Victor G. Romanenko

Tmem16A Encodes the Ca2+-activated Cl− Channel in Mouse Submandibular Salivary Gland Acinar Cells

Activation of an apical Ca2+-dependent Cl− channel (CaCC) is the rate-limiting step for fluid secretion in many exocrine tissues. Here, we compared the properties of native CaCC in mouse submandibular salivary gland acinar cells to the Ca2+-gated Cl− currents generated by Tmem16A and Best2, members from two distinct families of Ca2+-activated Cl− channels found in salivary glands. Heterologous expression of Tmem16A and Best2 transcripts in HEK293 cells produced Ca2+-activated Cl− currents with time and voltage dependence and inhibitor sensitivity that resembled the Ca2+-activated Cl− current found in native salivary acinar cells. Best2−/− and Tmem16A−/− mice were used to further characterize the role of these channels in the exocrine salivary gland. The amplitude and the biophysical footprint of the Ca2+-activated Cl− current in submandibular gland acinar cells from Best2-deficient mice were the same as in wild type cells. Consistent with this observation, the fluid secretion rate in Best2 null mice was comparable with that in wild type mice. In contrast, submandibular gland acinar cells from Tmem16A−/− mice lacked a Ca2+-activated Cl− current and a Ca2+-mobilizing agonist failed to stimulate Cl− efflux, requirements for fluid secretion. Furthermore, saliva secretion was abolished by the CaCC inhibitor niflumic acid in wild type and Best2−/− mice. Our results demonstrate that both Tmem16A and Best2 generate Ca2+-activated Cl− current in vitro with similar properties to those expressed in native cells, yet only Tmem16A appears to be a critical component of the acinar Ca2+-activated Cl− channel complex that is essential for saliva production by the submandibular gland.

Cholesterol and Ion Channels

A variety of ion channels, including members of all major ion channel families, have been shown to be regulated by changes in the level of membrane cholesterol and partition into cholesterol-rich membrane domains. In general, several types of cholesterol effects have been described. The most common effect is suppression of channel activity by an increase in membrane cholesterol, an effect that was described for several types of inwardly-rectifying K(+) channels, voltage-gated K(+) channels, Ca(+2) sensitive K(+) channels, voltage-gated Na(+) channels, N-type voltage-gated Ca(+2) channels and volume-regulated anion channels. In contrast, several types of ion channels, such as epithelial amiloride-sensitive Na(+) channels and Transient Receptor Potential channels, as well as some of the types of inwardly-rectifying and voltage-gated K(+) channels were shown to be inhibited by cholesterol depletion. Cholesterol was also shown to alter the kinetic properties and current-voltage dependence of several voltage-gated channels. Finally, maintaining membrane cholesterol level is required for coupling ion channels to signalling cascades. In terms of the mechanisms, three general mechanisms have been proposed: (i) specific interactions between cholesterol and the channel protein, (ii) changes in the physical properties of the membrane bilayer and (iii) maintaining the scaffolds for protein-protein interactions. The goal of this review is to describe systematically the role of cholesterol in regulation of the major types of ion channels and to discuss these effects in the context of the models proposed.

Purinergic P2X7 Receptors Mediate ATP-induced Saliva Secretion by the Mouse Submandibular Gland

Salivary glands express multiple isoforms of P2X and P2Y nucleotide receptors, but their in vivo physiological roles are unclear. P2 receptor agonists induced salivation in an ex vivo submandibular gland preparation. The nucleotide selectivity sequence of the secretion response was BzATP ≫ ATP > ADP ≫ UTP, and removal of external Ca2+ dramatically suppressed the initial ATP-induced fluid secretion (∼85%). Together, these results suggested that P2X receptors are the major purinergic receptor subfamily involved in the fluid secretion process. Mice with targeted disruption of the P2X7 gene were used to evaluate the role of the P2X7 receptor in nucleotide-evoked fluid secretion. P2X7 receptor protein and BzATP-activated inward cation currents were absent, and importantly, purinergic receptor agonist-stimulated salivation was suppressed by more than 70% in submandibular glands from P2X7-null mice. Consistent with these observations, the ATP-induced increases in [Ca2+]i were nearly abolished in P2X7–/– submandibular acinar and duct cells. ATP appeared to also act through the P2X7 receptor to inhibit muscarinic-induced fluid secretion. These results demonstrate that the ATP-sensitive P2X7 receptor regulates fluid secretion in the mouse submandibular gland.

Sensitivity of Volume-regulated Anion Current to Cholesterol Structural Analogues

Depletion of membrane cholesterol and substitution of endogenous cholesterol with its structural analogues was used to analyze the mechanism by which cholesterol regulates volume-regulated anion current (VRAC) in endothelial cells. Depletion of membrane cholesterol enhanced the development of VRAC activated in a swelling-independent way by dialyzing the cells either with GTPγS or with low ionic strength solution. Using MβCD–sterol complexes, 50–80% of endogenous cholesterol was substituted with a specific analogue, as verified by gas-liquid chromatography. The effects of cholesterol depletion were reversed by the substitution of endogenous cholesterol with its chiral analogue, epicholesterol, or with a plant sterol, β-sitosterol, two analogues that mimic the effect of cholesterol on the physical properties of the membrane bilayer. Alternatively, when cholesterol was substituted with coprostanol that has only minimal effect on the membrane physical properties it resulted in VRAC enhancement, similar to cholesterol depletion. In summary, our data show that these channels do not discriminate between the two chiral analogues of cholesterol, as well as between the two cholesterols and β-sitosterol, but discriminate between cholesterol and coprostanol. These observations suggest that endothelial VRAC is regulated by the physical properties of the membrane.

Regulation of membrane potential and fluid secretion by Ca2+-activated K+ channels in mouse submandibular glands.

We have recently shown that the IK1 and maxi‐K channels in parotid salivary gland acinar cells are encoded by the KCa3.1 and KCa1.1 genes, respectively, and in vivo stimulated parotid secretion is severely reduced in double‐null mice. The current study tested whether submandibular acinar cell function also relies on these channels. We found that the K+ currents in submandibular acinar cells have the biophysical and pharmacological footprints of IK1 and maxi‐K channels and their molecular identities were confirmed by the loss of these currents in KCa3.1‐ and KCa1.1‐null mice. Unexpectedly, the pilocarpine‐stimulated in vivo fluid secretion from submandibular glands was essentially normal in double‐null mice. This result and the possibility of side‐effects of pilocarpine on the nervous system, led us to develop an ex vivo fluid secretion assay. Fluid secretion from the ex vivo assay was substantially (about 75%) reduced in animals with both K+ channel genes ablated – strongly suggesting systemic complications with the in vivo assay. Additional experiments focusing on the membrane potential in isolated submandibular acinar cells revealed mechanistic details underlying fluid secretion in K+ channel‐deficient mice. The membrane potential of submandibular acinar cells from wild‐type mice remained strongly hyperpolarized (−55 ± 2 mV) relative to the Cl− equilibrium potential (−24 mV) during muscarinic stimulation. Similar hyperpolarizations were observed in KCa3.1‐ and KCa1.1‐null mice (−51 ± 3 and −48 ± 3 mV, respectively), consistent with the normal fluid secretion produced ex vivo. In contrast, acinar cells from double KCa3.1/KCa1.1‐null mice were only slightly hyperpolarized (−35 ± 2 mV) also consistent with the ex vivo (but not in vivo) results. Finally, we found that the modest hyperpolarization of cells from the double‐null mice was maintained by the electrogenic Na+,K+‐ATPase.

Apical maxi-K (KCa1.1) channels mediate K secretion by the mouse submandibular exocrine gland

The exocrine salivary glands of mammals secrete K+ by an unknown pathway that has been associated with HCO3(-) efflux. However, the present studies found that K+ secretion in the mouse submandibular gland did not require HCO3(-), demonstrating that neither K+/HCO3(-) cotransport nor K+/H+ exchange mechanisms were involved. Because HCO3(-) did not appear to participate in this process, we tested whether a K channel is required. Indeed, K+ secretion was inhibited >75% in mice with a null mutation in the maxi-K, Ca2+-activated K channel (KCa1.1) but was unchanged in mice lacking the intermediate-conductance IKCa1 channel (KCa3.1). Moreover, paxilline, a specific maxi-K channel blocker, dramatically reduced the K+ concentration in submandibular saliva. The K+ concentration of saliva is well known to be flow rate dependent, the K+ concentration increasing as the flow decreases. The flow rate dependence of K+ secretion was nearly eliminated in KCa1.1 null mice, suggesting an important role for KCa1.1 channels in this process as well. Importantly, a maxi-K-like current had not been previously detected in duct cells, the theoretical site of K+ secretion, but we found that KCa1.1 channels localized to the apical membranes of both striated and excretory duct cells, but not granular duct cells, using immunohistochemistry. Consistent with this latter observation, maxi-K currents were not detected in granular duct cells. Taken together, these results demonstrate that the secretion of K+ requires and is likely mediated by KCa1.1 potassium channels localized to the apical membranes of striated and excretory duct cells in the mouse submandibular exocrine gland.

Molecular identification and physiological roles of parotid acinar cell maxi-K channels.

The physiological success of fluid-secreting tissues relies on a regulated interplay between Ca2+-activated Cl– and K+ channels. Parotid acinar cells express two types of Ca2+-activated K+ channels: intermediate conductance IK1 channels and maxi-K channels. The IK1 channel is encoded by the KCa3.1 gene, and the KCa1.1 gene is a likely candidate for the maxi-K channel. To confirm the genetic identity of the maxi-K channel and to probe its specific roles, we studied parotid glands in mice with the KCa1.1 gene ablated. Parotid acinar cells from these animals lacked maxi-K channels, confirming their genetic identity. The stimulated parotid gland fluid secretion rate was normal, but the sodium and potassium content of the secreted fluid was altered. In addition, we found that the regulatory volume decrease in acinar cells was substantially impaired in KCa1.1-null animals. We examined fluid secretion from animals with both K+ channel genes deleted. The secretion rate was severely reduced, and the ion content of the secreted fluid was significantly changed. We measured the membrane potentials of acinar cells from wild-type mice and from animals with either or both K+ channel genes ablated. They revealed that the observed functional effects on fluid secretion reflected alterations in cell membrane voltage. Our findings show that the maxi-K channels are critical for the regulatory volume decrease in these cells and that they play an important role in the sodium uptake and potassium secretion process in the ducts of these fluid-secreting salivary glands.

Evidence for the role of cell stiffness in modulation of volume-regulated anion channels.

Aim:  To investigate the link between cell stiffness and volume‐regulated anion current (VRAC) in aortic endothelium.

Clcn2 encodes the hyperpolarization-activated chloride channel in the ducts of mouse salivary glands

Transepithelial Cl(-) transport in salivary gland ducts is a major component of the ion reabsorption process, the final stage of saliva production. It was previously demonstrated that a Cl(-) current with the biophysical properties of ClC-2 channels dominates the Cl(-) conductance of unstimulated granular duct cells in the mouse submandibular gland. This inward-rectifying Cl(-) current is activated by hyperpolarization and elevated intracellular Cl(-) concentration. Here we show that ClC-2 immunolocalized to the basolateral region of acinar and duct cells in mouse salivary glands, whereas its expression was most robust in granular and striated duct cells. Consistent with this observation, nearly 10-fold larger ClC-2-like currents were observed in granular duct cells than the acinar cells obtained from submandibular glands. The loss of inward-rectifying Cl(-) current in cells from Clcn2(-/-) mice confirmed the molecular identity of the channel responsible for these currents as ClC-2. Nevertheless, both in vivo and ex vivo fluid secretion assays failed to identify significant changes in the ion composition, osmolality, or salivary flow rate of Clcn2(-/-) mice. Additionally, neither a compensatory increase in Cftr Cl(-) channel protein expression nor in Cftr-like Cl(-) currents were detected in Clcn2 null mice, nor did it appear that ClC-2 was important for blood-organ barrier function. We conclude that ClC-2 is the inward-rectifying Cl(-) channel in duct cells, but its expression is not apparently required for the ion reabsorption or the barrier function of salivary ductal epithelium.

The role of cell cholesterol and the cytoskeleton in the interaction between IK1 and maxi-K channels

Recently, we demonstrated a novel interaction between large-conductance (maxi-K or K(Ca)1.1) and intermediate-conductance (IK1 or K(Ca)3.1) Ca(2+)-activated K channels: activation of IK1 channels causes the inhibition of maxi-K activity (Thompson J and Begenisich T. J Gen Physiol 127: 159-169, 2006). Here we show that the interaction between these two channels can be regulated by the membrane cholesterol level in parotid acinar cells. Depletion of cholesterol using methyl-beta-cyclodextrin weakened, while cholesterol enrichment increased, the ability of IK1 activation to inhibit maxi-K channels. Cholesterols stereoisomer, epicholesterol, was unable to substitute for cholesterol in the interaction between the two K channels, suggesting a specific cholesterol-protein interaction. This suggestion was strengthened by the results of experiments in which cholesterol was replaced by coprostanol and epicoprostanol. These two sterols have nearly identical effects on membrane physical properties and cholesterol-rich microdomain stability, but had very different effects on the IK1/maxi-K interaction. In addition, the IK1/maxi-K interaction was unaltered in cells lacking caveolin, the protein essential for formation and stability of caveolae. Finally, disruption of the actin cytoskeleton restored the IK1-induced maxi-K inhibition that was lost with cell cholesterol depletion, demonstrating the importance of an intact cytoskeleton for the cholesterol-dependent regulation of the IK1/maxi-K interaction.