Identification of TMEM206 proteins as pore of PAORAC/ASOR acid-sensitive chloride channels

Reviewer #3:

Minor points:

I personally like rather verbose Materials and methods sections, and I therefore would have wished for more information on a few occasions, specifically:

- How was the Z-score calculated, and what do the authors mean by "robust statistics"?

“Robust statistics” denotes the use of median and MAD instead of mean and SD, but we agree that this is somewhat ambiguous. To clearly explain how the Z-score was calculated, we now added a formula (subsection “Genome-wide siRNA Screen”, fourth paragraph).

- How was the solution exchanged in electrophysiological experiments, and did the precise way of exchanging solutions (potentially) have an impact on liquid junction potentials? Did the authors use an agar-bridge for the reference electrode?

- How was the liquid junction potential between different solution measured? As this is not an entirely trivial procedure (with potentially different outcomes depending on the method used), it would be desirable that the authors shared the values measured, especially for those experiments where anions were replaced completely.

We added more detailed descriptions of these procedures, as well as the values measured for LJPs to the Materiuals and methods section (subsection “Electrophysiology”, last paragraph).

Normally, I would consider not employing series resistance compensation an avoidable mistake when measuring currents of several nano-amperes with a series resistance of more than 5 MΩ. In this manuscript, the choice not to use this technique has no impact on the conclusions. It may, however, have affected the shape of some of the I/V curves (for example, the linearity of the curve in Figure 4H is perhaps due to voltage-clamp errors). Maybe the authors want to point this out in an appropriate place.

We thank the reviewer for pointing out this minor flaw.Series resistance in ourrecordings was generally low (2.5 – 4.5 MΩ) due to our use of patch pipettes with large openings. This should result in small voltage errors (<5 mV) when currents are in the low nA range, even without compensation. However, considering the often very high whole-cell current amplitudes in cells overexpressing TMEM206, there may indeed be a substantial voltage clamp error. We added a sentence in the Results section (subsection “Human TMEM206 mediates typical ASOR currents”, third paragraph) to caution the reader about this possibly confounding factor.

The authors point out in the Introduction that the name "PAORAC" provides a better description compared to "ASOR" and in addition, it is actually the older name. This, of course, is flattering to me as I coined the term PAORAC many years back. Maybe, the authors want to consider to use the term "PAORAC" also in the title, possibly together with the newer "ASOR".

To fairly acknowledge the earlier description of the current, we now added the PAORAC acronym (which in contrast to ASOR refers specifically to anion channels) to the title of our manuscript and to the Abstract.

In Figure 7E, it is hard to identify that the "three stars" of the L315D mutation belong to this panel, as they are plotted between panel E and F.

We changed the figure to clarify what the asterisks mean.

In Figure 7G and H, why is there no statistics reported?

We added statistics of the current densities with Na₂SO₄ as charge carrier in Figure 7H. However, we prefer not to perform statistical analysis on the data in Figure 7G as we believe this would mislead the reader into inferring meaningful differences between the mutants. Apparent differences are likely to be due to variable current amplitudes in Na₂SO₄ solutions with some mutants (see panel H), for which background currents (very negative reversal potential in our recording system) contribute more to the observed reversal potential. Also, we believe that a statistical comparison between measured reversal potentials and “not applicable” has little meaning. It is now clearly pointed out in the figure legend that no statistical analysis was performed for this panel to prevent misleading the reader.

In addition to the changes made to accommodate the suggestions of the reviewers, we also added a new figure supplement (Figure 3—figure supplement 1), which shows I_Cl,H measured in WT and TMEM206^-/- HeLa cells. Two additional HeLa KO clones not described in the initial submission have been measured and are now incorporated in Table 1.