Introduction Hille, 2001 Tamargo et al., 2004 Yost, 1999 Doyle et al., 1998 Jiang et al., 2002 2003 Long, Campbell & MacKinnon, 2005 Kavanaugh et al., 1991 MacKinnon & Yellen, 1990 Heginbotham & MacKinnon, 1992 Kavanaugh et al., 1992 Shaker Jarolimek et al., 1995 1996 Pascual et al., 1995 Crouzy, Berneche & Roux, 2001 Andalib et al., 2004 2+ Mozhayeva & Naumov, 1972 2+ MacKinnon, Latorre & Miller, 1989 Bretschneider et al., 1999 Shaker Quinn & Begenisich, 2001 Immke & Korn, 2000 Immke et al., 1999 Grissmer et al., 1994 Taglialatela et al., 1991 Shaker Materials and Methods HANNEL ONSTRUCTS Several K channel constructs were used in this study, including wild-type Kv2.1 and Kv3.1 channels (Chan Test Inc., Cleveland, OH). Three mutant Kv2.1 channels were also investigated: K356F, K382Q and the double mutant K356F/K382Q. The replacement amino acids were introduced into the Kv2.1 clone using a two-step polymerase chain reaction (PCR) protocol and the resulting mutants analyzed by DNA sequencing. OCYTE SOLATION AND ICROINJECTION Xenopus laevis Goldin (1992) 2+ M 2 M 2 2 Guide for the Care and Use of Laboratory Animals. LECTROPHYSIOLOGICAL ECORDINGS M V M SEM n M N D M 2 M 2 M 3 V V. 1 \documentclass[12pt]{minimal}
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\begin{document}$$ {\rm Fraction\ Blocked} = B_{max}{[B] \over [B] + K_{app}} $$\end{document} B B max K app Doyle et al., 1998 http://www.ca.expasy.org/spdbv/ http://www.accelrys.com/ Results HARGED ESIDUES IN THE UTER ESTIBULE OF HANNELS 1 Long et al., 2005 Shaker Shaker Shaker Kavanaugh et al., 1991 MacKinnon & Yellen, 1990 Figure 1. Bottom Top left Bottom left Right  All three channels contain several negative amino acids in this vestibule region (indicated in red in the alignment). Kv2.1 channels are unique in having two positively charged amino acids (K382 and K356) in the outer vestibule (blue in the alignment and illustrated as space-filling atoms in the structural diagrams). The influence of these lysine residues on TEA block is an important part of the present study. ONIC TRENGTH-DEPENDENT LOCK OF V HANNELS BY +  AND ALLAMINE 3+ Shaker Quinn & Begenisich, 2001 2A M V K app M M Grissmer et al., 1994 Jarolimek et al., 1995 Taglialatela et al., 1991 K app M, Figure 2. A Top V V Bottom V SEM K app M B max B Top V V Bottom V SEM  K app M B max  2B M 2B V K app M M Shaker Quinn & Begenisich, 2001 Shaker Shaker 3 Figure 3. Top V V Bottom V SEM  K app B max M  + LOCK OF V HANNELS AS OT ENSITIVE TO ONIC TRENGTH 3 M V K app M K app M Immke et al., 1999 Taglialatela et al., 1991 Shaker Shaker M n M + 2+ 2+ Begenisich, 1975 Frankenhaeuser & Hodgkin, 1957 Mozhayeva & Naumov, 1972 + 2+ V 4 Hartmann et al., 1991 Taglialatela et al., 1991 V Figure 4. A M B M  1 Taglialatela et al., 1991 1 3 5A Figure 5. A V SEM  K app M B max B V SEM  K app M B max  OSITIVE HARGE EUTRALIZATION IN V HANNELS 5A K app M K app M M n 1 5B K app M M V. 6A 6B K app M; M 2B Figure 6. A V SEM  K app M B max B V SEM  K app M B max  Discussion MacKinnon et al., 1989 Quinn & Begenisich, 2001 Kavanaugh et al., 1991 MacKinnon & Yellen, 1990 Shaker 1 Shaker 5 6 Shaker Shaker 4 7 Figure 7. Electrostatic potential map of the outer vestibule of Kv2.1 channels. Top view of the channel with the pore in the middle; the fourfold symmetry of these K channels is apparent. Electrostatic potentials computed as described in Materials and Methods and mapped onto the surface of the Kv2.1 channel. Negatively charged amino acid side chains are red, and blue represents positive charges. The two lysine residues that are the focus of this work (K336 and K382) as well as the other exposed charged amino acids are indicated in one of the four channel subunits.  Immke et al., 1999 Shaker 5B 6B Hartmann et al., 1991 Immke & Korn, 2000 Immke et al., 1999 + Zhou et al., 2001 Shaker + Thompson & Begenisich, 2005 MacKinnon et al., 1989 Hui et al., 2002 Shaker Shaker