Structure and mechanism of the bacterial K+/H+ exchanger KefC

Following their discovery of the inner workings of a unique voltage-gated transporter, SLC9C1, in sperm, David Drew’s lab continues to delight us with their remarkable findings. This time, the focus is on the K+ efflux transporter KefC.

Potassium homeostasis is crucial for the survival of bacteria as it is involved in fundamental cellular processes, such as the maintenance of cytosolic pH, membrane potential, and turgor pressure. Consequently, potassium transport is tightly regulated by a wide variety of systems, including ion channels, as well as potassium uptake and efflux transporters.

Among these, Kef transporters play a crucial role in protecting bacteria from toxic electrophilic substances. Kef transporters couple potassium efflux with proton influx in gram-negative bacteria, and the resulting acidification of the cytosol efficiently prevents bacterial killing by reactive electrophilic compounds.

Despite its importance, the structural and mechanistic understanding of Kef transporters has remained limited until now.

A recent study by Ashutosh Gulati et al., published in Nature Communications, sheds light on the structure and molecular basis for K+-selectivity of KefC, a glutathione-gated K+/H+ exchanger in Escherichia coli.

Employing high-resolution (3.1 Å) cryo-electron microscopy (cryo-EM), the team resolved the structure of KefC in complex with AMP, AMP/GSH, and an ion-binding variant. Similar to the inward-facing conformation of Na+/H+ antiporters, the KefC transporter forms a homodimer. It also possesses C-terminal regulator of K+ conductance (RCK) domains, as present in some bacterial K+ ion channels. The domain-swapped helices in the RCK domains bind AMP and GSH, inhibiting transport by directly interacting with the ion-transporter module. The authors propose that glutathione adducts disrupt AMP/GSH-mediated inhibition, causing RCK detachment from the transporter, thereby activating KefC.

Utilizing SSM-based electrophysiology (SURFE²R), the study confirms KefC’s selectivity for K+ over Na+, consistent with its physiological role in maintaining potassium efflux in response to intracellular electrophilic stress. The electrophysiological assays demonstrate KefC’s binding affinity for K+ (Kd ≈ 9.5 mM).

Overall, this study provides significant insights into the biophysical and structural basis of K+ selectivity in KefC, opening avenues for understanding similar transport mechanisms in other biological systems. The proposed model for KefC activation through RCK domain detachment and interaction with GSH adducts offers a novel perspective on transporter regulation, which could have broader implications for designing modulators that can mimic or disrupt these interactions for therapeutic purposes.

Find the original article here: https://www.nature.com/articles/s41467-024-49082-7

Learn more about SSM-Based Electrophysiology and SURFE²R devices here: https://www.nanion.de/products/surfe2r-n1/