The N terminus of a schistosome beta subunit regulates inactivation and current density of a Cav2 channel.
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.
Salvador-Recatalà
Vicenta
V
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. vicenta@vet.upenn.edu
Greenberg
Robert M
RM
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
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
Biochem Soc Trans. 2006 Nov;34(Pt 5):887-93
17052221
Biophys J. 1997 Sep;73(3):1355-63
9284303
Eur J Neurosci. 2005 Mar;21(6):1617-25
15845089
Parasitology. 2001 Nov;123(Pt 5):489-97
11719960
Neuron. 2003 Sep 11;39(6):951-60
12971895
Curr Opin Pharmacol. 2008 Jun;8(3):311-8
18329337
Neuron. 2003 Jul 31;39(3):483-96
12895422
Parasitology. 1983 Jun;86 (Pt 3):439-53
6877870
Nature. 1983 Dec 1-7;306(5942):436-41
6316158
Parasitol Today. 1998 Apr;14(4):157-60
17040734
Neuron. 1991 Jul;7(1):27-33
1712603
Am J Physiol. 1998 May;274(5):C1324-31
9612220
Biophys J. 1996 Nov;71(5):2509-21
8913590
J Comp Neurol. 2009 Mar 10;513(2):188-96
19127523
Eur J Neurosci. 2004 Jul;20(1):1-13
15245474
J Gen Physiol. 1995 Oct;106(4):641-58
8576701
J Neurosci. 2001 Nov 15;21(22):8715-21
11698583
Parasitology. 2006 Jul;133(Pt 1):67-74
16566851
Eur J Neurosci. 2003 Aug;18(4):841-55
12925010
J Neurosci. 2000 Nov 15;20(22):8323-30
11069939
Curr Opin Drug Discov Devel. 2009 Mar;12(2):231-9
19333868
FEBS Lett. 2000 Mar 24;470(2):135-8
10734222
Trends Pharmacol Sci. 2004 Dec;25(12):626-32
15530640
Cell Calcium. 2005 Sep-Oct;38(3-4):223-31
16098584
Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4720-5
9539805
Neuron. 2004 May 13;42(3):387-99
15134636
J Biol Chem. 2001 Oct 5;276(40):36873-6
11500482
J Gen Physiol. 2008 Dec;132(6):681-92
19029375
J Biol Chem. 2001 Dec 14;276(50):47163-70
11604404
Parasitology. 2003 Oct;127(Pt 4):349-56
14636021
Pest Manag Sci. 2001 Oct;57(10):877-88
11695180
J Biol Chem. 1999 Aug 6;274(32):22428-36
10428816
J Neurosci. 1987 Feb;7(2):418-31
2434628
Pflugers Arch. 2010 Feb;459(3):399-411
19821165
J Biol Chem. 1996 Oct 25;271(43):26465-8
8900112
Physiol Rev. 2006 Jan;86(1):369-408
16371601
Nature. 2004 Jun 10;429(6992):671-5
15141227
FASEB J. 2007 May;21(7):1527-38
17289923
Eur Biophys J. 2009 Dec;39(1):75-81
19455319
J Neurosci. 1999 Nov 1;19(21):9235-41
10531427
BMC Physiol. 2008 Mar 26;8:6
18366784
Mol Pharmacol. 1984 Jul;26(1):75-82
6087119
Pharmacol Rev. 2006 Dec;58(4):837-62
17132857
Nature. 1997 Jan 30;385(6615):446-50
9009193
PLoS Negl Trop Dis. 2010 Aug 10;4(8):e790
20706630
Parasitology. 2009 Nov;136(13):1825-35
19281637
J Biol Chem. 2000 Aug 11;275(32):24575-82
10823819
Biotechniques. 2002 Mar;32(3):536-8, 540
11911656
Biophys J. 1996 May;70(5):2144-54
9172738