified compounds, gambogic acid (GA), potently (EC50 ≤ 100 nm) inhibited Kir2.1 channels in mammalian cells when applied chronically for 3 h. This potent and slow inhibition was not seen with Kv2.1, HERG or Kir1.1 channels. However, acutely applied GA acted as a weak (EC50 = ∼10 μm) non-selective K+ channel blocker. Intracellular delivery of GA via a patch pipette did not potentiate the acute effect of GA on Kir2.1, showing that slow uptake is not responsible for the delayed, potent effect. Immunoblots showed that total Kir2.1 protein expression was not altered by GA. Similarly, immunostaining of intact cells expressing Kir2.1 with an extracellular epitope tag demonstrated that GA does not affect Kir2.1 surface expression. However, the 3-h treatment with GA caused redistribution of Kir2.1 and Kv2.1 from the Triton X-100-insoluble to the Triton X-100-soluble membrane fraction. Thus, GA changes the K+ channel membrane microenvironment resulting in potent, specific, and slow acting inhibition of Kir2.1 channels