The intermediate-conductance calcium/calmodulin-gated potassium channel KCa3.1 (encoded by the KCNN4 gene) is expressed in a variety of tissues such red and white blood cell lineage, epithelia, and endothelia (Wei et al., 2005). Notably, induction of KCa3.1 is suspected to drive abnormal cell proliferation, inflammation, and pathological organ remodeling (fibrosis) in heart, kidneys, lungs, some cancers, and atherosclerosis and neo-angiogenesis (for review see: Roach et al., 2013; Feske et al., 2015; Huang et al., 2015; Köhler et al., 2016). Accordingly, pharmacological modulation of the channel is considered a way to treat disease in humans. Hence, several small molecule blockers and positive and negative-gating modulators are available and Icagen compound, Senicapoc, was found clinically safe (for recent review see: Christophersen and Wulff, 2015). Also the endogenous omega-6 fatty, arachidonic acid (AA) and AA-metabolites such as some 11,12 epoxyeicosatrienoic acid and 20-hydroxyeicosatetraenoic acid (20-HETE), have been found to inhibit cloned human KCa3.1 that required mechanistically the inner pore lining amino acid residues, T250 and V275 as putative interaction sites (Hamilton et al., 2003; Kacik et al., 2014). However, the cell biological meaning of this modulation of KCa3.1 by omega-3/6 fatty acid is unclear.

Interestingly, KCa3.1-functions have been suggested to be compromised in Fabry disease (FD; Choi et al., 2014, 2015; Choi and Park, 2016), a X-linked lysosomal storage disorder (LSD), in which defective lysosomal targeting of mutated α-galactosidase A encoded by the GLA gene causes globotriaosyl-ceramide (Gb3) accumulation. Mechanistically, Gb3 has been suggested to downregulate KCa3.1-expression, fibroblast growth, differentiation into myofibroblasts, and collagen expression. These impairments could be reversed by channel activation (Choi et al., 2015).

In keeping with these previous findings, we characterized in the first part of this study KCa3.1 functions in a murine fibroblast cell line and human dermal fibroblasts and tested inhibitory actions of α-linolenic acid (α-LA) and docosahexaenoic acid (DHA) on channel functions and fibroblast mitogenesis.

In the second part, we tested whether disease-related impairments of fibroblast KCa3.1 are features of the two LSDs, FD and Niemann-Pick disease type C (NPC).

In this study, we found that murine and human dermal fibroblasts express amounts of functional KCa3.1 channels. We show strong sensitivity of fibroblast mitogenesis to α-LA and DHA.

KCa3.1 gene expression and KCa3.1 currents were reduced in two different LSDs.

KCa3.1 channel functions have been linked to abnormal cell proliferation, pathological tissue remodeling and fibrosis of a variety of organs, chronic inflammation, and autoimmune diseases (Wulff and Zhorov, 2008; Roach et al., 2013; Wulff and Köhler, 2013; Feske et al., 2015; Huang et al., 2015; Köhler et al., 2016). Accordingly, pharmacological inhibition of KCa3.1 has been suggested to be a treatment strategy for such disease states. In the present study we investigated: (1) the modulation of fibroblast KCa3.1 channels and fibroblast proliferation by omega-3 fatty acids, α-LA and DHA, as well as α-LA/γ-LA-rich and -low plant oils. (2) KCa3.1 functions in FD and NPC fibroblasts.

Here, we demonstrated KCa3.1 functions with the typical pharmacological fingerprint in murine 3T3-L1 fibroblasts and primary human dermal fibroblasts and showed inhibition of the channel by α-LA and DHA as well as potent anti-proliferative effects of these omega-3 fatty acids in vitro. Moreover, we demonstrated inhibition of KCa3.1 functions by α-LA- and γ-LA-rich plant oils and strong inhibition of 3T3-L1 fibroblast by linseed oil and borage oil, suggesting them as non-synthetic channel inhibitors with potential utilities for alleviating diseases states characterized by excessive cell proliferation, fibrosis, and chronic inflammation.

Impaired KCa3.1 functions have also been suggested to be a result of lipid overload in FD (Choi et al., 2014). Here, we demonstrated conserved but significantly reduced functional KCa3.1 channel membrane expression and mRNA expression in classical and atypical male FD patients and a NPC patient, KCa3.1 functions, suggesting impaired expression of KCa3.1 as potential biomarker of these LSDs.

Lipid regulation of K channels is a rather new field in ion channel research. So far, activation as well as inhibition of K channels has been described: Omega-6 and -3 fatty acids have been reported to activate KCa1.1 channels (a.k.a. BKCa encoded by the KCNMA1 and KCNMB1-3 genes; Kirber et al., 1992; Wei et al., 2005; Moreno et al., 2012). Likewise, the K2P channels, TWIK-2/2, TREK1/2, and TRAAK, were activated by omega-3/6 acids (Maingret et al., 1999; Wei et al., 2005; Nielsen et al., 2013). Regarding the KCa2/3 gene family, cloned KCa3.1 was, however, inhibited by omega-3/6 fatty acids (Hamilton et al., 2003; Kacik et al., 2014) and this inhibition required the inner-cavity lining amino acid residues T250 and V275 that are not present in corresponding sites in the above mentioned K channels families and in KCa2 channels. In the present study we provided new evidence for KCa3.1-inhibition by α-LA and also showed for the first time that also α-LA/γ-LA-containing plant oils (at 0.5%) were capable of efficiently inhibiting KCa3.1. However, inhibitory activity clearly depended on high α-LA or γ-LA-content (>30%) since oils with low α-LA/γ-LA content failed to produce significant inhibition, which could be explained by the high contents of saturated or mono-saturated fatty acids that were not active on KCa3.1 but competed with α-LA as suggested previously for P450-derived metabolites of arachidonic acid such as 5,6-EET, 8,9-EET, 5,6-DiHETE, and saturated arachidic acid (Kacik et al., 2014).

The possible consequences of KCa3.1-inhibtion by α-LA for fibroblast proliferation were not established. Here, our study extended the current knowledge by showing that the α-LA- and γ-LA-rich plant oils, linseed oil and borage oil, respectively, exerted strong anti-proliferative activity. The γ-LA-low onagra oil was inefficient, a finding that further fostered the notion that for efficient targeting KCa3.1 and inhibition of cell proliferation a high-α-LA-/γ-LA was needed. Concerning argan oil, we cannot judge on possible anti-proliferative actions because the oil was found to be cytotoxic at the concentration used here.

Concerning the magnitude of inhibition of fibroblast proliferation the effect of α-LA and α-LA-rich oils were similar to reported effects of a synthetic negative-gating modulator of KCa3.1 and a classical pore blocker (Olivan-Viguera et al., 2013; Roach et al., 2015). However, here we do not wish to exclude other mechanisms by which α-LA and α-LA-/γ-LA-rich oils reduce fibroblast proliferation.

Dietary supplementation with α-LA and α-LA-containing oils has been suggested since long to have therapeutic utilities in several autoimmune diseases of skin and bowel, multiple sclerosis, and rheumatoid arthritis (for review see: Macfarlane et al., 2011; Wergeland et al., 2012). Some of these effects have been proposed to rely on interference with e.g., pro-inflammatory prostaglandin production/metabolism (for review see Yates et al., 2014).

Here, we suggest that inhibition of KCa3.1 could be an additional mechanism of action. KCa3.1 inhibitors have been shown to suppress pro-inflammatory cytokine production in immune cells and to reduce pathological organ remodeling and fibrosis in experimental models. Therefore, from the perspective of phytopharmacology, our data fostered the view that inhibition of KCa3.1 in psoriatic lesions of skin or the chronically inflamed bowel by topically or systemically applied α-LA/γ-LA-rich plant oils and/or dietary supplementation with α-LA/γ-LA-rich plant oils or by synthetic blockers (Wulff and Köhler, 2013; Christophersen and Wulff, 2015) could be an alternative and economic treatment for patients with mild forms of disease or an adjuvant treatment in more severely affected patients receiving immune suppressive therapy.

It should also be considered that the plant oils used in this study are of complex composition and the nature of the active compounds is still unclear. So, we do not wish to exclude that other compounds than α-LA caused some of the anti-proliferative effects on 3T3 fibroblasts. Yet, it was striking that the anti-proliferative effects correlated with the quantity of α-LA or γ-LA suggesting that α-LA/γ-LA played a major role here. In this regard, it is worth speculating that the cytotoxic effects caused by argan oil likewise rely on so far undefined compounds in this oil.

Dysfunction of lysosomal lipid metabolism/transport has recently been shown to impair KCa3.1-functions and mRNA-expression and reduced membrane surface expression by lowered phosphatidylinositol 3-phosphate as reason for these alterations (Choi et al., 2015; Choi and Park, 2016). In the present study, we provided additional evidence for such defective KCa3.1-regulation in FD fibroblasts and also in NPC fibroblasts from a male patient, which seemed predominantly to rely on impaired de novo-KCa3.1 gene expression, thus similar to that was found in earlier studies (Choi et al., 2015; Choi and Park, 2016). To the contrast, membrane expression—albeit at lower density—and activation was still intact in most cells, suggesting no major disturbances in membrane trafficking or function in the fibroblasts from FD patients investigated in our study.

Interestingly, the alterations were exclusively present in dermal fibroblasts of male FD patients with a classical missense mutation of this X-linked disease and an atypical complex intronic haplotype that have been reported to produce FD pathologies. In contrast, heterozygous female carriers with classical and atypical mutations, who may or may not develop clinically relevant symptoms depending on whether they have the mutated allele or the healthy allele as active copy, showed largely conserved functions of the channel in their dermal fibroblasts. Together, this suggested that low KCa3.1-mRNA expression in skin fibroblasts could be at least an additional biomarker of FD-disease or disease activity in male FD patients and in NPC patients.

Interestingly, in another study on KCa3.1 functions in type-1 and -3 Gaucher patients we found a defect in the physiologically occurring up-regulation of KCa3.1 during monocyte-to-macrophage differentiation (Oliván-Viguera et al., 2015a), further suggesting that KCa3.1 dysregulation is perhaps a more general feature of LSDs and cellular pathophysiology. But again, the pathophysiological mechanism and significance remain unclear.

At this point, we would like to remind the reader that KCa3.1 functions in disease states have been so far related to pro-proliferative and -inflammatory processes and pathological organ remodeling such as kidney and lung fibrosis (Wulff and Zhorov, 2008; Roach et al., 2013; Wulff and Köhler, 2013; Feske et al., 2015; Huang et al., 2015; Köhler et al., 2016). Accordingly, pharmacological inhibition of KCa3.1 has been proposed as pharmacological strategy to treat chronic inflammation and organ fibrosis. So, whether or not KCa3.1—albeit reduced—is drug target in FD and associated pathologies such progressive heart and kidney disease, aneurism, and angiokeratoma cannot be decided at present. Still, there is the possibility that inhibition of KCa3.1 by small molecules or α-LA-/γ-LA-rich oils as dietary supplement may delay disease progression in LSD patients who develop organ pathologies characterized by chronic inflammation and fibrosis.

There are some limitations concerning our patient study: First, the number of patients studied here was relatively small, which is a general limitation when studying these rare LSD with complex and varying clinical pathology. However, the observed differences in KCa3.1-expression and functions were still considerable (ca. 75% reduction) between controls and male FD patients. Here additional, mRNA expression analysis in a larger number and other cohorts of patients will be needed to further establish KCa3.1 as biomarker of FD and/or other LSD. Whether these alterations were unique for FD disease remains unclear since a male NPC patient showed similarly reduced KCa3.1 functions. Secondly, in the present study we did not intent to reveal the pathomechanism linking defects in lipid metabolism and lipid overload to reduced KCa3.1 membrane functions, although lipid overload affecting de novo-gene expression is likely to be a major reason here.

In conclusion, the present study showed inhibition of KCa3.1 functions and fibroblast mitogenesis by the α-LA-/γ-LA-rich plant oils, in vitro. This suggests the utility of both as dietary supplement or topical ointments to target these pro-inflammatory and pro-mitogenic channels in cutaneous, neuronal or intestinal autoimmune disease.

AO and RK conceived the study, conducted research, analyzed data, and wrote the manuscript. JL, LL, and ÁLG conducted research and analyzed data. EA, YG, PG, AJG, JC, PI, conducted research on patients, analyzed data, provided materials, and compounds, and contributed to the writing of the manuscript.

This work was supported by the European Community (FP7-PEOPLE MC CIG ·”BrainIK,” to RK); Department of Industry and Innovation, Government of Aragon (GIPASC-B105 to ÁLG); and the Fondo de Investigacion Sanitaria, Instituto de Salud Carlos III (CB06/07/1036, to PG and RK).