LRMP inhibits the cAMP-dependence of HCN4 channels by disrupting intramolecular signal transduction

Reviewer #1 (Public Review):

Summary:
The authors use truncations, fragments, and HCN2/4 chimeras to narrow down the interaction and regulatory domains for LRMP inhibition of cAMP-dependent shifts in the voltage dependence of activation of HCN4 channels. They identify the N-terminal domain of HCN4 as a binding domain for LRMP, and highlight two residues in the C-linker as critical for the regulatory effect. Notably, whereas HCN2 is normally insensitive to LRMP, putting the N-terminus and 5 additional C-linker and S5 residues from HCN4 into HCN2 confers LRMP regulation in HCN2.

Strengths:
The work is excellent, the paper well written, and the data convincingly support the conclusions which shed new light on the interaction and mechanism for LRMP regulation of HCN4, as well as identifying critical differences that explain why LRMP does not regulate other isoforms such as HCN2.

Reviewer #2 (Public Review):

Summary:
HCN-4 isoform is found primarily in the sino-atrial node where it contributes to the pacemaking activity. LRMP is an accessory subunit that prevents cAMP-dependent potentiation of HCN4 isoform but does not have any effect on HCN2 regulation. In this study, the authors combine electrophysiology, FRET with standard molecular genetics to determine the molecular mechanism of LRMP action on HCN4 activity. Their study shows that parts of N- and C-termini along with specific residues in C-linker and S5 of HCN4 are crucial for mediating LRMP action on these channels. Furthermore, they show that the initial 224 residues of LRMP are sufficient to account for most of the activity. In my view, the highlight of this study is Fig. 7 which recapitulates LRMP modulation on HCN2-HCN4 chimera. Overall, this study is an excellent example of using time-tested methods to probe the molecular mechanisms of regulation of channel function by an accessory subunit.

Weaknesses:
1. Figure 5A- I am a bit confused with this figure and perhaps it needs better labeling. When it states Citrine, does it mean just free Citrine, and "LRMP 1-230" means LRMP fused to Citrine which is an "LF" construct? Why not simply call it "LF"? If there is no Citrine fused to "LRMP 1-230", this figure would not make sense to me.

2. Related to the above point- Why is there very little FRET between NF and LRMP 1-230? The FRET distance range is 2-8 nm which is quite large. To observe baseline FRET for this construct more explanation is required. Even if one assumes that about 100 amino are completely disordered (not extended) polymers, I think you would still expect significant FRET.

3. Unless I missed this, have all the Cerulean and Citrine constructs been tested for functional activity?

Reviewer #3 (Public Review):

Summary:
Using patch clamp electrophysiology and Förster resonance energy transfer (FRET), Peters and co-workers showed that the disordered N-terminus of both LRMP and HCN4 are necessary for LRMP to interact with HCN4 and inhibit the cAMP-dependent potentiation of channel opening. Strikingly, they identified two HCN4-specific residues, P545 and T547 in the C-linker of HCN4, that are close in proximity to the cAMP transduction centre (elbow Clinker, S4/S5-linker, HCND) and account for the LRMP effect.

Strengths:
Based on these data, the authors propose a mechanism in which LRMP specifically binds to HCN4 via its isotype-specific N-terminal sequence and thus prevents the cAMP transduction mechanism by acting at the interface between the elbow Clinker, the S4S5-linker, the HCND.

Weaknesses:
Although the work is interesting, there are some discrepancies between data that need to be addressed.

1. I suggest inserting in Table 1 and in the text, the Δ shift values (+cAMP; + LRMP; +cAMP/LRMP). This will help readers.

2. Figure 1 is not clear, the distribution of values is anomalously high. For instance, in 1B the distribution of values of V1/2 in the presence of cAMP goes from - 85 to -115. I agree that in the absence of cAMP, HCN4 in HEK293 cells shows some variability in V1/2 values, that nonetheless cannot be so wide (here the variability spans sometimes even 30 mV) and usually disappears with cAMP (here not).

This problem is spread throughout the manuscript, and the measured mean effects are indeed always at the limit of statistical significance. Why so? Is this a problem with the analysis, or with the recordings?

There are several other problems with Figure 1 and in all figures of the manuscript: the Y scale is very narrow while the mean values are marked with large square boxes. Moreover, the exemplary activation curve of Figure 1A is not representative of the mean values reported in Figure 1B, and the values of 1B are different from those reported in Table 1.

On this ground, it is difficult to judge the conclusions and it would also greatly help if exemplary current traces would be also shown.

3. "....HCN4-P545A/T547F was insensitive to LRMP (Figs. 6B and 6C; Table 1), indicating that the unique HCN4 C-linker is necessary for regulation by LRMP. Thus, LRMP appears to regulate HCN4 by altering the interactions between the C-linker, S4-S5 linker, and N-terminus at the cAMP transduction centre."

Although this is an interesting theory, there are no data supporting it. Indeed, P545 and T547 at the tip of the C-linker elbow (fig 6A) are crucial for LRMP effect, but these two residues are not involved in the cAMP transduction centre (interface between HCND, S4S5 linker, and Clinker elbow), at least for the data accumulated till now in the literature. Indeed, the hypothesis that LRMP somehow inhibits the cAMP transduction mechanism of HCN4 given the fact that the two necessary residues P545 and T547 are close to the cAMP transduction centre, remains to be proven.

Moreover, I suggest analysing the putative role of P545 and T547 in light of the available HCN4 structures. In particular, T547 (elbow) points towards the underlying shoulder of the adjacent subunit and, therefore, is in a key position for the cAMP transduction mechanism. The presence of bulky hydrophobic residues (very different nature compared to T) in the equivalent position of HCN1 and HCN2 also favours this hypothesis. In this light, it will be also interesting to see whether a single T547F mutation is sufficient to prevent the LRMP effect.