By James D. Stockand, Mark S. Shapiro
The varied purposes during this quantity diversity from the examine of allosteric rules of ion channel task utilizing a vintage mutagenesis strategy, to the learn of channel subunit stoichiometry utilizing a singular biophysical procedure according to fluorescence resonance strength move. Highlights comprise tools for heterologous expression of ion channels in cells, for making a choice on channel structure-function, and for learning channel rules.
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Extra resources for Ion Channels: Methods and Protocols
Phenol/chloroform/isoamyl alcohol 25:24:1 (Sigma Fluka, St. Louis, MO; use caution as this mix is caustic). 8. Chloroform/isoamyl alcohol 49:1 (Sigma Fluka). 9. Pellet Paint coprecipitant (Novagen, Madison, WI). 10. Millipore UltraFree FilterSpin columns (Fisher). 11. Rapid DNA Ligation Kit (Roche). 12. One Shot competent Escherichia coli (Invitrogen). 13. Luria-Bertani (LB) medium. 14. Bacto Agar for plates (BD Biosciences, Clontech). 15. LB with kanamycin (KAN; 50 µg/mL). 16. KAN plates: LB agar plates with kanamycin (50 µg/mL).
Beech, D. , Shapiro, M. , and Wollmuth, 1. L. P. (1995) Multiple G-protein-coupled pathways inhibit N-type Ca2+ channels of neurons. Life Sci. 56, 989–992. 2 Gamper, N. and Shapiro, M. S. (2003) Calmodulin mediates Ca 2+-dependent 2. modulation of M-type K+ channels. J. Gen. Physiol. 122, 17–31. , Stockand, J. , and Shapiro, M. S. (2003) Subunit-specific modula3. tion of KCNQ potassium channels by Src tyrosine kinase. J. Neurosci. 23, 84–95. , and Shapiro, M. S. (2004) Phos4. phatidylinositol 4,5-bisphosphate signals underlie receptor-specific Gq/11-mediated modulation of N-type Ca2+ channels.
14. Bacto Agar for plates (BD Biosciences, Clontech). 15. LB with kanamycin (KAN; 50 µg/mL). 16. KAN plates: LB agar plates with kanamycin (50 µg/mL). 17. QIAprep Spin Miniprep Kit (Qiagen). 3. Subcloning of Complementary DNA for POI-IRES-EGFP Into pSFV 1. 2. 2. pSFV and pSFVHelper2 vectors (Invitrogen; see Note 1). 3. Guanosine 5'-triphosphate (GTP)/cytidine 5'-triphosphate (CTP) mix: Mix of 1 mM each from PCR grade 100 mM dNTP set (Invitrogen) in MBG water. 4. Large (Klenow) fragment DNA polymerase I (Klenow; New England Biolabs).