Cardiovascular perspectives of the TRP channel polycystin 2

Abstract

Calcium release from the endoplasmic reticulum is predominantly driven by two key ion channel receptors, Inositol 1, 4, 5-triphosphate receptor (InsP₃R) in non-excitable cells and Ryanodine Receptor (RyR) in excitable and muscle-based cells. These calcium transients can be modified by other less-studied ion channels, including polycystin 2 (PC2), a member of the Transient Receptor Potential (TRP) family. PC2 is found in various cell types and is evolutionarily conserved with paralogs ranging from single-cell organisms to yeasts and mammals. Interest in the mammalian form of PC2 stems from its disease relevance, as mutations in the PKD2 gene, which encodes PC2, result in Autosomal Dominant Polycystic Kidney Disease (ADPKD). This disease is characterized by renal and liver cysts, and cardiovascular extrarenal manifestations. However, in contrast to the well-defined roles of many TRP channels, the role of PC2 remains unknown, as it has different subcellular locations, and the functional understanding of the channel in each location is still unclear. Recent structural and functional studies have shed light on this channel. Moreover, studies on cardiovascular tissues have demonstrated a diverse role of PC2 in these tissues compared to that in the kidney. We highlight recent advances in understanding the role of this channel in the cardiovascular system and discuss the functional relevance of PC2 in non-renal cells.

Graphical Abstract

The Transient Receptor Potential (TRP) channel polycystin 2 (PC2) allows for cation flux in a variety of subcellular locations including the primary cilia, plasma membrane and endoplasmic reticulum, ultimately contributing to cardiac and vascular contractility.

Introduction

The transient receptor potential (TRP) family of proteins is generally defined as non-selective cation channels characterized by six transmembrane segments and a TRP domain (Nilius & Owsianik, 2011). Within mammals, there are seven different TRP subfamilies with 28 proteins identified as constituent members (Ramsey et al., 2006). Structurally, TRP channels form homo- and hetero-tetramers that open in response to a wide range of stimuli, including pH, stretch, voltage, and temperature (Montell, 2001; Venkatachalam & Montell, 2007; Julius, 2013). Collectively, TRP channels have been the targets of therapeutic approaches, as many are known to localize to the plasma membrane and have been implicated in many diseases (Koivisto et al., 2022). However, there are a few notable exceptions, such as the mucolipin family, which predominantly localizes to endosomes and lysosomes, and the polycystin family, which resides on the endoplasmic reticulum and primary cilia (Cai et al., 1999; Venkatachalam et al., 2015).

One of the most enigmatic TRP channels is polycystin 2 (PC2), which belongs to the polycystin TRP family. (The designated IUPHAR name for PC2 is TRPP1; however, it is also referred to as TRPP2 in the literature (Clapham et al., 2005)). The biological significance of PC2 lies in its clinical relevance. Mutations in PKD2, which encodes the PC2 protein, are responsible for approximately 20% of all cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic kidney disease with no cure. The remaining 80% of ADPKD cases are primarily caused by germline mutations in the interacting partner of PC2, polycystin 1 (PC1), an 11 transmembrane segment protein. Although the last six transmembrane segments of PC1 are similar to those of PC2, PC1 is not considered a TRP channel. Like other TRP channels, PC2 is composed of six transmembrane domains and forms homo-and hetero-tetramer complexes that allow cation flux (Wu et al., 2010). Several features of PC2 distinguish it from other TRP channels. These include the unique structural folds within PC2 and its varied subcellular location of PC2 in different cell and tissue types, including the plasma membrane, primary cilia, and endoplasmic reticulum (ER). Another important aspect to highlight is that although mutations in PKD2 lead to ADPKD, the main cause of mortality in patients with ADPKD in the advent of renal replacement therapy is cardiac failure, suggesting an important role for these proteins in the heart and vasculature (Fick et al., 1995). Although the role of PC2 in renal dysfunction and cystogenesis has been thoroughly described, and other reviews that address this specific topic have been conducted (Harris & Torres, 2014; Bergmann et al., 2018), its cardiovascular role remains largely unknown. Thus, this perspective article will highlight recent advances in the structural and electrophysiological characterization of PC2 and place these findings in the context of recent studies within the heart and vasculature.

Structural features and ion conductivity of polycystin 2

Advances in cryo-electron microscopy have enabled elucidation of the structure and function of TRP channels, including recent high-resolution maps that have led the way for functional studies (Huang et al., 2018, 2020; Zhao et al., 2022; Diver et al., 2022). The structure of PC2 at the near-atomic resolution level was described by several groups in 2016 and 2017 (Shen et al., 2016; Grieben et al., 2016; Wilkes et al., 2017), in which the transmembrane structure of PC2, in the absence of cytosolic amino- or carboxy-termini, was resolved. They found that the overall PC2 architecture had typical TRP channel features, such as the classic TRP channel fold. For PC2, the TRP channel fold incorporated the first four transmembrane segments, forming the voltage sensor-like domain (VSLD), followed by the pore domain, consisting of transmembrane segments 5 and 6, separated by two pore helices and a selectivity filter (Shen et al., 2016; Grieben et al., 2016). This structure forms a voltage-sensitive cation-selective ion channel when reconstituted into artificial lipid membranes under voltage-clamp conditions (Grieben et al., 2016). However, unlike other typical TRP channel structures, the VSLD lacks arginine residues required for voltage sensing (Grieben et al., 2016). Instead, an extensive modification to the VSLD was observed, which in the PC2 structure was named the TOP domain (or polycystin domain) (Shen et al., 2016; Grieben et al., 2016). The TOP domain, which has little homology to other known TRP structures, is formed between transmembrane segments 1 and 2, and consists of a series of five beta strands and two alpha helices and is heavily glycosylated (Shen et al., 2016; Grieben et al., 2016). The TOP domain acts like a cap as it extends into the extracellular domain (if the channel is positioned in the plasma membrane) or into the cytosol (if the channel is positioned in the ER). Although mutations can be found throughout the PKD2 gene, cryo-EM structures revealed that the TOP domain harbors most of the missense mutations in ADPKD. The TOP domain of each polycystin molecule directly contacts both the pore region of the adjacent molecule and its own VSLD. Thus, the TOP domain can influence the opening of the pore filter and, consequently, control channel activity (Grieben et al., 2016). The necessity of the TOP domain for PC2 channel activity was recently demonstrated by pathological missense mutations in two cysteine residues within the TOP domain, leading to the loss of ion conductivity in the primary cilia of human embryonic kidney cells (HEK cells, Vien et al., 2020b). Structurally, the mutation of these two cysteine residues resulted in the collapse of the beta-sheets within the PC2’s TOP domain. Collectively, these studies demonstrate the essential nature of the TOP domain in PC2 channel function and that the loss of the TOP domain fold impairs ion conductivity, resulting in aberrant calcium signaling that ultimately contributes to a cystic phenotype in renal cells (Vien et al., 2020b).

Like other TRP channels, the closed structures of PC2 revealed two “gates” within the channel conduction pore: an upper gate formed by the selectivity filter, and a lower gate formed by segment 6 (Shen et al., 2016; Grieben et al., 2016). A subsequent structure with the N and C-termini of PC2 intact, including the regulatory C-terminal EF-hand motif (Ćelić et al., 2012; Yang et al., 2015), demonstrated a partially open conformation with cations in the ion conduction pathway (Wilkes et al., 2017). Since Ca²⁺ ions have been shown to block the ion conduction pathway (Wilkes et al., 2017), it was speculated that this may reflect the regulatory role of Ca²⁺ in the channel activity of PC2, whereby higher concentrations of cytosolic Ca²⁺ inhibit channel activity (Cai et al., 2004). These structural studies suggest a role for the cytoplasmic regions in the regulation of PC2 channel activity. However, mutations in the EF-hand motif resulting in ADPKD have not been reported, and no renal cysts have been observed in a transgenic mouse model with mutations that abrogated the Ca²⁺-binding properties of the EF-hand (Vien et al., 2020a). Moreover, in contrast to the lipid bilayer studies where mutation to the EF-hand motif prevented PC2 channel activity (Ćelić et al., 2012), there was no loss of channel activity in PC2 patched from the primary cilia with the EF-hand mutations (Vien et al., 2020a). Collectively, these data suggest that although Ca²⁺ binds to the EF-hand motif, whether the regulation of the channel by this region is cell-location-specific (i.e., primary cilia compared with the ER or plasma membrane) remains unknown. Nevertheless, the loss of the ability of PC2 to bind calcium by its EF-hand was not sufficient to cause cystogenesis.

Subsequent to the cryo-EM structure of PC2, the structure of human PC2 in complex with PC1 was previously described (Su et al., 2018). This structure revealed a 1:3 stoichiometry with one PC1 molecule and three PC2 molecules. Because the PC1/PC2 cryo-EM structure did not include N- or C-terminal domains, the fact that the transmembrane segments of PC1/PC2 formed a complex revealed new interaction sites between PC1 and PC2. The inclusion of the PC1 molecule offsets the four-fold symmetry of the pore domain (formed by a PC2 tetramer) owing to a different conformation of PC1’s S6. Interestingly, although both PC1 and PC2 contained TOP domains, the PC1 TOP domain caused a gap of 15° in the TOP domain of PC1 and its neighboring PC2. Although the cryo-EM structure revealed a possible channel complex formed by PC1/PC2 (Su et al., 2018), direct electrophysiological evidence corresponding to this structure is lacking. The co-expression of PC1 and PC2 can enhance the trafficking of both molecules to primary cilia; however, whether the two molecules functionally interact remains unclear (Su et al., 2015). The best evidence suggesting the direct regulation of PC1 on PC2 channel activity was recently demonstrated as a fragment of the PC1 N-terminus acted as a possible ligand for the PC2 TOP domain to enhance PC2 currents (Ha et al., 2020). However, as these studies required forced expression of PC2 in the plasma membrane, it remains unclear whether endogenous functional PC1-PC2 currents are formed. The following section focuses on the electrophysiological characteristics of PC2.

The electrophysiological characteristics of PC2 in different cellular locations

Understanding the structure of PC2 will allow targeted functional studies to reveal the functions and conditions that lead to PC2 channel activity. However, the cellular location of PC2 requires examination of its electrophysiological properties. The earliest studies on PC2 identified it as an ER-resident protein using classical biochemical assays, including endo-H digestion (Cai et al., 1999). Immunofluorescence studies by several laboratories have confirmed its location to the ER, with subsequent studies identifying and functionally characterizing the location of PC2 to the primary cilia (Pazour et al., 2002; Bae et al., 2006; Knobel et al., 2008). The C-terminal tail of PC2 contains an ER retention site, and truncation mutations in the C-terminus translocate the channel to the plasma membrane (Cai et al., 1999). In contrast, an RVxP motif within the N-terminus can transport PC2 to primary cilia, which explains the multiple locations of PC2 to the ER, plasma membrane, and primary cilia (Geng et al., 2006). PC2 also aids in trafficking PC1 to the ciliary membrane (Su et al., 2015). PC1 is localized to the plasma membrane (Hu & Harris, 2020) but there is no evidence that it can conduct ions on its own. Given the different subcellular locations of PC2, the distinct physiological role of PC2 at each of these locations remains unknown.

Electrophysiological characterization of PC2 has primarily relied on heterologous systems, such as expression in HEK293 cells or Xenopus oocytes, which do not reliably transport the protein to the membrane or encompass the full complement of proteins that interact with PC2. Unlike other TRP channels with defined agonists and antagonists, there is no clear consensus on the natural agonist of PC2 (Yuan et al., 2015; Delling et al., 2016), and no specific antagonists have been described. Thus, characterizing the channel properties of PC2 in the ER is challenging because of the lack of direct pharmacological tools to activate or inhibit PC2. Therefore, characterization of cationic currents conducted by ER-localized PC2 necessitates the reconstitution of ER-derived microsomes into lipid bilayers (Koulen et al., 2002). These studies demonstrated that PC2 can flux calcium, which is the dominant cation gradient in the ER (Koulen et al., 2002).

In contrast, the electrophysiological properties of PC2 in the ciliary membranes have been extensively studied. In this context, electrophysiological studies of the ciliary membrane have primarily used genetic approaches, such as gene deletion, to define PC2 cationic currents. In comparison to the ionic gradients in the ER, the primary ciliary ionic gradients differ, as luminal free calcium in the primary cilia is higher, calculated to be ~600 nM (compared to the cytoplasm, which is approximately 50-100 nM) (Delling et al., 2013). Various studies have proposed that ciliary PC2 can function either alone or in complex with other interacting proteins, including PC1 and other TRP channels, to allow cation influx (Tian et al., 2022). For example, the activation of the PC1/PC2 complex in primary cilia is thought to enable the cell to sense fluid flow, allowing for calcium influx, which could further activate downstream cytosolic signaling pathways (Nauli et al., 2003). However, this theory was questioned, as the calcium signal in cilia did not propagate into the cytoplasm (Delling et al., 2016). Measurements of PC2 cationic currents in the primary cilia of immortalized murine collecting duct (imCD3) cells have confirmed PC2 as a bonafide ion channel in the cilia (Kleene & Kleene, 2016, 2021; Liu et al., 2018; Kleene et al., 2019; Vien et al., 2020b; Kleene, 2022). Intriguingly, the current conductance of calcium via PC2 in different systems has been found to range between 4 and 50 pS depending on the experimental approach (Koulen et al., 2002; Márquez-Nogueras et al., 2021). Figure 1 summarizes the different electrophysiological approaches and ionic conductance’s determined experimentally for PC2. While the earliest studies on lipid bilayers and more contemporaneous studies on nuclear-ER patches suggest a conductance of ~95 pS for calcium, ciliary measurements suggest that PC2 is predominantly in a closed state with very low calcium conductance (4pS).

Figure 1. Characterization of ionic conductance of PC2 using electrophysiology.

The top panels highlight the different ionic gradients present in the extracellular environment, cilia, cytosol, and endoplasmic reticulum. (Left panel: calcium gradient; Middle panel: potassium gradient; Right panel: sodium gradient). Ionic conductance of PC2 has been examined using ciliary membrane patch, nuclear/ER patch, and lipid bilayer expression. PC2 is a non-selective cation channel that has been suggested to allow for sodium, potassium and/or calcium influx in the ciliary membrane or calcium release in the ER. Figure adapted from data from (Koulen et al., 2002; Kleene & Kleene, 2016, 2021; Liu et al., 2018; Márquez-Nogueras et al., 2021; Kleene, 2022).

Thus, the low conductance of calcium by PC2 in primary cilia raises the question of whether calcium is the ion being conducted by PC2. Ciliary patch studies of PC2 in imCD3 cells revealed the preferential selection of Na⁺ and K⁺ over Ca²⁺ (Liu et al., 2018). Nonetheless, it is possible to create conditions that maximize the PC2 calcium current in the cilia (Kleene & Kleene, 2021). Interestingly, to generate the conditions for obtaining a calcium current through PC2, a model was proposed in which calcium influx through PC2 requires a positive feedback loop from additional calcium- and potassium-conductive TRP channels that are also localized to the ciliary membrane (Kleene, 2022). An increase in intracellular calcium, mediated directly by PC2 or other calcium channels, further activates the channel activity of PC2, allowing additional calcium influx. Although numerous studies have demonstrated the ability of PC2 to conduct calcium within the ciliary context, the functional role of this calcium signal remains controversial. Studies using a transgenic mouse expressing ratiometric genetic indicators targeting the cilia found that, in a range of different tissues, including stereocilia and renal epithelial cilia, the calcium signal generated in the cilia was unable to propagate into the cytoplasm (Delling et al., 2016). Instead, it has been suggested that the cytoplasmic calcium signal invades primary cilia (Delling et al., 2016).

Polycystin 2 and cardiac development in nodal cilia

In contrast to the findings described by Delling et al. (2016), several studies have suggested that calcium conduction via PC2 in nodal cilia, which then enters the cytoplasm, is necessary to break symmetry during development (Yuan et al., 2015; Katoh et al., 2023). Thus, the regenerative model of calcium entry into primary cilia (Kleene, 2022) appears to be consistent with two recent studies demonstrating the importance of PC2 in the left-right patterning of the heart in nodal cilia (Djenoune et al., 2023; Katoh et al., 2023). Using complementary methodologies, including optical tweezers, to move nodal cilia in zebrafish (Djenoune et al., 2023) and mice (Katoh et al., 2023), it has been shown that a series of ~30 directional fluid flow bend to the nodal cilia, leads to increased membrane tension that activates the flux of calcium through PC2 (Djenoune et al., 2023). The proposed functional reason for the high number of ciliary bends before the activation of a ciliary calcium signal is to distinguish between false and true signals (Djenoune et al., 2023). Moreover, it has been suggested that PC2 preferentially localizes to the bent side of the cilium (Katoh et al., 2023), and that the ciliary calcium signal may then elicit a cytosolic wave; although it should be noted that there was not enough temporal resolution in the experiments to exclude the back propagation of a calcium signal from the cytoplasm to the primary cilia. These studies highlight the critical role of nodal ciliary-localized PC2 in heart development, as the deletion of PC2 prevents the appropriate breaking of symmetry (Djenoune et al., 2023).

Although structural and electrophysiological characterizations have provided insights into PC2 function, many important questions remain unanswered. To date, there are no definitive answers regarding the physiological conditions that activate PC2 cationic flux in primary cilia and the preferential ions conducted by PC2 under physiological conditions. Beyond the activation and ion conduction of PC2, it is still debatable whether a calcium signal generated by calcium flux through PC2 in cilia can propagate into the cytoplasm (Wachten & Mill, 2023). This leads to additional questions, including the specific role of PC2 in non-ciliary environments, and whether the specific activity of PC2 in each location is governed by the molecular partners in that location, which in turn can affect the resultant PC2 channel activity. The latter questions are the focus of the following sections, as we expand on the role of PC2 in cardiomyocytes and vasculature, where the functional characterizations of PC2 have focused on its ER/SR and plasma membrane locations of PC2, respectively.

Polycystin 2 and calcium regulation in the heart and vasculature

Interest in PC2 in cardiovascular settings is important as cardiovascular diseases are the main cause of mortality in patients with ADPKD (Kuo & Chapman, 2019). In contrast to the above discussion on the function of PC2 in the primary and nodal cilia, studies on PC2 in the heart and vasculature have mainly focused on the ER/SR and plasma membrane location of the molecule. Notably, whereas nodal cilia function in left-right patterning, only endocardial cardiomyocytes appear to have primary cilia, which may contribute to sensing pressure or flow for cardiac valve maintenance (Djenoune et al., 2022). Thus, PC2 in cardiomyocytes is predominantly expressed within the ER/SR (DiNello et al., 2020). As previously mentioned, understanding the role of PC2 in the ER is restricted because of the current limitations in directly activating or inactivating PC2 through pharmacological or electrophysiological approaches. Using a nuclear-ER patch approach, which has been established for channels, such as InsP₃R (Mak et al., 2000, 2013), we obtained the calcium currents of PC2 with a conductance of ~50 pS (Márquez-Nogueras et al., 2021). However, functional studies on cardiomyocytes, smooth muscle cells, and endothelial cells have largely relied on approaches based on manipulating the amount of PC2 through deletion, mutations, or overexpression. Although the direct electrophysiological properties of PC2 in the heart and vasculature have not been fully investigated, collective studies in the heart and vasculature have demonstrated a functional role of PC2 in calcium release. Calcium release mediated by ER/SR-localized PC2 regulates known signaling pathways and other calcium release channels that affect downstream pathways. The effects of PC2 on ion channels and its downstream signaling effects in cardiomyocytes and vasculature are discussed.

Clinical data suggest that the ADPKD patient population (made up of germline mutations in PC2 and PC1) has a five-fold higher risk of ventricular arrhythmias (Yu et al., 2016). Mechanistically, arrhythmias may arise due to calcium leakage from the Ryanodine Receptor (RyR) as mutations or aberrant phosphorylation of RyR leads to catecholamergic polymorphic ventricular tachycardia (Zhao et al., 2015; Kushnir et al., 2018). PC2 does not appear to be a leak channel, as an siRNA screen that included PC2 did not identify this molecule as a regulator of ER calcium leakage in HEK293 cultured cells (Bandara et al., 2013). In contrast, PC2 regulated RyR activity. The earliest studies on PC2 function in the heart examined its effect on RyRs using a combination of lipid bilayer approaches and PC2 knockout in embryonic cardiomyocytes (Anyatonwu et al., 2007). These studies demonstrate that the C-terminal tail has no impact on RyR function. However, the 220 amino acid N-terminus, which has been described to be unstructured and for which all four known cryo-EM structures of PC2 have been excluded, inhibits RyR in a dose-dependent manner (Anyatonwu et al., 2007). However, it is unclear how the N-terminus binds to the RyR to inhibit its activity. Examination of the amino acid sequence of the N-terminus revealed several acidic stretches that could potentially interfere with channel gating of the RyR; however, further studies are required to demonstrate the activity of the PC2 N-terminus in RyR inhibition. More recent studies have shown with higher resolution microscopy of adult mouse tissue that PC2 is closely associated with, but does not directly colocalize with, RyR (DiNello et al., 2020). Therefore, the regulation of RyR by PC2 becomes important, as the junctional triad formed between RyR and the L-type calcium channel (Ca_V1.2) leads to the release of calcium required for contractility (Bers, 2008).

Our own studies in adult mice demonstrated that inducible deletion of PC2 in cardiomyocytes leads to a higher electrically evoked cytosolic calcium increase. Under diastolic conditions, we observed a decreased RyR spark number without a change in amplitude in the PC2 knockout mice compared to controls (DiNello et al., 2020). SERCA and the phosphorylation status of its regulatory protein phospholamban remained unchanged following the acute deletion of PC2 (DiNello et al., 2020). However, a decrease in the phosphorylation of RyR at the Serine 16, a region that disassociates the binding of phospholamban from SERCA, was noted in whole-body Pkd2 heterozygous mice aged 5 months (Kuo et al., 2014). Collectively, these studies indicate that PC2 partially regulates the activity of RyR and that these changes may be dependent on the degree and timing of the PC2 knockout (Figure 2B). Molecularly, how PC2 specifically interacts with and regulates RyR activity and its effect on downstream signaling pathways remains to be determined. Notably, the regulatory activity of PC2 on RyR is opposite to that of another calcium release channel, InsP₃R. Whereas PC2 inhibits the amplitude of the evoked RyR calcium release, PC2 enhances the release from InsP₃R in porcine renal cells and HEK cells (Anyatonwu et al., 2007; Sammels et al., 2010; Mekahli et al., 2012).

Figure 2. Role of polycystin-2 in cardiac and vascular tissues.

A. PC2 localizes to the plasma membrane of both endothelial and smooth muscle cells where it allows for cation (possibly sodium) influx into the cell. PC2 has an opposing role in endothelial and smooth muscle cells. In endothelial cells, the activation of PC2 channels with flow causes a calcium current that in turn activates calcium activated potassium channels (MacKay et al., 2020). Upon stretch stimulus, a sodium current is activated in smooth muscle hindlimb which promotes depolarization (Bulley et al., 2018). A stretch stimulus also negatively regulates TREK1 via filamin A. (Sharif-Naeini et al., 2009) (Li Fraine et al., 2017). In mesenteric vessels, a sodium current is elicited by the addition of phenylephrine. B. PC2 regulates the activity of RYR in the SR of cardiomyocytes which regulates contractility (Anyatonwu et al., 2007) (DiNello et al., 2020). PC1 also regulates the trafficking of the DHPR (not shown, Pedrozo et al., 2015). Cilia reside on the endocardial side of cardiomyocytes, and are thought to contribute to valvular formation and function, although the exact mechanism of action is unknown (Djenoune et al., 2022).

In addition to PC2 enabling the regulation of the intracellular calcium release apparatus, ER-localized PC2, together with its partner protein PC1, which can localize to the mitochondria, can facilitate calcium exchange in organellar contact sites between the ER and mitochondria (Rowe et al., 2013; Chiaravalli et al., 2016; Padovano et al., 2017; Torres et al., 2019). Consistent with the effect of PC2 on cellular metabolism, PC2 regulates the autophagic pathway in myoblasts and cardiomyocytes (Peña-Oyarzun et al., 2017; Criollo et al., 2018; Kretschmar et al., 2019). In C2C12 myoblasts, calcium appears to be essential for the conversion of myoblasts to fused cells and ultimately for the maturation of an ER structure to a more sarcoplasmic structure (Márquez-Nogueras et al., 2022). However, the direct role of PC2 in the regulation of cardiomyocyte mitochondrial function remains to be elucidated. PC2 within the cardiac setting may also participate in maintaining ER-mitochondrial contact sites to facilitate calcium transfer and metabolic output, similar to that described in kidney cells (Kuo et al., 2019). Collectively, these recent scientific studies underscore the increased need to study signaling microdomains facilitated by channels, such as PC2.

In addition to the ER/SR and ciliary location, PC2 resides in the plasma membrane (Witzgall, 2005). However, unlike other TRP channels on the cardiomyocyte plasma membrane that have been identified to contribute to cardiovascular dysfunction (Sah et al., 2013; Seo et al., 2014; Yamaguchi et al., 2017; Jones et al., 2019), there is no clear evidence that PC2 functions on the plasma membrane in cardiomyocytes. This is in contrast to the functional location of PC2 in endothelial and smooth muscle cells of the vasculature, as discussed below (Bulley et al., 2018; MacKay et al., 2020, 2022). The function of PC2 in cardiomyocytes differs from that of PC1. PC1 has been demonstrated to functionally regulate the trafficking of the L-type calcium channel by negatively regulating the delta subunit of Ca_V1.2 in a from-birth murine knockout of PC2 in cardiomyocytes (Pedrozo et al., 2015). However, such an interaction between PC2 and L-type calcium channels has not been demonstrated for PC2 (Pedrozo et al., 2015).

Polycystin 2 in the vasculature

Recent studies have revealed that PC2 is a contender for vascular tone control. The function of PC2 in the vasculature is complex. Unlike the predominant endoplasmic location of PC2 observed in cardiomyocytes or epithelial cells, PC2 has prominent plasma membrane location in both endothelial and smooth muscle cells (Bulley et al., 2018; MacKay et al., 2020, 2022) (Figure 2). Initial studies demonstrated that aortic smooth muscle cells isolated from heterozygous PC2 knockout mice had diminished baseline calcium levels but enhanced contractility upon phenylephrine treatment (Qian et al., 2007). Subsequent studies examining the role of both polycystins in smooth muscle suggested that both PC1 and PC2 were localized to the plasma membrane and that a specific interaction between PC2 and the actin protein filamin A was required for pressure sensing (Sharif-Naeini et al., 2009). Follow-up studies have suggested that the interaction of PC2 and filamin A inhibits the opening of mechanically sensitive channels, identified as the potassium-conducting channel TREK1 (Li Fraine et al., 2017). Bulley et.al. suggested that the opening of PC2 in vascular smooth muscle cells enables sodium flux, leading to depolarization and vasoconstriction (Bulley et al., 2018). When PC2 was knocked out in smooth muscle cells (SMC PC2 KO), mice displayed a hypotensive phenotype. However, a region-based and tissue-specific response exists. In patched hind limb vascular smooth muscle cells, the outward sodium current became more prominent when the tonicity of the extracellular fluid decreased to 250 mOsm (from 300 mOsm). The current was blocked by trivalent gadolinium and was not observed in the cells isolated from the SMC PC2 KO animal. When the mesenteric vessels were examined, there was no difference in the pressure-induced myogenic tone in SMC PC2 KO mice, but there was an attenuation in phenylephrine-induced constriction (Bulley et al., 2018). Moreover, in an angiotensin II-induced hypertension model, PC2 was upregulated in the vasculature and smooth muscle PC2 KO mice were not as hypertensive as the controls (Bulley et al., 2018). Functionally, the swelling-induced current activated by PC2 in smooth muscle cells results in dilation owing to the conduction of sodium, which causes depolarization and promotes vascular tone (Bulley et al., 2018). Taken together, the findings of the Sharif-Naeini and Jaggar et al. suggests that PC2 plays a role in mechanosensation in smooth muscle; however, this role may be confined to specific vascular beds, such as the hindlimb, whereas PC2 may contribute to agonist-induced stimulation in the mesentery.

Finally, it has recently been suggested that in acutely dissociated vascular smooth muscle cells, STIM1 and PC2 form functional complexes that activate intracellular calcium signaling (Guo et al., 2020). The potential role of PC2 in regulating STIM1 in the vasculature is interesting, as the consensus role of store-operated calcium entry is more prominent in proliferative cell culture conditions or in disease conditions, where the levels of STIM and ORAI are increased (House et al., 2008; Potier et al., 2009; Bisaillon et al., 2010; Motiani et al., 2013; Fernandez et al., 2015). Loss of STIM1 in smooth muscle results in decreased vascular tone and hypotension, indicating that STIM1 plays a role in basal contractility (Krishnan et al., 2022). The knockout of PC2 in smooth muscle results in diminished contractility, although the functional interaction between STIM1 and PC2 remains unclear (Guo et al., 2020).

In contrast to the hypotensive phenotype observed when PC2 is selectively knocked out in the smooth muscle layers of the vasculature, polycystins in endothelial cells have been found to contribute to vasodilatory responses. Early studies examining endothelial cells cultured from neonatal mouse aortas suggested that ciliary-localized PC1 aids in facilitating the release of vasodilator nitric oxide (NO) under flow conditions (Nauli et al., 2008). However, to date, there are no ciliary studies on PC2 in endothelial cells. MacKay et al. found that the opening of plasma membrane-localized PC2 in murine endothelial cells via flow-mediated mechanisms caused an increase in intracellular calcium, which in turn activated calcium-activated potassium channels (small and intermediate conductance) (MacKay et al., 2020). This allows for the hyperpolarization of endothelial cells and leads to vessel relaxation (MacKay et al., 2020). Thus, when PC2 is specifically knocked out in the endothelial cells, mice become hypertensive (MacKay et al., 2020). How the same channel in two neighboring cell types (endothelial and smooth muscle cells) mediates different charge entries (calcium in endothelial cells vs. sodium in smooth muscle cells) and, ultimately, diametrically different effects on vascular tone remains to be elucidated (Figure 2). As the vast majority of patients with ADPKD have hypertension as the first symptom (Perrone & Miskulin, 2006), it appears that the contribution of endothelial cells outweighs that of smooth muscles.

One possible means of reconciling these disparate data is to consider the subcellular distribution of PC2 and post-translational modifications to PC2. Studies on HEK, imCD3, and vascular smooth muscle preparations have indicated that PC2 can be post-translationally modified through acetylation, SUMOylation, and phosphorylation (Cai et al., 2004; Plotnikova et al., 2011; Padovano et al., 2017; Hasan et al., 2019; Hu & Harris, 2020). Specifically, SUMOylation protects PC2 from internalization into the plasma membrane of smooth muscle cells (Hasan et al., 2019). Whether these modifications also alter other subcellular locations of PC2 or whether these modifications are present in cardiomyocytes is unknown. Finally, it has been noted in several pathological conditions, ranging from a high-salt diet to an ischemic kidney, heart failure, and cerebral diseases in both murine and human samples that PC2 is upregulated in these disease settings (Brill et al., 2020). Whether upregulated PC2 has the same function as endogenous PC2, or if there are additional roles for this protein, either conferred by preferred locations or by post-translational modifications, remains to be determined.

Conclusion and future perspectives

What makes PC2 a TRP channel? This structural revolution aided our understanding of the functionality of PC2. However, important functional questions remain unanswered. One aspect of PC2 biology that requires further elucidation is the identification of bona fide physiological agonists of PC2. Owing to the location of PC2 in primary cilia, considerable efforts have been made to determine whether PC2 is a mechanosensor that senses fluid flow or bending. However, the physiological environment in which PC2 predominantly resides has been overlooked. The kidney is the organ where salt and water are reabsorbed, and urine is formed. The apical side of the epithelium, where primary cilia reside, is exposed to urine. Surprisingly, despite the prominent location of renal cysts in the collecting duct of the kidney, where urine in the medulla can reach osmolarities of 1,200 mOsm in a dehydrated human or 50 mOsm in an overly hydrated human, few studies have examined the ability of osmotic stress to activate PC2. As discussed in the section on PC2 in smooth muscle, the presence or absence of PC2 alters the ability of the plasma membrane to sense stretching (Sharif-Naeini et al., 2009; Retailleau & Duprat, 2014; Bulley et al., 2018). Osmolarity has been reported to be a direct agonist of other TRP channels involved in urine concentration (Tomilin et al., 2019), and in concert with cells is associated with osmotic changes (Toft-Bertelsen & MacAulay, 2021). Future studies investigating the direct or indirect ability of osmolytes to activate this channel should provide more information on this unique protein.

Another aspect is the cellular location of PC2. The structural identity of PC2 and the PC1/PC2 complex has demonstrated the importance of missense TOP domain mutations in ADPKD pathogenesis; however, resolving the conditions under which PC2 is activated in-vivo without resorting to reductionist approaches remains a challenge. Thus, studies that distinguish between ER-localized PC2 and ciliary and plasma membrane PC2 channel functions are warranted to understand how this channel regulates critical signaling pathways within the cell. This distinction is needed as there is still no consensus regarding whether ciliary calcium can activate cytosolic calcium (Delling et al., 2016), and the biological implications of one governing the other (Yuan et al., 2015; Djenoune et al., 2023). The emerging role of PC2 in the plasma membrane of vascular cells provides an interesting comparison with the discussion of PC2 in the ER/SR in cardiomyocytes and the function of PC2 in the cilia of renal epithelial cells. This has important clinical relevance because of the high prevalence of cardiovascular defects in ADPKD, suggesting that the molecular dissection of PC2 in cardiovascular disease remains a key target for the future.

Biography

Dr. Karla M. Márquez-Nogueras is a postdoctoral researcher in Dr. Ivana Kuo's laboratory in the Department of Cell and Molecular Physiology at Loyola University Chicago. She is a postdoctoral fellow in the KUH Forward Program, an interdisciplinary training program across Chicago that focuses on nephrology, urology and hematology. She earned her bachelor’s and master’s degree in Microbiology from the University of Puerto Rico, Mayagüez and received her PhD in Microbiology from the University of Georgia. Dr. Márquez-Nogueras’s research examines the natural agonists of the polycystin complex in the kidney and the potential signaling pathways activated.