1. Structural Biology of Membrane Proteins Problems of structure determination & Membrane-specific solutions KcsA structure Mechanistic insights KvAP and voltage sensing Shaker (K V 1.2)

2. Key Difficulties in MP Structure Available amounts….expression systems? Solubility and solubilization; detergents Microheterogeneity; PTMs? Mutational alteration to facilitate crystallization; affinity tags Homologs; genomics

3. Channel Structures Provide.. Details of structure Familial relationships Structural basis of ion selectivity Mechanism of ion dehydration by channel Linkage between pore and voltage sensor (for VS channels); gating mechanisms???

4. Genomics and Channel Structures VSKC homolog (kcsA) discovered in S. lividans as ORF Monomer MW ~18kD Hydropathy analysis predicts 2 TM domains 32% identity to shaker in “signature” sequence KvAP

5. What Defines a K + Channel? Founder of VS cation channel family Widely distributed in nature; multiple isoforms in most organisms Monomer MW ~70kD (eukaryotes) Activated by membrane depolarization Most isoforms have 6 predicted tm domains/monomer; functional tetramers Mutagenic analysis of pore blockers: pore between S5 and S6

6. Schrempf, et al (1995) “A prokaryotic potassium ion channel with two predicted transmembrane segments from S. lividans,” EMBO J 14, 5170. Cortes & Perozo (1997) “Structural dynamics of the S. lividans K channel: oligomeric stoichiometry and stability,” Biochemistry 36, 10343. Doyle et al (1998) “The structure of the potassium channel: molecular basis of K- conduction and selectivity,” Science 280, 69. KcsA: The 1 st Structure

7. VSKC Architecture?

8. Sequences of K-channel Pore Regions

9. Genomic Identification of KcsA Signature region

10. Is KcsA a Channel? Single channel analysis Current-voltage relationship

11. KcsA Oligomerizes in Detergent stain western 1. Cell extract 2. Cell super 3. Membranes 4. DDM extract 5. NTA- purified DDM extract

12. KcsA Tetramers Maintained in DDM

13. Structural Organization of KcsA

14. KcsA Tetramer Defines Central Pore K+K+

15. Polarity of KcsA Pore Region Blue, cationic; red, anionic; yellow, hydrophobic

16. Aromatic Clustering at KcsA Membrane Interface

17. Proposed Mechanism of Ion Translocation Lake Kalemia: note charge stabilization by helix dipoles

18. Ionic Stabilization by Helix Dipole in ClC Channels

19. Comparison of KcsA and ClC Pores ClC KcsA

20. How Does Voltage-sensing Work? SCAM analyses consistent with transverse S4 movement

21. KvAP: Structure and a Proposed Mechanism for Voltage Sensing. Jiang, MacKinnon et al (2003) “X-ray structure of a voltage-dependent K- channel,” Nature 423, 33-41; “The principle of gating charge movement in a voltage-dependent K channel,” Nature 423, 42-48. The KvAP Bombshell

22. Sequence Homology in Voltage-dependent K-channels

23. KvAP: a Voltage-sensitive Channel

24. Backbone Structure of the KvAP Tetramer

25. KvAP: The Side View Putative voltage sensor paddle

26. Architecture of KvAP Monomer

27. Proposed Paddle Movement during Gating

28. Questions about the KvAP Structure Validity of KvAP as paradigm? Solubilization? Modification? Crystallization aids? Impact of any/all on overall structure?

29. Science, 309: 897-908

30. Shaker and KvAP: Not quite the Same Beast

31. Accessory Subunits in K- channels

32. K-channel Pores Are Conserved

33. Structure of the shaker Tetramer

34. Backbone Comparison: KvAP vs shaker

35. Pore Communication with Voltage Sensor in Kv Channel The Kv1.2 Tetramer: note that the nearest neighbor of S4 in the red subunit is S5 from the blue subunit Closeup view showing the weak interaction between S4 and the pore region

36. Revised View of Voltage-sensing Note S6 changes between open and closed states.