Defective Function of KCa3.1 Channels in Lysosomal Storage Disorders

Background: The calcium-calmodulin-gated KCa3.1 is considered a key regulator of membrane hyperpolarization, maintenance of cellular electrolyte balance, Ca²⁺-signalling, secretion of pro-inflammatory cytokines, neurodegeneration, pathological tissue remodelling. Osteonecrosis/-penia, vasculopathies, and neurodegeneration are chronic and progressive inflammatory complications of several inherited lysosomal storage diseases, such as Gaucher Disease (GD1/GD3), Fabry Disease(FD), and Niemann-Pick disease type C1 (NPC1). Here we hypothesized that KCa3.1 functions are impaired in a LSD-related fashion in patients' fibroblasts or the monocyte/macrophages lineage.

Methods: A total of25 subjects were analyzed (5 FD, 5 NPC, 5 GD1, 5 GD2, 5 healthy controls). Fibroblasts from patients' skin biopsies were cultured in DMEM supplemented with 10% FCS (Biochrom KG, Berlin, Germany) and cells were used at passages 1-4. For patch-clamp experiments, cells were seeded on coverslips and used within 24 h. Monocytes isolated and purified from peripheral blood from age-matched GD patients (GD1 and GD3) and healthy volunteers (Ctrl). Measurements were repeated in patient's and Ctrl's macrophages that were differentiated from monocyte precursors in vitro by exposure to patient's or Ctrl's erythrocyte lysates for 4 days, respectively.

Ca²⁺-activated K⁺ currents were measured in the whole-cell configuration using an EPC10-USB amplifier (HEKA, Electronics, Lambrecht-Pfalz, Germany) and a K pipette solution (intracellular) containing 1 μM Ca²⁺ free (in mM): 140 KCl, 1 MgCl₂, 2 EGTA, 1.71 CaCl₂ (1 μM [Ca²⁺]free), and 5 HEPES (adjusted to pH 7.2 with KOH). For data acquisition and analysis we used the patch-master program (HEKA). Ohmic leak currents of up to 1 nS were subtracted where appropriate. We quantified outward currents at a 0 mV holding potential (to avoid contamination with non-selective cation currents and chloride currents). Capacitance values (a measure of cell surface and thus cell size)

Results: Patch-clamp demonstrated lower fibroblast KCa3.1 membrane functions in NPC1 and male FD patients. KCa3.1-currents in monocytes were similar in GD-1/3 and Ctrls. In differentiated and activated macrophages, the overall larger KCa3.1 currents in GD-1 macrophages were reduced to 40% of controls while in GD-3 macrophages KCa3.1 currents were barely detectable. Glucosylceramide was found to produce KCa3.1-current desensitization. At the gene level, KCa3.1-mRNA expression was impaired in NPC1, GD1/3, and male FD patients.

Conclusions: Defective KCa3.1 functions are a feature and biomarker of cellular dysfunction in the LSD, GD1/3, FD, and NPC1 and supports the concept that biased lipid metabolism harms ion channel functions. Since KCa3.1 is involved in the mechanism of cellular differentiation, neurodegeneration, and inflammation, the impaired KCa3.1 -activity could be of pathomechanistic relevance in LSD cellular malfunction by promoting differentiation into pro-inflammatory cell types. Pharmacological targeting of KCa3.1 could be therapeutic utility to achieve neuroprotection in GD3 and NPC1 patients and bone complications in GD1.