A synthetic peptide that prevents cAMP regulation in mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels

Essential revisions:

1) What is the justification for utilizing reverse charge mutations as opposed to alanine mutation? It seems like a reverse charge mutation biases the system toward less interaction between the HCN and TRIP8b_nano.

As shown in Figure 3, the CNBD ‐ TRIP8b_nano interaction is mainly driven by an electrostatic network rather than single, isolated salt bridges. Therefore, our choice to invert charges instead of neutralize them was to overcome possible compensatory effects deriving from the network of electrostatic interactions. The reviewer is certainly right, a reverse charge mutation biases the system toward less interaction. But in the case of three negative charges (D₂₅₇, E₂₆₄, E₂₆₅) facing three positive ones (R₆₆₂, K₆₆₅, K₆₆₆), we considered the possibility of compensatory effects form the neighbors very likely. Similar consideration holds also for E₂₄₁ and E₂₄₀ although we could have performed R₆₅₀A. In this case we decided not to, for coherence with the rest.

2) Is there any data on how long the TRIP8b_nano is able to maintain an effect in vivo?

Cells were pretreated by adding the cell‐penetrating peptides TAT‐TRIP8b_nano and TAT‐ (SCRAMBLED) TRIP8b_nano (see below our reply to point 5) but also TRIP8b_nano (see our reply to point 6) to the extracellular solution, 30 minutes before the experiment. At the end of the pretreatment, cells were patched under constant perfusion of extracellular solution that did not contain the (TAT)‐peptides. Our patch recordings nevertheless show that TAT‐TRIP8b_nano was still effective even after 60 minutes from the beginning of the experiment. This indicates in our view that TAT‐TRIP8b_nano, once accumulated inside, is not released from the cell and maintains its effect up to 1 hours without becoming toxic.

We have added an explanatory paragraph on the modality of the experiment with TAT‐peptides (Materials and methods, subsection “TAT-peptides”).

3) In the Discussion it is stated that the TRIP8b_nano is entirely specific for HCN channels. Is this too strong of an assertion? Is there any data regarding off target effects on non-ion channel targets?

In agreement with the reviewer we have rephrased the sentence as follows:

“…it is selective for HCN and it does not interfere with other cAMP‐modulated channels present in the SAN, such as L‐type Ca²⁺channels. Collectively, this makes TRIP8b_nano a promising tool in targeted therapeutic interventions”.

4) Include legends in some of the figures for clarification. (Figure 4B, Figure 5B).

Not clear what the request was. We interpret it as the need to add, in the legends, the cross‐reference to data analysis in the Materials and methods (i.e. Boltzmann fit). Hope we got it right.

5) The peptide experiments should be supported by controls using scrambled peptides. This is particularly important for charged tat peptides, which can have non-specific effects.

We have tested the scrambled peptide on mouse SAN cells. The results are included as new Figure 6—figure supplement 1.

6) Related to this, what is the evidence that the TAT-fused peptide indeed translocates into the cell? Have control experiments with the non-TAT-fused peptide been performed? These should not be able to penetrate the cell.

We further tested the TRIP8b_nano peptide in HEK 293T cells. Also in this case, the peptide added to the extracellular solution for 30 minutes, did not affect the current. We added this result to Figure 4D.