Wang J , Dahl G .

GM48610 NIGMS NIH HHS , R01 GM048610 NIGMS NIH HHS

	Figure 1. Topology of mouse Panx1 based on hydrophobicity plots, site-specific antibodies, and glycosylation analysis (Locovei et al., 2006a; Boassa et al., 2007). (A) The topology of Panx1 in the membrane appears to be similar to that of connexins, with four predicted TM segments, two extracellular loops, and cytoplasmic localization of the amino and carboxy termini (Locovei et al., 2006a; Boassa et al., 2007; Penuela et al., 2007). Based on hydrophobicity analysis by the software programs TMpred, TMHMM, and DNA Star, the following boundaries for the TM segments are predicted: M38-I60 for TM1, F108-W127 for TM2, L217-236 for TM3, and L275-I296 for TM4. Because of uncertainty of the predictors, the boundaries in the figure are represented by a color gradient. Positions of endogenous cysteines are indicated with red numbers. The beginning (arrows) and end of stretches of amino acids substituted by cysteines for SCAM analysis are indicated by black numbers. (B) Localization of Panx1 SCAM mutants not forming functional channels. Clusters of these mutants are in the amino terminus, the first TM segment, and the third TM segment, indicating a structural importance of these moieties. (C) Positions of modified cysteines.
	Figure 2. Membrane currents induced by voltage steps from −60 to +60 mV at a rate of five/minute in oocytes expressing wt Panx1 (A and B) or Panx1 C426S. (A) 100 µM MBB induced a twofold response in wt Panx1 channels consisting of a reversible and an irreversible component (arrows). The reversible component was inducible by a subsequent application of MBB. The irreversible component was used to assess channel inhibition in all subsequent figures. (B) PEG400 and PEG600 at 1 mM caused a similar reversible channel inhibition as MBB. (C) 100 µM MBB applied to Panx1 C426S channels exclusively caused the reversible inhibition. (D) Quantitative analysis of irreversible inhibition of membrane currents by 100 µM MBB in oocytes expressing wt Panx1 and its cysteine replacement mutants C426S, C40S, C136S, C215S, C227S, and C346S.
	Figure 3. Micrographs of oocytes expressing Panx1 C40S or Panx1 C346S (top and bottom) incubated in OR2 medium (left) or in OR2 with 100 µM CBX (right). Pictures were taken 20 h after injection of mRNA.
	Figure 4. Dose-dependent modification of engineered cysteines in Panx1 by MBB (A) or MTSET (B). Membrane currents of oocytes expressing Panx1 I60C were attenuated by 10 or 100 µM MBB and by 100 µM or 1 mM MTSET. Both components, the reversible and the irreversible inhibition of the currents, were affected in a dose-dependent way. The reversible component was barely detectable at the lower MBB or MTSET concentrations.
	Figure 5. SCAM analysis of the four TM segments of Panx1, with MBB as thiol reagent. The amino acids are arranged in ascending order for TM1 and TM3 and in descending order for TM2 and TM4 to reflect their directional orientation in the membrane. Means ± SE of irreversible current inhibition by 100 µM MBB are plotted. The number of oocytes analyzed is indicated on the right. NC, no channel activity was detected. *, P < 0.05; **, P < 0.01 (vs. Panx1 C426S control).
	Figure 6. SCAM analysis of he four TM segments of Panx1, with MTSET as thiol reagent. Conditions were the same as described in Fig. 5, except that 1 mM MTSET was used as thiol reagent.
	Figure 7. Effects of various thiol reagents on Panx1 F54C currents. Oocytes expressing Panx1 F54C were held at −60 mV and pulses to +60 (A) or +20 mV (B–E) were applied to activate the channel. To test for cysteine accessibility, 10 µM MBB (A), 1 mM MTSET (B), 1 mM MTSET followed by 2 mM dithiothreitol (DTT; C), 1 mM MTSES (D), 1 mM MTSBn (E), 100 µM pyrenyl maleimide (F), or 100 µM MTS-fluorescein (G) was applied.
	Figure 8. SCAM analysis of NH2 and C terminus of Panx1. Conditions were the same as described in Fig. 5.
	Figure 9. Effect of 1 mM MBB on ATP release from oocytes expressing wt Panx1 (A) and Panx1 T62C (B). Oocytes were preincubated with 150 mM KGlu for 10 min, and then transferred to fresh KGlu solution for a 10-min incubation period before ATP measurement in the supernatant (left column). MBB was included in the preincubation solution (right column). The middle column in A shows data for oocytes preincubated in OR2 containing MBB, and then transferred to KGlu solution. The means of ATP released from uninjected oocytes subjected to identical treatments were subtracted.

Akabas, Acetylcholine receptor channel structure probed in cysteine-substitution mutants. 1992, Pubmed, Xenbase

Akabas, Acetylcholine receptor channel structure probed in cysteine-substitution mutants. 1992, Pubmed , Xenbase
Bao, Pannexin membrane channels are mechanosensitive conduits for ATP. 2004, Pubmed , Xenbase
Bao, Innexins form two types of channels. 2007, Pubmed , Xenbase
Bennett, Gap junctions: new tools, new answers, new questions. 1991, Pubmed
Boassa, Pannexin1 channels contain a glycosylation site that targets the hexamer to the plasma membrane. 2007, Pubmed
Boassa, Trafficking dynamics of glycosylated pannexin 1 proteins. 2008, Pubmed , Xenbase
Bruzzone, Pannexins, a family of gap junction proteins expressed in brain. 2003, Pubmed , Xenbase
Bruzzone, Pharmacological properties of homomeric and heteromeric pannexin hemichannels expressed in Xenopus oocytes. 2005, Pubmed , Xenbase
Chuang, An innexin-dependent cell network establishes left-right neuronal asymmetry in C. elegans. 2007, Pubmed , Xenbase
Dahl, Pannexin: to gap or not to gap, is that a question? 2006, Pubmed
Delmar, Structural bases for the chemical regulation of Connexin43 channels. 2004, Pubmed
Goodenough, Topological distribution of two connexin32 antigenic sites in intact and split rodent hepatocyte gap junctions. 1988, Pubmed
Holmgren, On the use of thiol-modifying agents to determine channel topology. 1996, Pubmed , Xenbase
Hu, Exchange of conductance and gating properties between gap junction hemichannels. 1999, Pubmed , Xenbase
Hu, Conductance of connexin hemichannels segregates with the first transmembrane segment. 2006, Pubmed , Xenbase
Iglesias, Pannexin 1: the molecular substrate of astrocyte "hemichannels". 2009, Pubmed
Kaplan, The yeast mitochondrial citrate transport protein. Probing the secondary structure of transmembrane domain iv and identification of residues that likely comprise a portion of the citrate translocation pathway. 2000, Pubmed
Kronengold, Single-channel SCAM identifies pore-lining residues in the first extracellular loop and first transmembrane domains of Cx46 hemichannels. 2003, Pubmed , Xenbase
Liu, Dynamic rearrangement of the outer mouth of a K+ channel during gating. 1996, Pubmed
Locovei, Pannexin 1 in erythrocytes: function without a gap. 2006, Pubmed , Xenbase
Locovei, Activation of pannexin 1 channels by ATP through P2Y receptors and by cytoplasmic calcium. 2006, Pubmed , Xenbase
Locovei, Pannexin1 is part of the pore forming unit of the P2X(7) receptor death complex. 2007, Pubmed , Xenbase
Lü, Silver as a probe of pore-forming residues in a potassium channel. 1995, Pubmed , Xenbase
Ma, Cosegregation of permeability and single-channel conductance in chimeric connexins. 2006, Pubmed , Xenbase
Maeda, Structure of the connexin 26 gap junction channel at 3.5 A resolution. 2009, Pubmed
Maroto, Brefeldin A block of integrin-dependent mechanosensitive ATP release from Xenopus oocytes reveals a novel mechanism of mechanotransduction. 2001, Pubmed , Xenbase
Oh, Charges dispersed over the permeation pathway determine the charge selectivity and conductance of a Cx32 chimeric hemichannel. 2008, Pubmed , Xenbase
Panchin, A ubiquitous family of putative gap junction molecules. 2000, Pubmed
Panchin, Evolution of gap junction proteins--the pannexin alternative. 2005, Pubmed
Penuela, Pannexin 1 and pannexin 3 are glycoproteins that exhibit many distinct characteristics from the connexin family of gap junction proteins. 2007, Pubmed
Pfahnl, Localization of a voltage gate in connexin46 gap junction hemichannels. 1998, Pubmed , Xenbase
Qiu, A permeant regulating its permeation pore: inhibition of pannexin 1 channels by ATP. 2009, Pubmed , Xenbase
Rabadan-Diehl, A connexin-32 mutation associated with Charcot-Marie-Tooth disease does not affect channel formation in oocytes. 1994, Pubmed
Ransford, Pannexin 1 contributes to ATP release in airway epithelia. 2009, Pubmed , Xenbase
Shi, Selected cysteine point mutations confer mercurial sensitivity to the mercurial-insensitive water channel MIWC/AQP-4. 1996, Pubmed , Xenbase
Silverman, The pannexin 1 channel activates the inflammasome in neurons and astrocytes. 2009, Pubmed , Xenbase
Silverman, Probenecid, a gout remedy, inhibits pannexin 1 channels. 2008, Pubmed , Xenbase
Skerrett, Identification of amino acid residues lining the pore of a gap junction channel. 2002, Pubmed , Xenbase
Trexler, Voltage gating and permeation in a gap junction hemichannel. 1996, Pubmed , Xenbase
Unwin, The structure of ion channels in membranes of excitable cells. 1989, Pubmed
Wang, Modulation of membrane channel currents by gap junction protein mimetic peptides: size matters. 2007, Pubmed , Xenbase
Yen, Gap junctional proteins of animals: the innexin/pannexin superfamily. 2007, Pubmed
Zhang, Identification of acetylcholine receptor channel-lining residues in the M1 segment of the beta-subunit. 1997, Pubmed , Xenbase
Zhou, Identification of a pore lining segment in gap junction hemichannels. 1997, Pubmed , Xenbase