Pannexin 1, an ATP release channel, is activated by caspase cleavage of its pore-associated C-terminal autoinhibitory region - PubMed (original) (raw)

Pannexin 1, an ATP release channel, is activated by caspase cleavage of its pore-associated C-terminal autoinhibitory region

Joanna K Sandilos et al. J Biol Chem. 2012.

Abstract

Pannexin 1 (PANX1) channels mediate release of ATP, a "find-me" signal that recruits macrophages to apoptotic cells; PANX1 activation during apoptosis requires caspase-mediated cleavage of PANX1 at its C terminus, but how the C terminus inhibits basal channel activity is not understood. Here, we provide evidence suggesting that the C terminus interacts with the human PANX1 (hPANX1) pore and that cleavage-mediated channel activation requires disruption of this inhibitory interaction. Basally silent hPANX1 channels localized on the cell membrane could be activated directly by protease-mediated C-terminal cleavage, without additional apoptotic effectors. By serial deletion, we identified a C-terminal region just distal to the caspase cleavage site that is required for inhibition of hPANX1; point mutations within this small region resulted in partial activation of full-length hPANX1. Consistent with the C-terminal tail functioning as a pore blocker, we found that truncated and constitutively active hPANX1 channels could be inhibited, in trans, by the isolated hPANX1 C terminus either in cells or when applied directly as a purified peptide in inside-out patch recordings. Furthermore, using a cysteine cross-linking approach, we showed that relief of inhibition following cleavage requires dissociation of the C terminus from the channel pore. Collectively, these data suggest a mechanism of hPANX1 channel regulation whereby the intact, pore-associated C terminus inhibits the full-length hPANX1 channel and a remarkably well placed caspase cleavage site allows effective removal of key inhibitory C-terminal determinants to activate hPANX1.

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Figures

FIGURE 1.

FIGURE 1.

C-terminal cleavage activates membrane-associated hPANX1 channels, independent of apoptosis. A, a CBX-sensitive current with _I_-V properties characteristic of hPANX1 was observed in Jurkat cells recorded under whole cell voltage clamp conditions with pipettes containing purified, activated Casp3 (400 n

m

). Inset, time series of peak current shows that the current developed slowly following whole cell access with pipettes containing active Casp3, but not with those containing heat-inactivated Casp3. B, when co-transfected with TEVp in HEK293T cells, hPANX1(TEV) generated robust whole cell CBX-sensitive currents with _I_-V properties characteristic of hPANX1. Inset, schematic of the hPANX1(TEV) construct with TEV protease site substituted for caspase site. C, averaged data (± S.E.) showing enhanced CBX-sensitive current in cells expressing both hPANX1(TEV) and TEVp, but not in those expressing hPANX1(TEV) alone or wild type channels with TEVp. *, p < 0.001. Inset, whole cell lysate and avidin pulldowns of biotinylated wild type hPANX1 and hPANX1(TEV). Note the surface localization of wild type and hPANX1(TEV) channels and loss of immunoreactivity to the C-terminal FLAG epitope when hPANX1(TEV) was co-expressed with TEVp. Data are representative of two independent experiments; mock indicates transfection with empty vector. D, TEVp expression induced To-Pro-3 dye uptake in HEK293T cells co-transfected with hPANX1(TEV) (blue) but not with wild type hPANX1 constructs (red); dye-loaded cells were not apoptotic, as indicated by the absence of annexin-V staining. % of max, percentage of maximum. E, the schematic depicts inside-out patch configuration with cytosolic face exposed to the bath solution containing Casp3 or TEVp. Upper and lower graphs, peak currents in inside-out patches from HEK293T cells expressing wild type hPANX1 (upper graph) or the hPANX1(TEV) construct (lower graph). The point of application of TEVp and caspase under stop flow conditions is indicated by arrows; the time during which CBX was superfused over the patch is indicated by the gray shading. F, CBX-sensitive current was induced by TEVp only in patches from cells expressing hPANX(TEV) and by Casp3 only in patches containing wild type PANX1. *, p < 0.05.

FIGURE 2.

FIGURE 2.

A C-terminal region immediately downstream of caspase cleavage site is essential for hPANX1 channel inhibition. A, schematic of hPANX1 C-terminal deletion constructs; the white box indicates FLAG epitope on the extreme C terminus, and the blue band indicates the caspase cleavage site. B, whole cell CBX-sensitive current density recorded from hPANX1 truncation mutants; only hPANX1Δ371 generated current. Inset, time series showing activation by Casp3 of basally inactive hPANX1Δ391 channels in inside-out patch. C, whole cell lysate and avidin pulldowns of biotinylated wild type hPANX1 and C-terminal truncation mutants (left); note that expression and surface localization for all silent truncation mutants was similar to wild type hPANX1 (n = 3). The highly active hPANX1Δ371 construct was expressed at lower levels, but could be detected with longer exposures (data not shown) or by performing the avidin pulldown on FLAG immunoprecipitates (IP, right; n = 2); mock indicates transfection with empty vector. D, alignment of mPanx1 and hPANX1, highlighting nonconserved residues in the critical region downstream of the caspase cleavage site. E, CBX-sensitive current density in cells expressing mPanx1, hPANX1, and the indicated point mutations in hPANX1. The upper boundary of the 95% confidence interval for hPANX1 current density is shown as a dashed line. *, p < 0.05. F, _I_-V relationships of CBX-sensitive current from mPanx1 and individual activating hPANX1 point mutants (normalized to peak current at +80 mV); note the more pronounced rectification, with little inward current at hyperpolarized potentials, in mutationally activated hPANX1 channels.

FIGURE 3.

FIGURE 3.

Isolated C terminus can act in trans to block hPANX1 current. A, whole cell CBX-sensitive current density from HEK293T cells co-expressing C-terminally truncated and activated hPANX1Δ371 along with the indicated constructs. Both the wild type hPANX1(Ct) and hPANX1(Ct)Δ391 strongly reduced averaged current amplitude and data dispersion, especially when expressed at the highest ratio (5:1), whereas the control constructs did not. *, p < 0.05. n.s., not significant. B, illustration of inside-out patch recording procedure (upper left). GST or purified, hPANX1 C-terminal GST fusion protein (GST-Cterm; see inset in panel C for Coomassie stained gel of GST fusion proteins) was applied to inside-out patches containing TEVp-activated hPANX1(TEV). C and D, _I_-V relationships of patch currents under the indicated conditions. control represents current following activation by TEVp (50 μg/ml); GST (C; ∼40 μ

m

) and C-terminal GST fusion protein (D; ∼20 μ

m

) were applied under stop flow conditions before CBX application (50 μ

m

). E, _I_-V relationship of CBX-sensitive current before and after C-terminal GST fusion protein application; note that the C-terminal protein only inhibits inward current in the hyperpolarized range (see arrows).

FIGURE 4.

FIGURE 4.

Relief of C-terminal block requires dissociation of C terminus from channel pore. A, whole cell CBX-sensitive current density was determined in HEK293T cells expressing hPANX1(TEV)F54C or hPANX1(TEV)F54C/C426S, with or without TEVp; where indicated, cells were pretreated with the reducing agent, TCEP, prior to recording (500 μ

m

for 2 h). In TEVp-expressing cells, currents were obtained from the cleaved hPANX1(TEV)F54C construct only after TCEP treatment; simultaneous mutation of Cys-426 abolished TCEP sensitivity of the truncated, F54C-mutated channel. *, p < 0.05. Insets, whole cell lysate and avidin pulldowns of biotinylated F54C and F54C/C426S channels, with or without TCEP pretreatment; expression and surface localization of the mutant channels were unaffected by TCEP treatment. Co-expressed GFP was used as a loading control; the antibody consistently recognized two bands in our expression system. Data are representative of three independent experiments; mock indicates transfection with empty vector. B, schematic interpretation of results. The pore-associated mutated TM1 residue, F54C, interacts with the endogenous C-terminal Cys-426 via a disulfide bond, such that the C terminus remains in the pore even after it has been cut by TEVp. Channel inhibition by the cleaved hPANX1F54C channel can be relieved by disrupting the cysteine cross-link, either by using TCEP or by mutation of Cys-426, the interacting residue.

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