Calcium-Activated Chloride Channels in the Corpus Cavernosum: Recent Developments and Future of a Key Cellular Component of the Erectile Process

REVIEW Calcium-activated chloride channels in the corpus cavernosum: recent developments and future of a key cellular component of the erectile process

DJ Linton1, LC Lau2 and PG Adaikan2

Calcium-activated chloride channels (CaCCs) are one of five families of chloride channels, ubiquitously expressed, and essential for a host of biological actions. CaCCs have key roles in processes as diverse as olfactory transduction and epithelial secretion, and also CaCCs are essential in smooth muscle contraction. The corpus cavernosum is a vascular smooth muscle that must relax to facilitate erections. Parasympathetic activation produces relaxation of the corpus cavernosum through a nitric oxide- dependent pathway, and sympathetic stimulation in both preventing and terminating erections by contracting the corpus cavernosum. Both these pathways affect activity of CaCCs. The past 5 years produced many successes in CaCC research. One key area of success was the identification of the elusive ‘molecular candidate’ of CaCCs, as the TMEM16A protein (dubbed anoctamin-1) and potentially other members of the anoctamin family of transmembrane proteins. However, enthusiasm has been somewhat tempered because of evidence that this family of proteins may not be responsible for calcium-activated chloride currents in certain epithelial tissues. Several studies identified specific inhibitors of CaCCs as well as specific inhibitors for anoctamin-1. Despite the number of recent achievements in this field there are many details that still need to be elucidated. Of particular value would be more details on the identity of the CaCCs in corpus cavernosum smooth muscle, using new inhibitors to gain insight into the signalling pathway, and the evaluation of whether inhibition of CaCCs provides any specific benefit in different models of ED.

International Journal of Impotence Research (2012) 24, 211--216; doi:10.1038/ijir.2012.22; published online 21 June 2012 Keywords: calcium-activated chloride channels; corpus cavernosum; penile erection; penis; smooth muscle

INTRODUCTION FIVE FAMILIES OF CHLORIDE CHANNELS Ion channels, most simply, are proteins that form pores in cell New developments in the research of calcium-activated chloride membranes that may permit the free passage of certain ions. channels (CaCCs) are relevant to sexual medicine. CaCCs have an These channels are broadly divided into those that selectively important physiological role in contraction of the corpus conduct positively charged ions and those that are selective for cavernosum, an action that is necessary for the penis to remain negatively charged ions. Channels involved in conducting ions flaccid. Inhibition of these channels thus holds promise as a with positive charges are logically termed ‘cation channels’ therapeutic target for ED. In addition to CaCCs there are four other whereas, channels that conduct negative ions have come to be families of chloride channels: ligand-gated chloride channels, the known as ‘chloride channels’, although it would be more accurate cystic fibrosis transmembrane regulator (CFTR), ClC voltage-gated to define the negative ion pores as ‘anion channels’. ‘Chloride chloride channels and volume-regulated anion channels (Table 1). channels’ is recognized as a doubly inaccurate name because These families are classified largely by homology and are diverse these channels are not only permeable to many anions other than in function.3 Members of the same family tend to share a common chloride but some ‘chloride channels’ are, in fact, not channels but mechanism, but there are many cases where the pharmacology antiporters facilitating anion movement.1,2 between multiple families can overlap.3 The misunderstanding of chloride channels has run deeper than Ligand-gated chloride channels are the only chloride channels a simple misnomer. For many years it was believed that chloride that were historically well researched. This family of chloride tended to be in electrochemical equilibrium across membranes channel is active in inhibitory neurotransmission in the central and that chloride channels were of little consequence to any nervous system by permitting a hyperpolarizing outward chloride cellular or physiological processes. This misconception likely current in response to binding by either gamma-aminobutyric contributed to the relative dearth of chloride channel research acid or glycine. This is in contrast to currents through other conducted until the 1990s.2 In the past two decades, however, families of channels, including those found in the smooth muscle significant inroads have been made toward understanding the of the corpus cavernosum, which tend to be inward and different types, roles, mechanisms, structures and pharmacology depolarizing. However the differing directions of chloride flow of chloride channels. are simply because of differences in equilibrium potential for

1Department of Anaesthiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada and 2Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University Hospital, Singapore. Correspondence: Professor PG Adaikan, Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University Hospital, NUHS Tower Block Level 12, 1E Kent Ridge Road, Singapore 119228. E-mail: [email protected] Received 11 August 2011; revised 9 March 2012; accepted 10 May 2012; published online 21 June 2012 CaCCs in the corpus cavernosum DJ Linton et al 212 Table 1. Families of chloride channels

Family Members Description

Ligand- GABA and glycine receptors These channels act as receptors for two inhibitory neurotransmitters---GABA and glycine. Activation gated of these channels by ligand binding allows a chloride current (generally an inward, hyperpolarizing current), thereby preventing the action potential required for post-synaptic activation. CFTR Cystic fibrosis transmembrane The cystic fibrosis transmembrane regulator is a chloride channel that opens in response to ATP and regulator contributes epithelial secretions through an efflux of Cl--. ClC Voltage-gated chloride channels These chloride channels (ClC-1 through ClC-9) are structurally similar and voltage gated. The functions, locations and mechanisms are diverse. Some of these channels are actually antiporters. VRAC Volume- or swelling-activated VRACs are activated when the volume of a cell increases and thereby have a role in homeostasis. chloride channels CaCC Calcium-activated chloride CaCCs are present in many different types of cells. They appear to have a role in epithelial secretion, channels olfactory transduction, as well as excitation of myocytes and neurons. Abbreviations: ATP, adenosine triphosphate; CaCC, calcium-activated chloride channel; CFTR, cystic fibrosis transmembrane regulator; GABA, g-aminobutyric acid; VRAC, volume-regulated anion channel.

chloride ions between neurons and smooth muscle cells. The smooth muscle.21,22 In the decades following those early presence of inhibitory hyperpolarizing chloride currents, and the investigations, smooth muscle CaCCs in the corpus cavernosum ligand-activated nature of these currents was identified in the were shown to respond to a number of different agonists 1950s and 1960s.4--6 The majority of the research into other families (including norepinephrine, histamine and endothelin-1) all of of chloride channels, however, was performed decades later. which ultimately lead to contraction of the corpus cavernosum In 1989, it was found that the mutation responsible for cystic smooth muscle.23 Karkanis et al.24 used the patch-clamp fibrosis occurs in a gene encoding a chloride channel, and the technique to demonstrate the presence of calcium-activated CFTR channel was successfully cloned.7--9 The efflux of anions depolarizing currents in both rat and human corpus cavernosum controlled by this channel is very important for ionic and fluid myocytes. In addition, they undertook in vivo experiments on rats homeostasis at epithelial membranes.10 Research into cystic to determine the effects of intracavernosal administration of fibrosis, as well as research independent of cystic fibrosis in the known chloride channel blockers. Inhibition of chloride channels years following this discovery helped make the 1990s a decade of produced dose-dependent increases in intracavernosal pressure in considerable advances in the field of chloride channel research. male rats. Ensuing investigations have demonstrated the im- These discoveries include many developments regarding three portance of CaCCs in the control of corporeal tissue contraction additional families of chloride channels. through both isolated tissue experiments using rabbit corpus ClC channels are a family of nine chloride channels and cavernosum, as well as through the effects of intracavernous transporters---denoted as ClC-1 through to ClC-7, ClC-Ka and ClC- injection of chloride channel and chloride transport blockers on Kb---that are structurally similar, and are all voltage gated.11 The intracavernosal pressure in male rats.25,26 Although CaCCs are nine members of this family found in mammals were initially known to be essential for noradrenaline-induced smooth muscle identified based on homology to a widely studied chloride contraction, research suggests that the increases or decreases in channel (now called ClC-0) responsible for developing prey- activity of CaCCs may have a role in modulation of smooth muscle shocking current in the electric ray Torpedo marmorata.12,13 contraction through other signalling cascades. Volume-regulated anion channels, also called swelling-activated The mechanism by which CaCCs affect smooth muscle chloride channels, are thought to function mainly in the regulation contraction can be summarized into four key steps: (i) intracellular of cell volume in response to hypotonic solution by releasing calcium levels increase, (ii) CaCCs open in response to the calcium anions to produce an osmotic efflux.14 A member of the ClC family increase, (iii) depolarization results as anions effuse from the cell (ClC-3) has been proposed as the channel responsible for volume- and (iv) voltage-dependent calcium channels (VDCCs) on the activated anion conductance,15,16 but this is a matter of some plasma membrane open because of the change in voltage, dispute.3,17 allowing the large increase in calcium required for contraction. CaCCs were first discovered in the early 1980s because of their However, both the initiation and the inhibition of this signalling presence in large numbers in Xenopus oocytes.18,19 In oocytes, process can be affected by three different physiological pathways: CaCCs open to depolarize the membrane with a chloride stimulation by the sympathetic nervous system, spontaneous efflux, which prevents the entry of multiple sperm, but these calcium release and inhibition through the erectrogenic nitric channels perform a diverse array of functions in other tissues, oxide (NO) pathway (Figure 1). including signal and sensory transduction, membrane depolariza- Perhaps the most significant is the integral role played by tion, epithelial secretion and smooth muscle contraction. A review CaCCs in producing smooth muscle contraction following by Hartzell et al.20 describes these various functions in detail, sympathetic stimulation. Activation of alpha-1 adrenoreceptors and also raises the possibility that there are as many as four by noradrenaline leads to the intracellular release of calcium different subtypes of CaCCs because of differences in certain from the sarcoplasmic reticulum thereby activating the CaCCs physical properties---particularly conductance---between individual and leading to the contraction of corpus cavernosum smooth channels. muscle. Continuous activation by noradrenaline is one mechanism that has long been implicated in the constant smooth muscle tone required to maintain flaccidity, and may also have a CACCS IN THE CORPUS CAVERNOSUM role in detumescence during the termination of erections.27 It is the role of CaCCs in the contraction of the corpus cavernosum Interestingly, increased alpha-adrenoceptor sensitivity in the smooth muscle that is of particular interest to sexual medicine penis has been correlated with age and although activity of researchers. The presence of chloride currents activated by a rise CaCCs has not been examined in aged individuals, an increase in in intracellular calcium in smooth muscle cells was established in CaCC activity may contribute to the higher rates of ED in older 1987 by the current-clamp investigations of rat anococcygeus populations.28

International Journal of Impotence Research (2012), 211 -- 216 & 2012 Macmillan Publishers Limited CaCCs in the corpus cavernosum DJ Linton et al 213

Figure 1. CaCCs and signalling pathways in corpus cavernosum. CaCC activity is likely modified by several signalling pathways. Some of the signalling pathways that have been implicated in modulating the activity of CaCCs are shown. NA released by nerves of the sympathetic nervous system binds to a1 adrenoceptors (a), producing increases in IP3 (b), which in turn promote intracellular calcium release (c). Intracellular calcium is released (1), binds to calmodulin (2) and this complex increases anion efflux through CaCC (3). The depolarization of the membrane (4) activates VDCC, allowing the Ca2 þ influx (5) necessary for muscle contraction. NO released from parasympathetic nerves and vascular endothelium during sexual arousal stimulates guanylyl cyclase (I); the activated guanylyl cyclase converts GTP to cGMP (II). PKG is activated by cGMP (III), and alters several products through phosphorylation resulting in inhibition of Ca2 þ release from the sarcoplasmic reticulum (IV) and activation of BK channels (V). The resulting Ca2 þ release and hyperpolarization of the membrane (VI) inhibit contraction. a1, alpha-1 receptor; BK, large-conductance potassium channel; CaCC, calcium-activated chloride channel; cGMP, cyclic guanosine monophosphate; Gq, Gq heteromeric protein; GTP, guanosine triphosphate; IP3, inositol triphosphate; NA, noradrenaline; NO, nitric oxide; PKG, protein kinase G; VDCC, voltage-dependent calcium channel.

Another process by which intracellular calcium may be released contrary, recent data show that cGMP was capable of inhibiting leading to activation of CaCCs is the spontaneous release of chloride efflux during noradrenaline-induced contraction re- calcium from intracellular stores in the form of ‘sparks’, or sponse in the corpus cavernosum.37 ‘waves’.29--31 The electrical activity resulting from these sponta- CaCCs are of vital importance to erectile function, but neous releases of calcium may also contribute to the resting tonic investigations into mechanisms and roles of CaCCs have been contraction of the corpus cavernosum.32 hampered by not knowing the molecular identity of CaCCs and It is also important to note that parasympathetic stimulation of the absence of any selective inhibitors of CaCCs.10,20,38,39 However, corpus cavernosum tissue by nitrergic nerves and endothelial- recent efforts have led to discoveries that may remedy both of derived NO produces erections and inhibition of chloride channels these challenges. may have a crucial role in this process.33 The NO/cyclic guanosine monophosphate (cGMP) pathway leads to the activation of a cGMP-dependent kinase I (also known as protein kinase G). This ADVANCES IN THE IDENTIFICATION OF THE MOLECULAR kinase promotes relaxation through several different actions: IDENTITY OF CACCS desensitization to calcium, the activation of large conductance Although chloride currents characteristic of CaCCs were first potassium channels (BK) and the inhibition of calcium release described almost 30 years ago, efforts to elucidate the specific from the sarcoplasmic reticulum.34 The latter two actions of a cellular component responsible have proven problematic. The cGMP-dependent kinase I both have an effect on intracellular identification of the molecular identity of classical CaCCs has calcium and, by extension, could be responsible for the effect on undergone constant evolution. Two different proposed identities CaCCs. However, BK channels are also sensitive to calcium (CLCA and bestrophin proteins) emerged and subsequently fell out activation, so the net effect on BK channels is somewhat of favor as a new, more promising identity (the anoctamin protein ambiguous. The activation of BK channels leads to an efflux of family) came to prominence in 2008. However, research published in potassium ions, which hyperpolarizes the cell membrane, thereby 2011 suggests that anoctamins may not be the sole molecular making it less likely that VDCCs will open and produce muscle structure responsible for classical calcium-activated chloride currents, contraction. Although CaCC-mediated depolarization is important and are even not the principal type of CaCC in certain epithelial to facilitate the opening of VDCCs, VDCCs and CaCCs are involved tissues, which leaves the question of the identity of CaCCs open.40 in positive feedback and the opening of VDCCs may be essential to propagate the depolarization of the membrane.29 Perhaps even more significantly, the inhibition of intracellular calcium CLCA release affects a key step leading to chloride channel activation, An early candidate for the CaCC was the CLCA gene, which was and could promote relaxation of the corpus cavernosum by identified through antibody screening and found to produce interfering with the contractile tone dependent on CaCCs. It has calcium-sensitive chloride currents when expressed in bovine also been demonstrated that vascular smooth muscle exhibits a tracheal epithelia.41 Despite exerting some important regulatory non-classical CaCC that is dependent on cGMP.35,36 On the effects on CaCCs, the CLCA proteins have biophysical properties,

& 2012 Macmillan Publishers Limited International Journal of Impotence Research (2012), 211 -- 216 CaCCs in the corpus cavernosum DJ Linton et al 214 such as pharmacology, calcium sensitivity, voltage sensitivity and cystic fibrosis,60 asthma,61 hypertension,53 secretory diarrhea and pattern of expression, which were inconsistent with classical even as a cancer treatment.49 Despite this enormous potential, the CaCCs.20,38,42,43 pharmacological ‘toolkit’ for CaCCs has, until recently, consisted of only a few poorly selective chloride channel inhibitors and activators, and it has been frequently noted that effective, BESTROPHINS selective inhibitors could be invaluable to researchers investigat- In the early 2000s, proteins known as bestrophins were found by ing the physiology of these ion channels.10,20,38,39 several studies to be chloride channels.38,44 Further studies using Some well-characterized inhibitors of chloride channel activity heterologous expression, structural analysis, RNA interference include 5-nitro-2-(3-phenylpropylamino) benzoic acid, 4,40-dii- knock-down, as well as comparison of some functional properties sothiocyanatostilbene-2,20-disulphonic acid, niflumic acid, meflo- suggested that bestrophins formed CaCCs or at least a required quine, tamoxifen and n-phenyl-anthranilic acid; all of which have subunit of the molecular CaCCs.45--48 However, owing to evidence also been demonstrated to inhibit anoctamin-1.55 These com- that wild-type bestrophins exhibit neither the voltage depen- pounds do not, however, show very much specificity for CaCCs: dence nor outward rectification of CaCCs,20 the theory that many of them are active on other families of chloride channels bestrophins are classical CaCCs has recently fallen out of favor.49,50 (often CFTR and volume-regulated anion channels), and some also It was suggested that bestrophins may actually be regulators of have activity on non-chloride ion channels.10 For example, many calcium channels.51 There is also promising evidence based on of the chloride channel blockers also act as activators of calcium- patch-clamp investigations that bestrophins are responsible for activated potassium channels. These drugs (particularly the the non-classical cGMP-dependent calcium-activated chloride fenamates such as niflumic acid) are often used in experiments currents in vascular smooth muscle cells.52 despite the fact that it can be difficult to determine whether effects should be attributed to the block of CaCCs or activation of calcium-activated potassium channels; a challenge that has been ANOCTAMINS discussed in detail by Greenwood and Leblanc.62 Three studies with different methodologies performed by separate In the past few years, efforts to identify CaCC-modifying research groups in 2008 identified transmembrane protein 16A compounds have begun to come to fruition. CaCC modulators (also known as TMEM16A, anoctamin-1 and ANO1) as a CaCC.53--55 currently in development include denufosol and lancovutide This led to the current theory that suggests CaCCs are formed by (also called duramycin), both of which are CaCC activators TMEM16A and other members of the TMEM16 family of ten undergoing clinical trials for the treatment of cystic fibrosis.3,59 transmembrane proteins---recently dubbed ‘anoctamin-1 through However, neither denufosol nor lancovutide act directly to activate anoctamin-10’.55 This theory is supported by considerable experi- CaCCs. It is believed that CaCC activity is increased through the mental evidence and has been expertly summarized as part of a activation of purinergic receptors or an increase in intracellular review of chloride channels by Duran et al.2 Studies have calcium, respectively.60 As activators of chloride channels these demonstrated that anoctamins produce calcium-triggered chloride compounds could be expected to be inhibitory to erections and currents when heterologously expressed,53,55 and exhibit proper- only potentially have clinical benefit to less common disorders such ties very similar to those of classical CaCCs.2 as priapism. The theory that additional subunits are required for CaCCs or, Another CaCC modulator currently under development is alternatively, indirect pathways of calcium activation are utilized crofelemer, a compound originally discovered as a component (such as binding with calmodulin) has been suggested because of of the bark latex of Croton lechleri used by indigenous South structural evidence that anoctamins lack a calcium-binding Americans as a remedy for a variety of diseases.63,64 Crofelemer domain.56 A recently published study also indicates that the acts not only as an inhibitor at CaCCs (and has been confirmed to anoctamin-1 protein associates with calmodulin, although the inhibit anoctamin-1) but also as a partial antagonist at CFTR authors were unable to show precisely how this interaction channels.64 Inhibition of CaCCs and CFTR reduces chloride and occurs.57 fluid secretion from the intestinal epithelia,64 and could be However, the history of misidentifying CaCCs may be continuing. valuable in the treatment of secretory diarrheas.65 Phase III clinical It has been frequently noted that it is possible that anoctamins do trials on crofelemer were recently completed with positive results, not form the entire chloride channel alone, and the anoctamin but it has not been evaluated for activity on smooth muscle cells. component may be a subunit of a larger channel assembly for Crofelemer is not absorbed, so oral crofelemer is unlikely be useful CaCCs.3,53,58 Another group identified several specific, small- as a potential treatment for ED. molecule inhibitors of anoctamin-1, however, these inhibitors not The development of specific laboratory techniques (even before only failed to prevent a calcium-activated chloride current in the identification of the putative molecular identity of the intestinal and pulmonary epithelial cells, but knock-down of channels) has facilitated significant advances. A recent review anoctamin-1 expression with small interfering RNA also had no discussed useful techniques (among other topics) in considerable effect on CaCCs.40 It is important to note, however, that calcium- detail and the technology will not be discussed in detail here.3 activated chloride current in salivary gland epithelial cells was As mentioned above, a very recent study discovered a few completely abolished by the same inhibitors and small interfering specific inhibitors of anoctamin-1.40 However, several other papers RNA.40 Also, another similar study successfully blocked calcium- have also recently performed successful screens for CaCC activated chloride currents in pulmonary arterial smooth muscle inhibitors. Using high-throughput screening, De La Fuente cells.59 Despite the fact that anoctamins are by far the most et al.66 recently identified six novel classes of compounds (from promising of all molecular candidates to date, the history of a screen of 50 000 compounds) with inhibitory activity on incorrectly identifying molecules as CaCCs should serve as a intestinal epithelial CaCCs. These classes were designated as reminder to be cautious about embracing these proteins as the classes A through F. Classes A and B were specifically found to be true answer to the elusive identity of CaCCs in smooth muscle cells. likely to act directly on the CaCCs, as opposed to any subcellular signalling processes. The most potent inhibitors of the two direct inhibitor classes were ‘CaCCinh-A01’ (from ‘class A’, the 3-acyl-2- PHARMACOLOGY aminothiophen class) and ‘CaCCinh-B01’ (from ‘class B’, the 5-aryl- It has been proposed that inhibitors and activators of CaCC activity 2-aminothiazole class). Both of these compounds appear to have could prove to be therapeutically valuable for the treatment of selectivity toward CaCC receptors, and CaCCinh-A01 has been used many different disorders other than refractory ED,25 including: in other studies as a tool for blocking CaCCs.67

International Journal of Impotence Research (2012), 211 -- 216 & 2012 Macmillan Publishers Limited CaCCs in the corpus cavernosum DJ Linton et al 215 A few other compounds have been shown to have activity on sexual medicine, modulation of the CaCC family of channels CaCCs. Liang et al.68 performed a high-throughput screen of a occurs both by chloride current activation through sympathetic drug library and found that spiperone, a psychoactive drug, stimulation, and inhibition through the parasympathetic NO/ could elicit the release of calcium ions from the sarcoplasmic cGMP pathway. Inhibition of CaCCs by pharmacological agents reticulum, and activate CaCCs. A different study performed by could theoretically be very valuable to the treatment of ED Namkung et al.69 found that gallotannins (natural antioxidant because, unlike current treatment with PDE5 inhibitors, the compounds that are found in green tea and red wine) could relaxation produced would not be dependent on NO signalling. inhibit anoctamin-1 chloride conduction in cultured epithelial cells Despite the potential of chloride channel modulators as ther- and prevent contraction of rat arterial smooth muscle cells. apeutics for ED and countless other diseases, chloride channel Although these are just small preliminary studies, they clearly inhibitors have historically been plagued by very poor specificity, illustrate not only the growing interest in CaCC-targeted and, until recently, little has been known about the identity of the therapeutics but the success of new screening methods at CaCCs themselves. The recent identification of a very promising isolating compounds with specific inhibitory activity. candidate (the anoctamin family), and deliberate efforts to screen for specific chloride channel modulators have produced promising results. However, many of these studies focused on epithelial AREAS FOR FUTURE WORK chloride channels and not the smooth muscle cell chloride The importance of chloride channels in the corpus cavernosum channels such as those in the corpus cavernosum. In the future, has been clearly demonstrated, but there are many directions for research may confirm the identity of chloride channels in smooth future research to advance our understanding of these ion muscle and discern whether compounds active on CaCCs in channels and move toward the development of new chloride epithelial cells or arterial smooth muscle cells also have activity on channel-targeted therapeutics for ED. A quantitative PCR expres- CaCCs in the smooth muscle of the corpus cavernosum. sion study performed by Schreiber et al.58 indicated that various proteins of the anoctamin family are expressed in all murine tissues, with predominantly anoctamin-5 being primarily ex- CONFLICT OF INTEREST pressed in skeletal muscle, but there was little focus on smooth The authors declare no conflict of interest. muscle and no mention of corpus cavernosum tissue. One next step could be performing expression studies to confirm the expression of anoctamin-1---or perhaps other members of the ACKNOWLEDGEMENTS anoctamin family---in corpus cavernosum tissue. Exchange student fellowship for DJL from the University of British Columbia, Canada Another potential avenue for research is the genetic manipula- to the Department of OBGYN, National University of Singapore was supported by a tion of the proposed genes encoded CaCCs. 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