The effect of the antimycotic drug 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-state method based on fast solution exchange on a solid supported membrane that adsorbs the protein. Both techniques are suitable for monitoring 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 E(2) state, E(2)(CLT)(X(2)) 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 approximately (1/3) 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.

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2483785PMC
http://dx.doi.org/10.1529/biophysj.108.133546DOI Listing

Publication Analysis

Top Keywords

pump cycle
16
cycle nak-atpase
8
clotrimazole pump
4
cycle
4
nak-atpase
4
nak-atpase antimycotic
4
antimycotic drug
4
drug clotrimazole
4
clt
4
clotrimazole clt
4

Similar Publications

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!