1. High Resolution Structure of the Open NaK Channel Joon Sik Kang

2. Background Opening & Closing of ion channel pores in response to ligand or voltage is the fundamental studies of channel physiology. Most of the studies have been from the K+ channels of which KcsA(closed) and MthK(open) have been accepted. Closed Conformation: Straight line helices and bundle crossing Open Conformation: Inner helices bent at glycine residue(gating hinge)

3. Objective/Method Nak Channel chosen, because it shares overall sequence and structural similarities with KcsA. Crystallized the truncated construct of NaK(NaKND19), which lacks N-terminal M0 helix, and viewed at 1.6 A Resolution. Crystallized in the presence of monovalent cations. Unexpectidly, NaKND19 revealed intracellular gate in an open conformation.

4. Result

5. Overall NaK Structure

6. 2 subunits, each use 3 four-fold counterparts to form a functional channel tetramer. Region surrounding selectivity filter are similar for all channels, suggesting that this region remain static during channel gating. Major Structure change occur in the pore lining inner-helices just below the selectivity filter.

7. Overall NaK Structure Inner helices of NaK straight, as those of KcsA whereas NaKND19 develops bend (1D), occuring at Gly 87 in NaK. Inner helices undergo bending and twisting around the helical axis.

8. Conservation of Pore Opening Mechanism In the closed conformation, inner and pore helices superimpose well, but outer helices are slightly shifted translationally. The fact that all 4 of these have similarities in structure indicate that there is conserved gating mechanics on tetrameric cation channel. But. Outer helices do not need same level of conservation.

9. Inter&Intrasubunit interaction in closed/Open Pore

10. The intrasubunit interaction between inner&outer helices remain similar between closed and open state, because of the concurrent movement of both helices and twisting. However, interaction is disrupted upon pore opening (zoom 3).

11. Continued In Closed conformation, Phe 92 from each inner helix form contact with neighboring Phe 92. (Zoom 2A) Inner helix bending in open conformation causes Phe 92 to swing away and point its side chain toward the central ion-conducting pathway.

12. Effect Of ion-conduction pathway size on channel conduction In a closed NaK channel, side chain resides around bundle crossing form constriction points along the ion-conduction pathway. Bundle of inner helices in open form, disrupts the bundle crossing. However a new point is created through the Phe 92.

13. Continued In most cases, Positions of Phe 92 is occupied by smaller residues such as alanine. Positioning its aromatic ring toward the ion pathway, Phe-92 form constriction point about size of 6.5 A in diameter. However, MthK, being occupied by smaller alanines, have larger diameter, allowing higher conduction rate.

14. Effect of Ion-Conduction Pathway size on channel conduction

15. Continued F92A mutant shows an pathway that is less hindered with the diameter of 10.5 A Smaller the side chain residue, wider the ion passageway, thus easier access to the selectivity filter in the cation channels.

16. Discussion The region surrounding the selectivity filter remain static during the channel gating. However, inner helices at Gly 87, just below the filter, show major conformational change. NaKND19 and MthK similar in the use of Glycine as a gating hinge to allow for inner helix bending.

17. Discussion Overall, intra/intersubunit interaction in the open state is not as extensive as closed state due to helix bundle crossing, MO helices, use of detergent, and protein packing. In conclusion, KcsA in open state would be similar to that of NaKND19 or MthK. NaK (closed) and NaKND19(open) represent the general structure of tetrameric cation channel pore.