A mysterious channel: new insights into proton channel functioning raise new questions

Voltage-gated proton channels were described for the first time in snail neurons more than 25 years ago (Thomas & Meech, 1982). They have been found subsequently in a wide variety of mammalian cells, including epithelial cells, oocytes, skeletal muscle cells, lymphocytes and various phagocytes. Proton channels of all cell preparations are characterized by high proton selectivity, strong pH and voltage dependence, and high sensitivity to Zn2+. Proton channels open only when the electrochemical gradient for protons is outward, so they carry only outward current in the steady state. Depending on the cell type studied, proton channel currents differ to some extent with respect to their activation and deactivation time constants and their Ca2+ dependence (for review, see DeCoursey, 2003). To date, it remains unclear whether all proton channels are encoded by the same genes, while the observed differences in proton currents reflect channel modulation due to the specific microenvironment in certain cell types, or whether different proton channel types exist which are encoded by different genes. Genes coding for a protein that resembles a voltage sensor domain that is capable of functioning as a voltage-gated proton channel have recently been identified in human and mouse (Ramsey et al. 2006; Sasaki et al. 2006). Due to these findings, the long-lasting debate of whether a voltage-gated proton current results from the activity of either channels or transporters (for review, see DeCoursey, 2003) has finally reached an end. These new findings convincingly show that protons have their own channels. Furthermore, the newly identified voltage sensor domain proteins enable researchers now to study proton channel properties and modulation in more detail. In this issue of The Journal of Physiology, Musset et al. (2008) investigated proton currents of human Hv1 and mouse mVSOP channels expressed in HEK-293 and COS-7 cells. Intriguingly, the authors found that currents of these expressed proton channels exhibit similarities as well as differences in comparison with currents of native proton channels. Similar to currents of native proton channels, currents of expressed human and mouse proton channels are highly selective for protons and are strongly dependent on pH and voltage. The most striking difference between the expressed and native proton channels is the capability of expressed channels to generate inward proton currents, which has not been observed before in recordings of native channel currents in unstimulated phagocytes or in any other cell preparation. As clearly demonstrated by Musset et al. (2008), a negative shift in the voltage-dependent gating of expressed proton channels explains why these channels are capable of generating inward currents. The authors found that the proton current reversal potentials were similar in native and expressed channels, whereas the current activation threshold and the normalized conductance–voltage relationship of expressed proton channels were shifted by about 30 mV in the negative direction compared with those of native channels. In the attempt to find an explanation for the differences between native and expressed proton channels, Musset et al. (2008) tested carefully a number of hypotheses. As a result, they excluded the possibility that the unusual behaviour of expressed proton channels is due to the expression systems used. Furthermore, the authors hypothesized that expressed proton channels are already in an ‘activated’ state, because a negative shift in the voltage dependence of proton channel gating has previously been observed when phagocytes were activated either by superfusion with the phorbol ester PMA or arachidonic acid, or by intracellular perfusion with GTPγS or high Ca2+-containing solution (Banfi et al. 1999; Morgan et al. 2007). Because, in activated phagocytes, PMA- and arachidonic acid-induced proton channel modulation could be reversed by protein kinase C (PKC) inhibitors (Morgan et al. 2007), Musset et al. (2008) tested whether dephosphorylation induced by a phosphatase or by protein kinase inhibition leads to a positive shift in the voltage-dependent gating of expressed proton channels, and whether expressed proton channels can additionally be modulated by PMA. However, in contrast to native proton channels of phagocytes, expressed proton channel currents were unaffected by either dephosphorylation or phosphorylation. This finding raises again an old, yet unanswered question: do phagocytes express more than one type of proton channels (Banfi et al. 1999)? It seems to be possible that phagocytes express two types of proton channels, i.e. one type exhibiting properties similar to those channels described in other cell preparations and a second type which is activated only following cell stimulation and is characterized by a more negative activation threshold and the capability to generate inward currents. Based on their structure it can be ruled out that the expressed proton channels are related to the gp91phox subunit of the NADPH oxidase, which has previously been suggested as a proton channel (Henderson et al. 1995). However, it cannot be excluded that the expressed Hv1/mVSOP proton channels resemble a proton channel type that is normally quiescent in unstimulated phagocytes, but becomes activated by a not yet fully understood mechanism in activated phagocytes. Investigations of proton currents in Hv1/mVSOP knockout mice may answer this question. Alternatively, it has previously been suggested by DeCoursey's group that phagocytes express only one type of proton channels, which change their properties and gating behaviour following PKC phosphorylation (Morgan et al. 2007). It is possible that the expressed proton channels lack a sequence or subunit that is required for channel modulation by PKC. To explain the differences between expressed and native proton channels, Musset et al. (2008) additionally propose that gating of native proton channels is regulated by an as yet unidentified protein, which is either absent or underexpressed in the expression systems used. In summary, this intriguing study by Musset et al. (2008) raises a number of questions that need to be addressed in future experiments.