GFP-tagged channel subunit, the red fluorescence of the Alexa Fluor 546 antibody as well as the overlay of each, respectively. The yellow-brown colour in the overlay indicates colocalization. Acknowledgments We thank Tine Bruyns and Gerda Van de Vijver for technical help, Dr. Andrew Shepherd for linguistic corrections and Dr. Miguel Holmgren for valuable discussions around the manuscript. Author Contributions Conceived and developed the experiments: EB DJS. Performed the experiments: EB EM AVT GR. Analyzed the information: EB. Contributed reagents/materials/analysis tools: JPT. Wrote the paper: EB JPT DJS. References 1. Gutman GA, Chandy KG, Grissmer S, Lazdunski M, McKinnon D, et al. International Union of Pharmacology. LIII. Nomenclature and molecular relationships of voltage-gated potassium channels. Pharmacol Rev 57: 473508. 2. Long SB, Campbell EB, MacKinnon R Crystal structure of a mammalian voltage-dependent Shaker household K+ channel. Science 309: 897903. 3. Lee TE, Philipson LH, Kusnetsov A, Nelson DJ Structural determinant for assembly of mammalian K+ channels. Biophys J 66: 667673. four. Shen NV, Pfaffinger PJ Molecular recognition and assembly sequences involved within the subfamily-specific assembly of voltage-gated K+ channel subunit proteins. Neuron 14: 625633. five. Xu J, Yu W, Jan YN, Jan LY, Li M Assembly of voltage-gated potassium channels. J Biol Chem 270: 2476124768. 6. Kobertz WR, Miller C K+ channels lacking the ‘tetramerization’ domain: implications for pore structure. Nat Struct Biol six: 11221125. 7. Tu LW, Santarelli V, Sheng ZF, Skach W, Discomfort D, et al. Voltage-gated k+ channels include multiple intersubunit association web pages. J Biol Chem 271: 1890418911. 8. Zerangue N, Jan 
YN, Jan LY An artificial tetramerization domain restores effective assembly of functional Shaker channels lacking T1. Proc Natl Acad Sci U S A 97: 35913595. 9. Bocksteins E, Snyders DJ Electrically silent Kv subunits: their molecular and functional characteristics. Physiology 27: 7384. 27/2/73 10. Ottschytsch N, Raes A, Van Hoorick D, Snyders DJ Madecassoside Obligatory heterotetramerization of three previously uncharacterized Kv channel alpha subunits identified in the human genome. 
Proc Natl Acad Sci U S A 99: 7986 7991. 11. Hugnot JP, Salinas M, Lesage F, Guillemare E, de Weille J, et al. Kv8.1, a brand new neuronal potassium channel subunit with precise inhibitory properties towards Shab and Shaw channels. EMBO J 15: 33223331. 12. Stocker M, Hellwig M, Kerschensteiner D Subunit assembly and domain analysis of electrically silent K+ channel alpha-subunits of the rat Kv9 subfamily. J Neurochem 72: 17251734. 13. Mohapatra DP, Siino DF, Trimmer JS Interdomain cytoplasmic interactions govern the intracellular trafficking, gating, and modulation from the Kv2.1 channel. J Neurosci 28: 49824994. 14. Bocksteins E, Labro AJ, Mayeur E, Bruyns T, Timmermans JP, et al. Conserved damaging charges within the N-terminal tetramerization domain mediate efficient assembly of Kv2.1 and Kv2.1/Kv6.4 channels. J Biol Chem 284: TA 02 3162531634. 15. Shen NV, Chen X, Boyer MM, Pfaffinger PJ Deletion evaluation of K+ channel assembly. Neuron 11: 6776. 16. Li M, Jan YN, Jan LY Specification of 23977191 subunit assembly by the hydrophilic amino- terminal domain from the Shaker potassium channel. Science 257: 12251230. 17. Bixby KA, Nanao MH, Shen NV, Kreusch A, Bellamy H, et al. Zn2+binding and molecular determinants of tetramerization in voltage-gated K+ channels. Nat Struct Biol 6: 3843. 18. Kreusch A, Pfaffinger PJ, Stevens CF, Cho.GFP-tagged channel subunit, the red fluorescence on the Alexa Fluor 546 antibody plus the overlay of both, respectively. The yellow-brown colour in the overlay indicates colocalization. Acknowledgments We thank Tine Bruyns and Gerda Van de Vijver for technical help, Dr. Andrew Shepherd for linguistic corrections and Dr. Miguel Holmgren for useful discussions on the manuscript. Author Contributions Conceived and developed the experiments: EB DJS. Performed the experiments: EB EM AVT GR. Analyzed the data: EB. Contributed reagents/materials/analysis tools: JPT. Wrote the paper: EB JPT DJS. References 1. Gutman GA, Chandy KG, Grissmer S, Lazdunski M, McKinnon D, et al. International Union of Pharmacology. LIII. Nomenclature and molecular relationships of voltage-gated potassium channels. Pharmacol Rev 57: 473508. 2. Lengthy SB, Campbell EB, MacKinnon R Crystal structure of a mammalian voltage-dependent Shaker family members K+ channel. Science 309: 897903. three. Lee TE, Philipson LH, Kusnetsov A, Nelson DJ Structural determinant for assembly of mammalian K+ channels. Biophys J 66: 667673. four. Shen NV, Pfaffinger PJ Molecular recognition and assembly sequences involved in the subfamily-specific assembly of voltage-gated K+ channel subunit proteins. Neuron 14: 625633. 5. Xu J, Yu W, Jan YN, Jan LY, Li M Assembly of voltage-gated potassium channels. J Biol Chem 270: 2476124768. 6. Kobertz WR, Miller C K+ channels lacking the ‘tetramerization’ domain: implications for pore structure. Nat Struct Biol six: 11221125. 7. Tu LW, Santarelli V, Sheng ZF, Skach W, Pain D, et al. Voltage-gated k+ channels include several intersubunit association internet sites. J Biol Chem 271: 1890418911. 8. Zerangue N, Jan YN, Jan LY An artificial tetramerization domain restores efficient assembly of functional Shaker channels lacking T1. Proc Natl Acad Sci U S A 97: 35913595. 9. Bocksteins E, Snyders DJ Electrically silent Kv subunits: their molecular and functional traits. Physiology 27: 7384. 27/2/73 ten. Ottschytsch N, Raes A, Van Hoorick D, Snyders DJ Obligatory heterotetramerization of 3 previously uncharacterized Kv channel alpha subunits identified in the human genome. Proc Natl Acad Sci U S A 99: 7986 7991. 11. Hugnot JP, Salinas M, Lesage F, Guillemare E, de Weille J, et al. Kv8.1, a new neuronal potassium channel subunit with specific inhibitory properties towards Shab and Shaw channels. EMBO J 15: 33223331. 12. Stocker M, Hellwig M, Kerschensteiner D Subunit assembly and domain analysis of electrically silent K+ channel alpha-subunits from the rat Kv9 subfamily. J Neurochem 72: 17251734. 13. Mohapatra DP, Siino DF, Trimmer JS Interdomain cytoplasmic interactions govern the intracellular trafficking, gating, and modulation of your Kv2.1 channel. J Neurosci 28: 49824994. 14. Bocksteins E, Labro AJ, Mayeur E, Bruyns T, Timmermans JP, et al. Conserved negative charges in the N-terminal tetramerization domain mediate efficient assembly of Kv2.1 and Kv2.1/Kv6.4 channels. J Biol Chem 284: 3162531634. 15. Shen NV, Chen X, Boyer MM, Pfaffinger PJ Deletion evaluation of K+ channel assembly. Neuron 11: 6776. 16. Li M, Jan YN, Jan LY Specification of 23977191 subunit assembly by the hydrophilic amino- terminal domain from the Shaker potassium channel. Science 257: 12251230. 17. Bixby KA, Nanao MH, Shen NV, Kreusch A, Bellamy H, et al. Zn2+binding and molecular determinants of tetramerization in voltage-gated K+ channels. Nat Struct Biol 6: 3843. 18. Kreusch A, Pfaffinger PJ, Stevens CF, Cho.
