Rebuttal from Yosuke Funato, Kazuharu Furutani, Yoshihisa Kurachi and Hiroaki Miki

Arjona and de Baaij (2018) nicely summarize the history of cyclin M (CNNM) research. However, we do not agree with their conclusion that CNNM proteins are not Na⁺/Mg²⁺ exchangers. Their conclusion is primarily based on the following observations and reports: (1) CNNM proteins form dimers of three transmembrane domains, which would be too small to function as a channel/transporter, (2) two groups have reported that Mg²⁺ efflux was not observed in Mg²⁺‐imaging experiments, and (3) Mg²⁺ influx was observed in the authors’ experiments using ²⁵Mg²⁺, irrespective of the quinidine treatment or absence of extracellular Na⁺. We here make our rebuttal to each of these points.

CNNM oligomers

We reported that CNNM2 proteins (∼100 kDa) form SDS‐resistant oligomers, which migrated far above the 250 kDa marker by SDS‐PAGE (Funato et al. 2017). Sponder et al. (2016) showed similar results. Furthermore, very large CNNM2‐containing complexes (>500 kDa) were observed by native PAGE. More experiments are necessary to determine the number of CNNM protomers within each complex.

Mg²⁺‐imaging experiments

Hardy et al. (2015) performed Mg²⁺‐imaging experiments by expressing CNNM3. However, only the effects of mutations that abolished the interaction with PRL were shown. No control experiments were performed (e.g. transfection of empty vector or non‐functional mutants), and their experiments do not provide information regarding CNNM3 function itself. Sponder et al. (2016) expressed CNNM2, but immunofluorescence analyses showed the expressed proteins primarily localized at the intracellular membrane, not the plasma membrane. Therefore, effects on Mg²⁺ efflux, which occurs across the plasma membrane, cannot be evaluated.

Mg²⁺ influx observed in the authors’ experiments

We propose that the influx of extracellular ²⁵Mg²⁺ through TRPM7 was stimulated by Mg²⁺ depletion caused by CNNM2 expression. Quinidine can inhibit many proteins, such as various cation channels, and is not a general inhibitor for Na⁺/Mg²⁺ exchangers. Therefore, the inability of quinidine to inhibit ²⁵Mg²⁺ influx does not indicate that Na⁺/Mg²⁺ exchangers are not involved in the process. Also, removal of extracellular Na⁺ would not significantly impact Mg²⁺ influx if it was performed simultaneously with ²⁵Mg²⁺ addition, because intracellular Mg²⁺ levels would have already been reduced significantly by Mg²⁺ efflux by CNNM2 before measurements were recorded.

Finally, the K _d value of the interaction between CNNM2 cystathionine‐β‐synthase domains and Mg‐ATP is 0.16 mm (Hirata et al. 2014), which is far below the intracellular concentration of Mg‐ATP (∼5 mm). Therefore, contrary to the authors’ model, the ability of CNNM proteins to sense intracellular Mg‐ATP fluctuations through this interaction is unlikely.

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Additional information

Competing interests

None declared.

Author contributions

All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

This work was supported by JSPS KAKENHI JP26460364 and JP17K19396 to Y.F., and JP26291042, JP26111007, JP16K14723 and JP17H04041 to H.M.