Report

PAC, an evolutionarily conserved membrane protein, is a proton-activated chloride channel

See allHide authors and affiliations

Science  26 Apr 2019:
Vol. 364, Issue 6438, pp. 395-399
DOI: 10.1126/science.aav9739
  • Fig. 1 RNA interference (RNAi) screen identifies PAC as being essential for the PAC currents.

    (A) Acid-induced and I-dependent fluorescence change (normalized to the baseline) in HEK293-YFP cells. (B) Acid-induced quenching response-to-pH relationship (n = 12 wells). Unapparent error bars are smaller than symbols. (C) Z-score (the number of standard deviations from the mean) of individual siRNAs (PAC siRNAs are in red) plotted according to the rank order. See supplementary materials and methods for details. (D) Acid-induced fluorescence change, (E) quenching response (n = 6 wells), and (F) PAC mRNA knockdown (n = 3 wells) in HEK293-YFP cells transfected with control or PAC siRNA. (G) Whole-cell currents induced by extracellular pH 4.6 in WT HEK293 cells. The transient inward current at −100 mV is blocked by 100 μM amiloride, an ASIC channel blocker. (H) ICl,H monitored by voltage-step (left) and voltage-ramp (right) protocols in WT HEK293 cells. (I) Whole-cell currents induced by extracellular pH 4.6 in PAC KO HEK293 cells. The normal amiloride-sensitive ASIC current is apparent at −100 mV. (J) ICl,H monitored by voltage-step (left) and voltage-ramp (right) protocols in PAC KO HEK293 cells. (K) Current densities at +100 mV induced by extracellular pH 4.6 in WT and two PAC KO HEK293 cells. KO1 was used throughout the study unless stated otherwise. Data points or bars represent mean ± SEM. **P < 0.01, ***P < 0.001 [two-tailed Student’s t test in (E) and (F); one-way analysis of variance (ANOVA) with Bonferroni post hoc test in (K)].

  • Fig. 2 Human PAC recapitulates the properties of the endogenous PAC channel.

    (A) Hydrophobicity plot (top) and predicted 2 TM topology (bottom) of hPAC. Red circles indicate FLAG insertion sites. (B) Nonpermeabilized and permeabilized HEK293 cells expressing FLAG-tagged hPAC were immunostained with antibody against FLAG. Scale bars: 10 μm. (C) Time courses (left) and current densities (right) of extracellular pH 4.6–induced whole-cell currents at +100 mV in vector- or hPAC-transfected PAC KO HEK293 cells. (D) hPAC-mediated currents at different pH values monitored by voltage-step (left) and voltage-ramp (right) protocols. (E) Normalized current-to-pH relationship of hPAC recorded at RT (n = 10 to 15 cells) or 37°C (n = 11 to 12 cells). Current at pH 4.6 and +100 mV is set to 1.0. (F) pH 6.0-induced hPAC current recorded at 37°C. (G) Representative IV relationship recorded in extracellular I, Br, or Cl pH 4.6 solution and (H) anion selectivity in WT (n = 10 cells) and PAC KO HEK293 cells expressing hPAC (n = 14 cells). Arrows in (G) indicate the reversal potentials. Data points or bars represent mean ± SEM. n.s., not significant [two-tailed Student’s t test in (H)].

  • Fig. 3 Zebrafish PAC encodes a PAC channel with distinct properties.

    (A) Phylogenetic tree of PAC from different vertebrate animals (sequence similarity to human) generated with the ClustalW program. (B) fPAC-mediated currents at different pH values monitored by voltage-step (left) and voltage-ramp (right) protocols. Arrows point to the tail currents compared with those of hPAC (Fig. 2D). (C) Normalized current-to-pH relationship of hPAC (current at pH 4.6 is set to 1.0, n = 10 to 15 cells) and fPAC (current at pH 4.0 is set to 1.0, n = 5 to 12 cells) recorded at RT. (D) Examples and (E) time constant values (τ) of time-dependent facilitation at +20- to +100-mV voltage steps for hPAC (n = 7 cells) and fPAC (n = 11 cells). τ values were determined by fitting the currents with a single exponential function. (F) Representative IV relationship recorded in extracellular pH 4.6 containing I or Cl solution and (G) I versus Cl permeability for hPAC and fPAC. Arrows in (F) indicate the reversal potentials. Data points or bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 [two-way ANOVA with Bonferroni post hoc test in (E); two-tailed Student’s t-test in (G)].

  • Fig. 4 PAC contributes to acid-induced cell death and ischemic brain injury.

    (A) Transcripts per million (TPM) of PAC gene in selective human tissues from RNA-sequencing data (n = 2 to 7 biological replicates) (15). (B) ICl,H and (C) baseline-subtracted pH 4.6–induced currents at +100 mV in WT or Pac KO primary mouse neurons. Recordings were performed in the presence of 100 μM amiloride to block ASICs. (D) Images of neurons stained with Hoechst 33342 (blue) for nuclei of all neurons and with propidium iodide (red) for nuclei of dead neurons. Scale bar: 50 μm. (E) Percentage of acid-induced neuronal cell death (n = 8 wells, 1-hour acid treatment and 24-hour recovery in culture medium) (9). (F) Triphenyltetrazolium chloride staining (necrotic tissue in white), (G) total infarct volume (normalized to total volume of the ipsilateral hemisphere as 100%), and (H) neurological score 1 day after pMCAO (n = 12 and 13 for WT and Pac KO mice, respectively). Bars represent mean ± SEM. *P < 0.05, ***P < 0.001 [two-tailed Student’s t test in (E) and (G); Mann–Whitney U test in (H)].

Supplementary Materials

  • PAC, an evolutionarily conserved membrane protein, is a proton-activated chloride channel

    Junhua Yang, Jianan Chen, Maria del Carmen Vitery, James Osei-Owusu, Jiachen Chu, Haiyang Yu, Shuying Sun, Zhaozhu Qiu

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Materials and Methods 
    • Figs. S1 to S8 
    • References 

Stay Connected to Science

Navigate This Article