Wang J and Dahl G (2010), SCAM analysis of Panx1 suggests a peculiar pore...

XB-ART-42964

J Gen Physiol 2010 Nov 01;1365:515-27. doi: 10.1085/jgp.201010440.

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SCAM analysis of Panx1 suggests a peculiar pore structure.

Wang J , Dahl G .

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Vertebrates express two families of gap junction proteins: the well-characterized connexins and the pannexins. In contrast to connexins, pannexins do not appear to form gap junction channels but instead function as unpaired membrane channels. Pannexins have no sequence homology to connexins but are distantly related to the invertebrate gap junction proteins, innexins. Despite the sequence diversity, pannexins and connexins form channels with similar permeability properties and exhibit similar membrane topology, with two extracellular loops, four transmembrane (TM) segments, and cytoplasmic localization of amino and carboxy termini. To test whether the similarities extend to the pore structure of the channels, pannexin 1 (Panx1) was subjected to analysis with the substituted cysteine accessibility method (SCAM). The thiol reagents maleimidobutyryl-biocytin and 2-trimethylammonioethyl-methanethiosulfonate reacted with several cysteines positioned in the external portion of the first TM segment (TM1) and the first extracellular loop. These data suggest that portions of TM1 and the first extracellular loop line the outer part of the pore of Panx1 channels. In this aspect, the pore structures of Panx1 and connexin channels are similar. However, although the inner part of the pore is lined by amino-terminal amino acids in connexin channels, thiol modification was detected in carboxyterminal amino acids in Panx1 channels by SCAM analysis. Thus, it appears that the inner portion of the pores of Panx1 and connexin channels may be distinct.

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Species referenced: Xenopus laevis
Genes referenced: panx1 tbx2 tpm3

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	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.

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