Intracellular permeant anions, and not extracellular protons, are the predominant driver of fast gating in the hyperpolarization-activated CLC-2 chloride channel.

Virus-like particles (VLPs) are being used for therapeutic developments such as vaccines and drug nanocarriers. Among these, plant virus capsids are gaining interest for the formation of VLPs because they can be safely handled and are noncytotoxic. A paradigm in virology, however, is that plant viruses cannot transfect and deliver directly their genetic material or other cargos into mammalian cells. In this work, we prepared VLPs with the CCMV capsid and the mRNA-EGFP as a cargo and reporter gene. We show, for the first time, that these plant virus-based VLPs are capable of directly transfecting different eukaryotic cell lines, without the aid of any transfecting adjuvant, and delivering their nucleic acid for translation as observed by the presence of fluorescent protein. Our results show that the CCMV capsid is a good noncytotoxic container for genome delivery into mammalian cells.

We previously reported that mouse parotid acinar cells display an anion conductance (IATPCl) when stimulated by external ATP in Na+-free extracellular solutions. It has been suggested that the P2X7 receptor channel (P2X7R) might underlie IATPCl. In this work we show that IATPCl can be activated by ATP, ADP, AMP-PNP, ATPγS and CTP. This is consistent with the nucleotide sensitivity of P2X7R. Accordingly, acinar cells isolated from P2X7R−/− mice lacked IATPCl. Experiments with P2X7R heterologously expressed resulted in ATP-activated currents (IATP-P2X7) partially carried by anions. In Na+-free solutions, IATP-P2X7 had an apparent anion permeability sequence of SCN− > I− ≅ NO3− > Br− > Cl− > acetate, comparable to that reported for IATPCl under the same conditions. However, in the presence of physiologically relevant concentrations of external Na+, the Cl− permeability of IATP-P2X7 was negligible, albeit permeation of Br− or SCN− was clearly resolved. Relative anion permeabilities were not modified by addition of 1 mM Carbenoxolone—a blocker of Pannexin-1. Moreover, Cibacron Blue 3GA, which blocks the Na+ current activated by ATP in acinar cells but not IATPCl, blocked IATP-P2X7 in a dose-dependent manner when Na+ was present, but failed to do so in TEA+-containing solutions. Thus, our data indicate that P2X7R is fundamental for IATPCl generation in acinar cells and that external Na+ modulates ion permeability and conductivity as well as drug affinity in P2X7R.

We investigated whether pannexin-1, a carbenoxolone-sensitive hemichannel activated in erythrocytes by swelling, could contribute to swelling-activated chloride currents (ICl,swell) in HEK-293 cells. We used ethidium bromide uptake as an index of pannexin-1 activation and IC,swell activation as an index of plasma membrane stretching. ICl swell activated by a hypotonic solution was reversible inhibited by carbenoxolone (IC50 98 ± 5 μM), a blocker of pannexin-1. However, the hypotonic solution that activated ICl,swell did not induce ethidium bromide uptake indicating that pannexin-1 was not activated by cell swelling. The mimetic peptide 10panx1, a pannexin-1 antagonist, did not affect ICl,swell activation but completely inhibited the ATP-induced ethidium bromide uptake coupled to P2X7 receptors activation. We conclude that carbenoxolone directly inhibited ICl,swell independent of pannexin-1 and that pannexin-1 hemichannels are not activated by swelling in HEK-293 cells.

The volume-sensitive chloride current (IClVol) exhibit a time-dependent decay presumably due to channel inactivation. In this work, we studied the effects of Cl- and H+ ions on IClVol decay recorded in HEK-293 and HL-60 cells using the whole-cell patch clamp technique. Under control conditions ([Cl-]e = [Cl-]i = 140 mM and pHi = pHe = 7.3), IClVol in HEK cells shows a large decay at positive voltages but in HL-60 cells IClVol remained constant independently of time. In HEK-293 cells, simultaneously raising the [Cl-]e and [Cl-]i from 25 to 140 mM (with pHe = pHi = 7.3) increased the fraction of inactivated channels (FIC). This effect was reproduced by elevating [Cl-]i while keeping the [Cl-]e constant. Furthermore, a decrease in pHe from 7.3 to 5.5 accelerated current decay and increased FIC when [Cl-] was 140 mM but not 25 mM. In HL-60 cells a slight IClVol decay was seen when the pHe was reduced from 7.3 to 5.5. Our data show that inactivation of IClVol can be controlled by changing either the Cl- or H+ concentration or both. Based on our results and previously published data we have built a model that explains VRAC inactivation. In the model the H+ binding site is located outside the electrical field near the extracellular entry whilst the Cl- binding site is intracellular. The model depicts inactivation as a pore constriction that happens by simultaneous binding of H+ and Cl- ions to the channel followed by a voltage-dependent conformational change that ultimately causes inactivation.