Determinants of ion channel versus transporter mechanism in the K(+) transporter superfamily

• Applicant: Professorin Dr. Inga Hänelt
• Subject Area: Biochemistry ; Biophysics ; Structural Biology
• Term: from 2014 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 266161834

• Final Report Year: 2023

Final Report Abstract

The exchange of substances across membranes is controlled by two classes of proteins: channels and transporters. Traditionally, these classes were considered distinct due to their differentiated functions and varying translocation rates. However, at the time of the proposal various data challenged this separation. To explore the commonalities and differences between channels and transporters, we suggested to study the structure-function relationship of bacterial members of the K+ transporter superfamily. These systems appeared to function as active transporters with regulatory subunits, yet their central subunits resembled K+ channels. Specifically, the KtrAB system, initially proposed as a K+/Na+ symporter, and the KdpFABC complex, a primary active K+ pump, were investigated. In the course of our studies, we showed that KtrAB in fact functions as Na+- and nucleotidedependent K+ channel. Na+, likely binding in proximity to KtrBs’ selectivity filters, increases the selectivity for K+ binding and modulates the open probability of the gates. ATP and ADP binding to the regulator KtrA gating ring, controls channel gating at a distance. In the presence of ADP, extended helices of the KtrB dimer are interacting with KtrA, stabilizing the closed gate. In the presence of ATP, the KtrA ring undergoes a conformational change, likely breaking the helices and forming helical hairpins, which triggers gate opening. The latter probably is accompanied by an interaction of KtrBs’ N-termini with the surrounding membrane. KdpFABC in contrast is a true chimera of a K+ channel and a P-type ATPase. We showed that K+ selectively binds to KdpA’s selectivity filter, from where it is forwarded through an intersubunit tunnel to KdpB’s canonical binding site. After a transition from an E1-P to an E2-P state, K+ is released into the cytosol via a second half-channel. Further, we elucidated the structural basis for KdpFABC inhibition by a serine phosphorylation. At elevated external K+, KdpFABC is trapped in an off-cycle E1-P state avoiding the further pumping of K+. The state is required to alleviate electrostatic clashes occurring in the preceding high-energy E1-P state. In summary, our studies of the bacterial K+ transport systems KtrAB and KdpFABC have revealed remarkable nuances in their functions and mechanisms. KtrAB challenges conventional norms derived from canonical 2-TM K+ channels. Similarly, KdpFABC emerges as an extraordinary chimera of a K+ channel and a P-type ATPase that ensures highly affine, selective, and active K+ transport. The intriguing question remains whether the fusion of a K+ channel and a P-type ATPase occurred to serve this particular function, or whether such unions were the precursors for today's predominant P-type ATPases but repelled the additional channel subunit during evolution.

Publications

• Author response: Helical jackknives control the gates of the double-pore K+ uptake system KtrAB.
  Diskowski, Marina; Mehdipour, Ahmad Reza; Wunnicke, Dorith; Mills, Deryck J.; Mikusevic, Vedrana; Bärland, Natalie; Hoffmann, Jan; Morgner, Nina; Steinhoff, Heinz-Jürgen; Hummer, Gerhard; Vonck, Janet & Hänelt, Inga
  
• Cryo-EM structures of KdpFABC suggest a K+ transport mechanism via two inter-subunit half-channels. Nature Communications, 9(1).
  Stock, C.; Hielkema, L.; Tascón, I.; Wunnicke, D.; Oostergetel, G. T.; Azkargorta, M.; Paulino, C. & Hänelt, I.
  
• Native mass spectrometry goes more native: investigation of membrane protein complexes directly from SMALPs. Chemical Communications, 54(97), 13702-13705.
  Hellwig, Nils; Peetz, Oliver; Ahdash, Zainab; Tascón, Igor; Booth, Paula J.; Mikusevic, Vedrana; Diskowski, Marina; Politis, Argyris; Hellmich, Yvonne; Hänelt, Inga; Reading, Eamonn & Morgner, Nina
  
• A channel profile report of the unusual K+ channel KtrB. Journal of General Physiology, 151(12), 1357-1368.
  Mikušević, Vedrana; Schrecker, Marina; Kolesova, Natalie; Patiño-Ruiz, Miyer; Fendler, Klaus & Hänelt, Inga
  
• Membrane Protein Solubilization and Quality Control: An Example of a Primary Active Transporter. Methods in Molecular Biology, 93-103.
  Stock, Charlott & Hänelt, Inga
  
• Structural basis of proton-coupled potassium transport in the KUP family. Nature Communications, 11(1).
  Tascón, Igor; Sousa, Joana S.; Corey, Robin A.; Mills, Deryck J.; Griwatz, David; Aumüller, Nadine; Mikusevic, Vedrana; Stansfeld, Phillip J.; Vonck, Janet & Hänelt, Inga
  
• Two Ways To Convert a Low-Affinity Potassium Channel to High Affinity: Control of Bacillus subtilis KtrCD by Glutamate. Journal of Bacteriology, 202(12).
  Krüger, Larissa; Herzberg, Christina; Warneke, Robert; Poehlein, Anja; Stautz, Janina; Weiß, Martin; Daniel, Rolf; Hänelt, Inga & Stülke, Jörg
  
• Deciphering ion transport and ATPase coupling in the intersubunit tunnel of KdpFABC. Nature Communications, 12(1).
  Silberberg, Jakob M.; Corey, Robin A.; Hielkema, Lisa; Stock, Charlott; Stansfeld, Phillip J.; Paulino, Cristina & Hänelt, Inga
  
• Molecular Mechanisms for Bacterial Potassium Homeostasis. Journal of Molecular Biology, 433(16), 166968.
  Stautz, Janina; Hellmich, Yvonne; Fuss, Michael F.; Silberberg, Jakob M.; Devlin, Jason R.; Stockbridge, Randy B. & Hänelt, Inga
  
• Inhibited KdpFABC transitions into an E1 off-cycle state. eLife, 11.
  Silberberg, Jakob M.; Stock, Charlott; Hielkema, Lisa; Corey, Robin A.; Rheinberger, Jan; Wunnicke, Dorith; Dubach, Victor R. A.; Stansfeld, Phillip J.; Hänelt, Inga & Paulino, Cristina