Background: Proton activated chloride (PAC) channels, encoded by the PACC1 gene, mediate outwardly rectifying chloride currents activated by acidification of the extracellular environment (1, 2). PAC is expressed in a plethora of mammalian cell types, but the physiological mechanisms of PAC modulation are incompletely understood. Recently, phosphatidylinositol-4,5-bisphosphate (PIP₂) was reported to bind and inhibit PAC channel from the extracellular side (3).  However, PIP₂ is predominantly an inner-leaflet lipid. Here, we explore whether physiological variations in inner-leaflet PIP₂ levels, such as those associated with activation of G_q-protein coupled receptors (G_qPCRs) may influence PAC activity.

Methods: Whole-cell patch-clamp recordings of PAC currents in human embryonic kidney 293T (HEK293T) cells were used in conjunction with heterologous expression of the acid sensing GqPCR ovarian cancer G-protein coupled receptor 1 (OGR1), the a₁-adrenergic receptor (a₁R) or the Danio rerio voltage sensitive protein phosphatase (DrVSP). In some experiments, DrVSP with a C302S mutation (DrVSP(C302S)) to abolish phosphatase activity was used as control. Data are given as mean ± SEM alongside the number of experiments (n). P‐values < 0.05 were considered significant.

Results: A chloride current was recorded in HEK293T cells at extracellular pH (pH_e) below 5.5. At pH_e 5 and 7.4, the current was 100.7±8.6 pA/pF (n=18) and 4.5±0.5 pA/pF (n=18) when measured at + 95 mV, respectively. The pH_e giving the half-maximal activation (EC₅₀) was 5.2±0.0 (n=5). HEK293 cells in which the PACC1 gene was deleted (2) presented negligible currents even at pH_e 5 (4.9±2.9 pA/pF (n=5)), and reintroduction of PACC1 cDNA (transcript variant 2) restored PAC currents (838.6±119.7 pA/pF at pH 5).

The PAC current measured at 5 pH was not affected by heterologous expression of OGR1 or a₁R (stimulated with phenylephrine (1 mM)), suggesting that the PAC current may not be modulated by the G_qPCR signaling pathway in HEK293T cells. The role of second messengers associated with G_qPCR signalling, Ca²⁺ and PIP₂, was further investigated. PAC current amplitude was not affected when free Ca²⁺ in the intracellular recording solution ([Ca²⁺]_i ) was raised from 0 to 1 mM. Inclusion of the PIP₂ scavenger neomycin (1 mM) in the intracellular solution had no effect on PAC current magnitude. In cells transfected with DrVSP the current at a negative potential (-60 mV) did not differ from that measured in cells expressing DrVSP(C302S). However, at a supra-physiological hyperpolarizing potential (+100 mV), the steady-state current was reduced by a factor ~1.8 from 152.0±24.6 pA/pF (n=11) in DrVSP(C302S) to  86.3±9.1 pA/pF (n=14) with DrVSP.

Conclusions: G_qPCR and PIP₂ signaling did not produce significant modulation of the PAC current in HEK293T in a physiological range of membrane potentials. The data suggest that cellular responses to extracellular acidification that are mediated by OGR1 and PAC may involve different pathways. While further work will be required to establish the crosstalk of pH_e sensing mechanisms in native cells, the data highlight new aspects of the cellular responses to variations in pH_e homeostasis.