Abstract

The Na+,K+-ATPase (NKA) is a vital membrane protein responsible for maintaining cellular ion gradients, and it is the pharmacological target of cardiotonic steroids (CTS) such as ouabain, digoxin, and marinobufagenin. While these compounds are known to inhibit NKA activity, the precise structural and conformational changes induced by different CTS on various NKA isoforms, particularly those exhibiting resistance, remain a critical area of study. This work investigates the binding of ouabain, digoxin, and marinobufagenin to the kidney $\alpha$1-Na$^+$,K$^+$-ATPase isoforms, specifically comparing the CTS-sensitive ($\alpha$1S) and CTS-resistant ($\alpha$1R) variants. Using biophysical techniques, we demonstrate that the binding of each CTS induces distinct conformational changes in the $\alpha$1 subunit. Ouabain, digoxin, and marinobufagenin do not act as simple, uniform inhibitors; instead, they each stabilize a unique enzyme conformation. Crucially, these differential conformational shifts are observed in both the CTS-sensitive and CTS-resistant $\alpha$1 isoforms. The findings suggest that the mechanism of CTS action involves a complex allosteric modulation of the NKA structure, which is dependent on the specific steroid ligand. These results provide new structural insights into the differential pharmacological effects of CTS and highlight the importance of ligand-specific conformational dynamics in the function and regulation of the Na$^+$,K$^+$-ATPase.