Effects of calmodulin and Ca2+ channel blockers on omega-conotoxin GVIA binding to crude membranes from alpha1B subunit (Cav2.2) expressed BHK cells and mice brain lacking the alpha1B subunits.
Characteristics for the specific binding of 125I-omega-CTX GVIA and 125I-omega-CTX MVIIC to crude membranes from BHKN101 cells expressing the alpha1B subunits of Cav2.2 channels and from mice brain lacking the alpha1B subunits of Cav2.2 channels, particularly, the effects of CaM and various Ca2+ channel blockers on these specific bindings were investigated. Specific binding of 125I-omega-CTX GVIA to the crude membranes from BHKN101 cells was observed, but not from control BHK6 cells. omega-CTX GVIA, omega-CTX MVIIC and omega-CTX SVIB inhibited the specific binding of 125I-omega-CTX GVIA to crude membranes from BHKN101 cells, and the IC50 values for omega-CTXGVIA, omega-CTX MVIIC and omega-CTX SVIB were 0.07, 8.5 and 1.7 nM, respectively. However, omega-agatoxin IVA and calciseptine at concentrations of 10(-9)-10(-6) M did not inhibit specific binding. Specific binding was also about 80% inhibited by 20 microg protein/ml CaM. The amount of 125I-omega-CTX GVIA (30 pM) specifically bound to membranes from brain of knockout mice lacking alpha1B subunits of Cav2.2 channels was about 30% of that to the crude membranes from brain of wild-type. On the other hand, specific binding of 125I-omega-CTX MVIIC (200 pM) was observed on the crude membranes of both BHKN101 and control BHK6 cells. The specific binding of 125I-omega-CTX MVIIC (200 pM) was not inhibited by omega-CTX GVIA and omega-CTX SVIB, and also omega-Aga IVA and calciseptine at concentrations of 10(-9)-10(-7) M, although specific binding was almost completely dose dependently inhibited by non-radiolabeled omega-CTX MVIIC (IC50 value was about 0.1 nM). 20 microg protein/ml CaM did not inhibit specific binding. Therefore, these results suggest that BHKN101 cells have a typical Cav2.2 channels which are also inhibited by CaM and have not specific binding sites for omega-CTX MVIIC, although omega-CTX MVIIC is a blocker for both Cav2.1 (alpha1A; P/Q-type) and Cav2.2 channels.
Wada
Tetsuyuki
T
Department of Biological Chemistry, School of Pharmaceutical Sciences, Kinki University , Kowakae 3-4-1, 577-8502, Higashiosaka, Japan.
Imanishi
Takashi
T
Kawaguchi
Akinori
A
Mori
Masayuki X
MX
Mori
Yasuo
Y
Imoto
Keiji
K
Ichida
Seiji
S
D000818:Animals; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020864:Calcium Channels, N-Type / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology*; D002460:Cell Line; D006224:Cricetinae; D007457:Iodine Radioisotopes / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D020866:omega-Conotoxin GVIA / Q000378:metabolism*
C500963:Cacna1b protein, mouse; D002121:Calcium Channel Blockers; D020864:Calcium Channels, N-Type; D002147:Calmodulin; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D020866:omega-Conotoxin GVIA