CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING |
Effect of Clotrimazole on the Pump Cycle of the
Na,K-ATPase
Gianluca Bartolommei 1, Nadège Devaux 2, Francesco Tadini-Buoninsegni 1, Maria Rosa Moncelli 1 and Hans-Juergen Apell 2*
1 University of Florence
2 University of Konstanz
* To whom correspondence should be addressed. E-mail: h-j.apell{at}uni-konstanz.de.
Submitted on March 14, 2008
Revised on April 12, 2008
Accepted on 22 April 2008
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Abstract |
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The effect of the antimycotic Clotrimazole (CLT) on the Na,K-ATPase was investigated using fluorescence and electrical measurements. The results obtained by steady state fluorescence experiments with the electrochromic styryl dye RH421, were combined with those achieved by a pre-steady method based on fast solution exchange on a solid supported membrane that adsorbs the protein. Both techniques are suitable to monitor the electrogenic steps of the pump cycle and are in general complementary, yielding distinct kinetic information. The experiments show clearly that CLT affects specific partial reactions of the pump cycle of the Na,K-ATPase with an affinity in the low micromolar range and in a reversible manner. All results can be consistently explained by proposing the CLT-promoted formation of an ion-occluded-CLT-bound conformational E2 state, E2CLT(X2) that acts as a "dead-end" side track of the pump cycle, where X stands for H+ or K+. Na+ binding, enzyme phosphorylation, and Na+ transport were not affected by CLT, and at high CLT concentrations about one third of the enzyme remained active in the physiological transport mode. The presence of Na+ and K+ destabilized the inactivated form of the Na,K-ATPase.
Key Words:
charge movements, electrochromic fluorescent dyes, electrogenicity, inhibition mechanism, kinetics, solid supported membranes
