Sweating chloride bullets: understanding the role of calcium in eccrine sweat glands and possible implications for hyperhidrosis

Eccrine (atrichial) sweat glands are large, active cutaneous end-organs vital to human thermal and fluid homeostasis, but regional overactivity of these glands can lead to focal hyperhidrosis. Eccrine sweat glands can expulse over 10 nl/min in a single isolated gland and as much as 3.7 l/h systemically in response to thermal and non-thermal stimuli (1, S1, S2). How do these glands produce such copious secretions? Sweating involves a two-stage process of first producing an isotonic primary solution within the bulbous coiled region of the gland and then modifying (primarily via reabsorbing NaCl) this fluid within the ductal portion as it travels to the skin surface. Thus, production of this primary fluid requires careful scrutiny to understand hyperhidrosis. Epithelial transport in these glands appears to be primarily mediated via clear (agranular) cells, which are also the proposed cell type associated with hyperhidrosis 2. In the current understanding of clear cell function, Cl− is transported through the clear cell, Na+ is transported through tight junctions via a transepithelial voltage gradient, and finally water is pulled through aquaporin channels via osmotic forces. Thus, Cl− is a principal player in the formation of the isotonic primary solution. It is, however, another ion, Ca2+, that appears to be the primary regulator within clear cells. The majority of sudorific agonists (e.g. acetylcholine, norepinephrine via α1-adrenergic receptors and ATP) increase cytosolic Ca2+ via efflux from cellular stores and influx from extracellular fluid. Ca2+ release from intracellular stores is derived via IP3-activated Ca2+ and Ca2+-induced Ca2+ release channels. The influx into the clear cells is a bit more complex, likely involving TRPV1, store-operated Ca2+ entry (Orai1 and TRPC1) and L-type voltage-gated Ca2+ channels (3-5, S3). Highlighting the importance of Ca2+ influx, secretions are abated when isolated glands are placed into Ca2+-free bath (S4, S5), and recent in vivo experiments identified right shifts in the cholinergic agonist to sweating relation with both EDTA (used as an interstitial Ca2+ chelator) and verapamil (L-type channel blocker) 4. The regulatory role of cytosolic Ca2+ is to activate many of the ion channels involved in epithelial transport. This is where the study by Ertongur-Fauth and colleagues 6 enters the picture. Anoctamin 1 (ANO1, also termed TMEM16A), is a Ca2+-activated Cl− channel, which is an anion-selective membrane protein that allows passive Cl− flow and is activated by intracellular Ca2+ as well as being voltage-activated at low Ca2+ concentrations (S6). Ertongur-Fauth and colleagues 6 identified 3 TMEM16A splice variants in the NCL-SG3 sweat gland epithelial cell line as well as in hyperhidrotic and control skin. Investigating the function of a novel splice variant, TMEM16A(acΔe3), authors found it lacks the dimerization domain but still forms a functional channel. Previous studies indicated that TMEM16A subunits appeared to dimerize before reaching the plasma membrane and that dimerization was required for functionality 7. Overexpression of TMEM16A(acΔe3) increased basal Cl− transport even without the dimerization domain 6. This may indicate that the novel TMEM16A splice variant is more constitutively active and thus could possibly produce primary solution during non-stimulated conditions. This, in theory, could lead to increases in production of primary fluid. Alternately, it is possible that sudorific modulators such as galanin and CGRP 8-10, which appear to accentuate sweat formation once transport is initiated, may now be more functionally important due to this increased basal Cl− secretion. Overexpression of the novel TMEM16A splice variant had another interesting result: unlike in basal conditions, expressing either the novel TMEM16A(acΔe3) or the canonical TMEM16A(ac) splice variant enhanced Ca2+-induced Cl− secretion during activated conditions, as compared to the parental cells containing only endogenously expressed TMEM16A splice variants 6. Overexpression of TMEM16A(acΔe3), however, decreased Ca2+-dependent Cl− transport compared to the canonical variant 6. This seems to suggest that a certain mix of TMEM16A splice variants may enhance Ca2+-regulated transport and that the novel variant may be less regulated and less responsive to activators than other splice variants. Thus, overexpression of either splice variant in disease states, such as hyperhidrosis, could yield greater sweating per agonist activation of Ca2+. TMEM16A Cl− channels are not the only channels involved in the formation of isotonic primary solution. Another Ca2+-activated Cl− channel, bestrophin 2 (Best2), is expressed in basolateral and apical membranes of sweat gland secretory cells (S7) and among other functions regulates L-type voltage-gated Ca2+ channels (S8). Clear cells are arranged with a basolateral membrane that contains Na+-K+ ATPase, Na+-Cl−-K+ cotransporters (NCCK1), Ca2+-activated K+ channels, Best2, aquaporin-5 channels (AQP5) and various channels that allow for the Ca2+ influx discussed above. The apical membrane contains TMEM16A and Best2 Ca2+-activated Cl− channels, Ca2+-activated K+ channels, vacuolar H+-ATPase and AQP5. A conceptual model of the role of Ca2+ in activating basolateral Cl− transport, primarily via NCCK1, and then apical Cl− transport, via TMEM16A and Best2, is depicted in Fig. 1. Both known and proposed transporters in clear cells are included in the model to show not only ion transport but also the potential interactions, regulatory points and redundancies in the system. The NCL-SG3 cell line (S9) has dramatically increased our understanding of sweating, but there are some inherent limitations in the use of the cell line. In addition to not knowing precisely which eccrine sweat gland cells are included in the line, cultured cells cannot interact with surrounding cells of other types in cell–cell interactions as would occur in intact human skin. Cultured cells also do not have the neural influences that drive responses in vivo. This specifically relates to agonist-induced sweating in which the traditional neural agonists such as acetylcholine do not cause secretion (S9, S10). Thus, the normal IP3 response to release Ca2+ from intracellular stores cannot occur. While in vitro work such as that described by Ertongur-Fauth and colleagues 6 tells an important story, there is also a need for in vivo studies to determine whether agonist-induced Cl− secretion is similar in intact human skin. It would have been nice for the purpose of telling this story if the novel TMEM16A splice variant would have only been expressed in hyperhidrotic skin and if overexpression would have yielded extremely high agonist-induced Cl− secretion, but, alas, it appears more complex. It is interesting that a combination of isoforms may cause greater function (more Cl− transport and therefore more sweating) than any one isoform. Speculations on the data from Ertongur-Fauth and colleagues 6 could include TMEM16A splice variant differences leading to different rates of sweating and perhaps even differences between hyperhidrotic and normal skin. One of the most effective hyperhidrosis treatments consists of blocking neural activation of sweat glands through cholinergic presynaptic release inhibitors like botulinum toxin, but perhaps the root cause of focal hyperhidrosis is not only at the neural activation level but also includes downstream components such as at the Ca2+-activated Cl− or other Ca2+-activated ion channel level. Both authors contributed to all aspects of background research, interpretation and manuscript preparation. The authors have declared no conflict of interests. Data S1-S10: Supplemental References. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.