The N terminus of a schistosome β subunit regulates inactivation and current density of a Cav2 channel

Abstract

The β subunit of high voltage-activated Ca2+ (Ca v) channels targets the pore-forming α1 subunit to the plasma membrane and tunes the biophysical phenotype of the Cav 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 Cavβ subunit of the human parasite Schistosoma mansoni (βSm), a motif that does not occur in other known Cavβ subunits. When expressed in human embryonic kidney cells stably expressing Cav2.3, βSm accelerates Ca 2+/calmodulin-independent inactivation of Cav2.3. Deleting the first 44 amino acids of βSm, a region that includes NPAM, significantly slows the predominant time constant of inactivation (τfast) under conditions that prevent Ca2+/CaM- dependent inactivation (βSm: τfast = 66 ms; βSmΔ2-44: τfast = 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 (τfast = 42 ms with βSmΔ2-17; τfast = 40 ms with βSmΔ2-24, 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 βSm attached to mammalian β1b, β2a, and β3 subunits. For any given mammalian β subunit, inactivation was faster if it contained the N terminus of βSm than if it did not. Co-expression of the mammalian α2δ-1 subunit resulted in doubling of the inactivation rate, but the effects of NPAM persisted. Thus, it appears that the schistosome Cav channel complex has acquired a new function that likely contributes to reducing the amount of Ca2+ that enters the cells in vivo. This feature is of potential interest as a target for new antihelminthics. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.