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

Abstract

Severe local acidosis causes tissue damage and pain, and is one of the hallmarks of many diseases including ischemia, cancer, and inflammation. However, the molecular mechanisms of the cellular response to acid are not fully understood. We performed an unbiased RNA interference screen and identified PAC (TMEM206) as being essential for the widely observed proton-activated Cl^- (PAC) currents (I _Cl,H). Overexpression of human PAC in PAC knockout cells generated I _Cl,H with the same characteristics as the endogenous ones. Zebrafish PAC encodes a PAC channel with distinct properties. Knockout of mouse Pac abolished I _Cl,H in neurons and attenuated brain damage after ischemic stroke. The wide expression of PAC suggests a broad role for this conserved Cl^- channel family in physiological and pathological processes associated with acidic pH.

Fig. 1.. RNAi screen identifies PAC as essential for the proton-activated Cl⁻ 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 in red) are plotted according to the rank order. See 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) I_{Cl, H} monitored by voltage step (left) and ramp (right) protocols in WT HEK293 cells. (I) Whole-cell currents induced by extracellular pH 4.6 in PAC KO HEK293 cells. Note the normal amiloride-sensitive ASIC current is apparent at −100 mV. (J) I_{Cl, H} monitored by voltage step (left) and 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 ANOVA with Bonferroni post hoc test in (K)].

Fig. 2.. Human PAC recapitulates the properties of endogenous proton-activated Cl⁻ channel.

(A) Hydrophobicity plot (top) and a 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 anti-FLAG antibody. 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 monitored by voltage step (left) and ramp (right) protocols. (E) The normalized current-to-pH relationship of hPAC recorded at RT (n = 10–15 cells) or 37°C (n = 11–12 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 I-V 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 proton-activated Cl⁻ 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 monitored by voltage step (left) and ramp (right) protocols. Arrows point to the tail currents compared with those of hPAC (Fig. 2D). (C) The normalized current-to-pH relationship of hPAC (current at pH 4.6 is set to 1.0, n = 10–15 cells) and fPAC (current at pH 4.0 is set to 1.0, n = 5–12 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 I-V relationship recorded in extracellular pH 4.6 containing I⁻ or Cl⁻ solution and (G) I⁻ vs. 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) TPM (transcripts per million) of PAC gene in selective human tissues from RNA-seq data (n = 2–7 biological replicates) (15). (B) I_{Cl, 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) Percent of acid-induced neuronal cell death (n = 8 wells, 1-hour acid treatment and 24-hours recovery in culture medium) (9). (F) Triphenyltetrazolium chloride (TTC) 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 permanent middle cerebral artery occlusion (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)].