Multi-omic profiling reveals the ataxia protein sacsin is required for integrin trafficking and synaptic organization

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a childhood-onset cerebellar ataxia caused by mutations in SACS, which encodes the protein sacsin. Cellular ARSACS phenotypes include mitochondrial dysfunction, intermediate filament disorganization, and progressive death of cerebellar Purkinje neurons. It is unclear why the loss of sacsin causes these deficits or why they manifest as cerebellar ataxia. Here, we perform multi-omic profiling in sacsin knockout (KO) cells and identify alterations in microtubule dynamics and mislocalization of focal adhesion (FA) proteins, including multiple integrins. Deficits in FA structure, signaling, and function can be rescued by targeting PTEN, a negative regulator of FA signaling. ARSACS mice possess mislocalization of ITGA1 in Purkinje neurons and synaptic disorganization in the deep cerebellar nucleus (DCN). The sacsin interactome reveals that sacsin regulates interactions between cytoskeletal and synaptic adhesion proteins. Our findings suggest that disrupted trafficking of synaptic adhesion proteins is a causal molecular deficit in ARSACS

The crosstalk between the tumour microenvironment (TME) and cancer cells: the role of ion channels.

The tumour microenvironment (TME) is a dynamic and intricate network of cells and molecules that evolves in response to cancer invasion, influencing tumour biology and treatment responses. This study investigates the role of ion channels, particularly hERG1, in the communication between cancer cells and the TME. We elucidate a mechanistic pathway involving hERG1 interaction with the β1 integrin receptor, unveiling its profound impact on cell migration, response to mechanical stimuli, and stroma interaction. Our findings reveal that integrin engagement triggers a biphasic response in hERG1 ion channels. Initial stimulation leads to the translocation of hERG1 channels to the plasma membrane, resulting in increased current amplitude and membrane hyperpolarization. Subsequently, hERG1 forms a complex with integrin, maintaining a closed conformation on the plasma membrane, which gradually restores the initial state. This hERG1/β1 integrin complex, modulated by the Gαi3 signalling pathway, significantly influences cancer cell migration and response to mechanical cues, in part due to its impact on cytoskeletal elements like cortical f-actin. In addition, we demonstrate the role of hERG1 in mediating the response of cancer cells to changes in extracellular matrix stiffness, implicating YAP mechanotransduction. This connection adds to the growing understanding of the role of mechanotransduction in cancer progression and supports the potential for targeted therapies in this context. Overall, this research provides crucial insights into the complex interplay between ion channels, the TME, and cancer cells, offering new avenues for anti-metastatic strategies and personalized treatment approaches in the field of oncology. The mechanistic insights provided by our findings, indeed, suggest the potential for targeting hERG1 and β1 integrin interaction during the initial phases of cancer cell migration as an anti-metastatic strategy. This approach may offer therapeutic benefits without affecting hERG1 function in non-cancerous tissues, thus circumventing cardiotoxic side effects associated with hERG1 blockers. Our results also highlight the potential use of Ectica plates for conducting preclinical research and personalized treatment in patient-derived primary culture

High throughput clone screening on overexpressed hERG1 and Kv1.3 potassium channels using ion channel reader (ICR) label free technology

Pharmacological studies aimed at the development of newly synthesized drugs directed against ion channels (as well as genetic studies of ion channel mutations) involve the development and use of transfected cells. However, the identification of the best clone, in terms of transfection efficiency, is often a time consuming procedure when performed through traditional methods such as manual patch-clamp. On the other hand, the use of other faster techniques, such as for example the IF, are not informative on the effective biological functionality of the transfected ion channel(s). In the present work, we used the high throughput automated ion channel reader (ICR) technology (ICR8000 Aurora Biomed Inc.) that combine atomic absorption spectroscopy with a patented microsampling process to accurately measure ion flux in cell-based screening assays. This technology indeed helped us to evaluate the transfection efficiency of hERG1 and hKv1.3 channels respectively on the HEK-293 and CHO cellular models. Moreover, as proof of the validity of this innovative method, we have corroborated these data with the functional characterization of the potassium currents carried out by the same clones through patch-clamp recordings. The results obtained in our study are promising and represent a valid methodological strategy to screen a large number of clones simultaneously and to pharmacologically evaluate their functionality within an extremely faster timeframe

The Interaction between hERG1 and β1 Integrins Modulates hERG1 Current in Different Pathological Cell Models

Ion channels are implicated in various diseases, including cancer, in which they modulate different aspects of cancer progression. In particular, potassium channels are often aberrantly expressed in cancers, a major example being provided by hERG1. The latter is generally complexed with β1 integrin in tumour cells, and such a molecular complex represents a new druggable hub. The present study focuses on the characterization of the functional consequences of the interaction between hERG1 and β1 integrins on different substrates over time. To this purpose, we studied the interplay alteration on the plasma membrane through patch clamp techniques in a cellular model consisting of human embryonic kidney (HEK) cells stably transfected with hERG1 and in a cancer cell model consisting of SH-SY5Y neuroblastoma cells, endogenously expressing the channel. Cells were seeded on different substrates known to stimulate β1 integrins, such as fibronectin (FN) for HEK-hERG1 and laminin (LMN) for SH-SY5Y. In HEK cells stably overexpressing hERG1, we observed a hERG1 current density increase accompanied by Vrest hyperpolarization after cell seeding onto FN. Notably, a similar behaviour was shown by SH-SY5Y neuroblastoma cells plated onto LMN. Interestingly, we did not observe this phenomenon when plating the cells on substrates such as Bovine Serum Albumin (BSA) or Polylysine (PL), thus suggesting a crucial involvement of ECM proteins as well as of β1 integrin activation