Functionally-Coupled Ion Channels Begin Co-assembling at the Start of Their Synthesis
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eLife Assessment
This valuable manuscript provides convincing evidence that BK and CaV1.3 channels can co-localize as ensembles early in the biosynthetic pathway, including in the ER and Golgi. The findings, supported by a range of imaging and proximity assays, offer insights into channel organization in both heterologous and endogenous systems. However, mechanistic questions remain unresolved, particularly regarding the specificity of mRNA co-localization, the dynamics of ensemble trafficking, and the functional significance of pre-assembly at the plasma membrane. While the data broadly support the central claims, certain conclusions would benefit from more restrained interpretation and additional clarification to enhance the manuscript's impact and rigor.
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Abstract
Calcium binding to BK channels lowers their activation threshold. Hence, BK channels are functionally coupled to calcium-permeable channels. This functional coupling requires proximity of two different types of channels. Formation of an ensemble at nanometer distances between BK and CaV1.3 channels exemplifies this unique organization. We investigated the mechanism underlying their structural organization, testing the hypothesis that the assembly between BK and CaV1.3 channels is formed before their insertion at the plasma membrane. Our design used four approaches: 1) to detect the interaction between BK and CaV1.3 proteins inside the cell, 2) to identify the membranes where intracellular ensembles reside, 3) to measure the proximity between their mRNAs, and 4) to look for protein interaction at the plasma membrane soon after the start of their translation. These measures showed that a percentage of transcripts for BK and CaV1.3 channels are close and that the newly synthesized proteins interact through the endoplasmic reticulum and Golgi. Comparisons were made with other proteins and transcripts, as well as with predictions of randomized localizations of BK channels. We found evidence of assembly between BK and CaV1.3 ensembles before their insertion at the plasma membrane.
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Author Response:
We thank the reviewers for their thoughtful feedback and appreciate their recognition of the value of our findings. In response, we are refining the manuscript to clarify key terminology, more clearly describe our image analysis workflows, and temper the interpretation of our results where appropriate. We are planning to perform additional experiments to further investigate the specificity of mRNA co-localization between BK and CaV1.3 channels. We acknowledge the importance of understanding ensemble trafficking dynamics and the functional role of pre-assembly at the plasma membrane, and we plan to explore these questions in future work. We look forward to submitting a revised manuscript that addresses the reviewers’ comments in detail.
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eLife Assessment
This valuable manuscript provides convincing evidence that BK and CaV1.3 channels can co-localize as ensembles early in the biosynthetic pathway, including in the ER and Golgi. The findings, supported by a range of imaging and proximity assays, offer insights into channel organization in both heterologous and endogenous systems. However, mechanistic questions remain unresolved, particularly regarding the specificity of mRNA co-localization, the dynamics of ensemble trafficking, and the functional significance of pre-assembly at the plasma membrane. While the data broadly support the central claims, certain conclusions would benefit from more restrained interpretation and additional clarification to enhance the manuscript's impact and rigor.
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Joint Public Review:
This study presents a valuable contribution to our understanding of ion channel complex assembly by investigating whether BK and CaV1.3 channels begin to form functional associations early in the biosynthetic pathway, prior to reaching the plasma membrane. Using a combination of proximity ligation assays, single-molecule RNA imaging, and super-resolution microscopy, the authors provide convincing evidence that these channels co-localize intracellularly within the ER and Golgi, in both overexpression systems and a relevant endogenous cell model. The study addresses an important and underexplored aspect of membrane protein trafficking and organization, with broader implications for how ion channel signaling complexes are assembled and regulated. The experimental approaches are generally appropriate and the imaging …
Joint Public Review:
This study presents a valuable contribution to our understanding of ion channel complex assembly by investigating whether BK and CaV1.3 channels begin to form functional associations early in the biosynthetic pathway, prior to reaching the plasma membrane. Using a combination of proximity ligation assays, single-molecule RNA imaging, and super-resolution microscopy, the authors provide convincing evidence that these channels co-localize intracellularly within the ER and Golgi, in both overexpression systems and a relevant endogenous cell model. The study addresses an important and underexplored aspect of membrane protein trafficking and organization, with broader implications for how ion channel signaling complexes are assembled and regulated. The experimental approaches are generally appropriate and the imaging data are clearly presented, with a commendable number of control experiments included. However, several limitations temper the interpretation of the results. The mechanisms underlying mRNA co-localization, and the role of co-translation in complex formation, remain insufficiently defined. Similarly, while intracellular colocalization is convincingly demonstrated, the study does not establish whether such early assembly is the predominant pathway for generating functional complexes at the plasma membrane. More rigorous quantification of channel co-association across compartments, and clarification of key terminology and image analysis methods, would strengthen the overall conclusions. Some of the language in the manuscript would also benefit from a more measured tone to avoid overstating the novelty of the findings. Despite these limitations, the study offers meaningful insights into intracellular ion channel organization and will be of interest to researchers in cell biology, membrane trafficking, and neurophysiology. With focused revisions addressing the outlined points, the manuscript has the potential to make a solid contribution to the field.
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