title: The N terminus of a schistosome beta subunit regulates inactivation and current density of a Cav2 channel.
abstract: The β subunit of high voltage-activated Ca(2+) (Ca(v)) channels targets the pore-forming α(1) subunit to the plasma membrane and tunes the biophysical phenotype of the Ca(v) channel complex. We used a combination of molecular biology and whole-cell patch clamp to investigate the functional role of a long N-terminal polyacidic motif (NPAM) in a Ca(v)β subunit of the human parasite Schistosoma mansoni (β(Sm)), a motif that does not occur in other known Ca(v)β subunits. When expressed in human embryonic kidney cells stably expressing Ca(v)2.3, β(Sm) accelerates Ca(2+)/calmodulin-independent inactivation of Ca(v)2.3. Deleting the first 44 amino acids of β(Sm), a region that includes NPAM, significantly slows the predominant time constant of inactivation (τ(fast)) under conditions that prevent Ca(2+)/CaM-dependent inactivation (β(Sm): τ(fast) = 66 ms; β(SmΔ2-44): τ(fast) = 111 ms, p < 0.01). Interestingly, deleting the amino acids that are N-terminal to NPAM (2-24 or 2-17) results in faster inactivation than with an intact N terminus (τ(fast) = 42 ms with β(SmΔ2-17); τ(fast) = 40 ms with β(SmΔ2-24), p < 0.01). This suggests that NPAM is the structural determinant for accelerating Ca(2+)/calmodulin-independent inactivation. We also created three chimeric subunits that contain the first 44 amino acids of β(Sm) attached to mammalian β(1b), β(2a), and β(3) subunits. For any given mammalian β subunit, inactivation was faster if it contained the N terminus of β(Sm) than if it did not. Co-expression of the mammalian α(2)δ-1 subunit resulted in doubling of the inactivation rate, but the effects of NPAM persisted. Thus, it appears that the schistosome Ca(v) channel complex has acquired a new function that likely contributes to reducing the amount of Ca(2+) that enters the cells in vivo. This feature is of potential interest as a target for new antihelminthics.
authors.affiliation: Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. vicenta@vet.upenn.edu
authors.forename: Vicenta
authors.initials: V
authors.lastname: Salvador-Recatalà
authors.forename: Robert M
authors.initials: RM
authors.lastname: Greenberg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D020908:Calcium Channels, R-Type / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002147:Calmodulin / Q000378:metabolism; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D018613:Microscopy, Confocal; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000235:genetics / Q000502:physiology; D012550:Schistosoma mansoni / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: C423009:CACNA1E protein, human; D015220:Calcium Channels; D020908:Calcium Channels, R-Type; D002147:Calmodulin; D027682:Cation Transport Proteins; D002614:Chelating Agents; D015801:Helminth Proteins; D021122:Protein Subunits; D049452:Green Fluorescent Proteins; D004533:Egtazic Acid; D002118:Calcium
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