Clotrimazole inhibits the Ca2+-ATPase (SERCA) by interfering with Ca2+ binding and favoring the E2 conformation.
Clotrimazole (CLT) is an antimycotic imidazole derivative that is known to inhibit cytochrome P-450, ergosterol biosynthesis and proliferation of cells in culture, and to interfere with cellular Ca(2+) homeostasis. We found that CLT inhibits the Ca(2+)-ATPase of rabbit fast-twitch skeletal muscle (SERCA1), and we characterized in detail the effect of CLT on this calcium transport ATPase. We used biochemical methods for characterization of the ATPase and its partial reactions, and we also performed measurements of charge movements following adsorption of sarcoplasmic reticulum vesicles containing the ATPase onto a gold-supported biomimetic membrane. CLT inhibits Ca(2+)-ATPase and Ca(2+) transport with a K(I) of 35 mum. Ca(2+) binding in the absence of ATP and phosphoenzyme formation by the utilization of ATP in the presence of Ca(2+) are also inhibited within the same CLT concentration range. On the other hand, phosphoenzyme formation by utilization of P(i) in the absence of Ca(2+) is only minimally inhibited. It is concluded that CLT inhibits primarily Ca(2+) binding and, consequently, the Ca(2+)-dependent reactions of the SERCA cycle. It is suggested that CLT resides within the membrane-bound region of the transport ATPase, thereby interfering with binding and the conformational effects of the activating cation.
Bartolommei
Gianluca
G
Department of Chemistry, University of Florence, 50019 Sesto Fiorentino, Florence, Italy.
Tadini-Buoninsegni
Francesco
F
Hua
Suming
S
Moncelli
Maria Rosa
MR
Inesi
Giuseppe
G
Guidelli
Rolando
R
D000327:Adsorption; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D040761:Biomimetic Materials; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000378:metabolism*; D002462:Cell Membrane; D003022:Clotrimazole / Q000494:pharmacology*; D004789:Enzyme Activation; D018656:Muscle Fibers, Fast-Twitch; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases
D000935:Antifungal Agents; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D003022:Clotrimazole