The cell cycle regulates cell-penetrating peptide cytosolic uptake via differential expression of KCNH1 potassium channel
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Curated by eLife
eLife Assessment
This study reveals valuable links between cell-penetrating peptide uptake and the cell cycle, membrane voltage and an endogenous potassium channel. The evidence is solid, and further experiments would strengthen the proposed mechanism and the degree of the mechanism's generalization. The findings will interest researchers working on delivery of therapeutic molecules into cells.
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Abstract
Membrane potential fluctuations, driven by ion channel activity, are important regulators of processes such as action potential propagation in neurons and muscle cell contraction. These variations also occur in non-excitable cells, influencing various cellular processes, including, but not limited to, direct translocation of cell-penetrating peptides (CPPs) into the cytosol. CPPs are cationic peptide vectors that facilitate the delivery of cargos into cells. The efficiency of CPP direct translocation varies significantly within the same cell population, a phenomenon that is not understood mechanistically. In this study, we used an unbiased transcriptomic approach to identify genes involved in efficient CPP cytosolic uptake. This revealed a close correlation between the expression of cell cycle-related genes and the ability of cells to take up CPPs by direct translocation. Monitoring phases of the cell cycle with the PIP-FUCCI system showed that CPP direct translocation was most efficient in the G2/M and S phases and least efficient during the G1 phase. This was correlated with the cells’ plasma membrane potential: cells in G2/M and S phases being more polarized than cells in the G1 phase. The expression of the KCNH1 voltage-gated potassium channel was found to be highest in the phases of the cell cycle most permissive for CPP direct translocation. Pharmacological inhibition of KCNH potassium channels efficiently blocked CPP direct translocation in these cell cycle phases. These data indicate that KCNH1 controls the cell membrane potential in a cell cycle-dependent manner and consequently the ability of cells to take up CPPs by direct translocation. Our findings highlight the importance of the cell cycle in regulating CPP uptake. Variations in the plasma membrane potential along the cell cycle should be considered in the development of CPP-based therapeutics and their applications.
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eLife Assessment
This study reveals valuable links between cell-penetrating peptide uptake and the cell cycle, membrane voltage and an endogenous potassium channel. The evidence is solid, and further experiments would strengthen the proposed mechanism and the degree of the mechanism's generalization. The findings will interest researchers working on delivery of therapeutic molecules into cells.
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Reviewer #1 (Public review):
Summary:
Ribeiro et al. investigate the molecular mechanisms affecting the efficiency of synthetic cell-penetrating peptide (CPP) uptake by cultured mammalian cells. CPPs are frequently coupled to cargoes that can change cell function and are highly useful to investigators studying cell biology. Moreover, CPPs are potentially amenable to delivery of cargoes that can affect cells in clinically useful ways, with several under investigation as potential anti-cancer treatments. A challenge to the therapeutic use of CPPs is their inefficient delivery to their intended molecular targets, with poor cellular uptake being a major barrier.
The present study presents new insights into the cellular and plasma membrane properties that can affect CPP uptake. In the studied cell line, the authors find that cell …
Reviewer #1 (Public review):
Summary:
Ribeiro et al. investigate the molecular mechanisms affecting the efficiency of synthetic cell-penetrating peptide (CPP) uptake by cultured mammalian cells. CPPs are frequently coupled to cargoes that can change cell function and are highly useful to investigators studying cell biology. Moreover, CPPs are potentially amenable to delivery of cargoes that can affect cells in clinically useful ways, with several under investigation as potential anti-cancer treatments. A challenge to the therapeutic use of CPPs is their inefficient delivery to their intended molecular targets, with poor cellular uptake being a major barrier.
The present study presents new insights into the cellular and plasma membrane properties that can affect CPP uptake. In the studied cell line, the authors find that cell cycle-dependent changes in the plasma membrane potential correlate with uptake of a CPP conjugated to a fluorescent dye. These changes correlate with mRNA abundance of a voltage-gated potassium channel, KCNH1. RNA silencing of KCNH1 expression or pharmacological blockade of KCNH1 currents impairs CPP uptake, supporting the proposed model.
Strengths:
(1) Identified KCNH1 expression and function as a specific modulator of CPP uptake in U2OS cells and LAN1 cells.
(2) Data show a clear correlation between the plasma membrane potential, cell cycle stage, and efficiency of intracellular CPP delivery in these cells.
Weaknesses:
(1) Some of the claims are more generalized than warranted by the presented evidence. Is KCNH1-dependent modulation of cell cycle-dependent changes in the plasma membrane potential and ensuing effect on CPP uptake a niche property of this channel in some cell lines? Or a more broadly generalized function? I think this can be addressed merely through more careful or caveated language in the manuscript. However, an additional experiment that would be potentially informative and complementary to the chronic high-potassium depolarization experiments would be to examine the impact of heterologous expression of a hyperpolarizing potassium channel (for example, Kir2.1).
(2) Please provide more details on how endosomes were identified and measured.
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Reviewer #2 (Public review):
Summary:
The article by Ribeiro et al. provides further mechanistic understanding of the abilities of cell-penetrating peptides (CPPs) to cross directly through the membrane. The authors performed a transcriptomic evaluation to conclude that CPPs enter via direct translocation through cells undergoing cell division, particularly in G2/M phase cells. They reasoned that this is due to the expression of KCNH1, a potassium channel, which is hypothesized to fluctuate throughout the cell cycle and peak at G2/M phase. This expression, in turn, hyperpolarizes the cells, enabling the direct translocation of CPPs.
The uptake mechanism of CPPs has long been debated. The authors and other groups have previously mentioned the importance of cell membrane potential and the involvement of specific ion channels in its …
Reviewer #2 (Public review):
Summary:
The article by Ribeiro et al. provides further mechanistic understanding of the abilities of cell-penetrating peptides (CPPs) to cross directly through the membrane. The authors performed a transcriptomic evaluation to conclude that CPPs enter via direct translocation through cells undergoing cell division, particularly in G2/M phase cells. They reasoned that this is due to the expression of KCNH1, a potassium channel, which is hypothesized to fluctuate throughout the cell cycle and peak at G2/M phase. This expression, in turn, hyperpolarizes the cells, enabling the direct translocation of CPPs.
The uptake mechanism of CPPs has long been debated. The authors and other groups have previously mentioned the importance of cell membrane potential and the involvement of specific ion channels in its regulation. The current work further adds a layer of understanding by connecting the occurrence of direct translocation to a specific cell cycle phase, which may be of great relevance when considering the use of CPPs for cells that are constantly dividing, such as cancer cells.
Strengths:
The work took advantage of transcriptomics and the unique use of the PIP-FUCCI system to determine the different uptake of CPPs in different cell cycle stages. All the data are in agreement with each other, especially when observing the membrane potential change through the cell cycle stages. The experiments are well executed and are all complementary to the authors' hypothesis.
Weaknesses:
The article could be further strengthened through:
(1) Implementation of additional confirmatory experiments
(2) Positioning against other recently reported channel proteins
(3) Confirming whether CPPs with different physicochemical properties are also affected by the fluctuation of KCNH1 expression
(4) Determining whether the concept of cell cycle-dependent membrane potential change is applicable not just in model cells, but also in cells that undergo rapid cell cycles, and
(5) Relating CPP direct translocation to known biological phenomena that are physiologically similar.
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The cell cycle regulates cell-penetrating peptide cytosolic uptake via differential expression of KCNH1 potassium channel https://www.biorxiv.org/content/10.64898/2026.09.01.748619v1
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The cell cycle regulates cell-penetrating peptide cytosolic uptake via differential expression of KCNH1 potassium channel https://www.biorxiv.org/content/10.64898/2026.09.01.748619v1
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