Jorge Arreola

Voltage- and calcium-dependent gating of TMEM16A/Ano1 chloride channels are physically coupled by the first intracellular loop

Ca2+-activated Cl− channels (CaCCs) are exceptionally well adapted to subserve diverse physiological roles, from epithelial fluid transport to sensory transduction, because their gating is cooperatively controlled by the interplay between ionotropic and metabotropic signals. A molecular understanding of the dual regulation of CaCCs by voltage and Ca2+ has recently become possible with the discovery that Ano1 (TMEM16a) is an essential subunit of CaCCs. Ano1 can be gated by Ca2+ or by voltage in the absence of Ca2+, but Ca2+- and voltage-dependent gating are very closely coupled. Here we identify a region in the first intracellular loop that is crucial for both Ca2+ and voltage sensing. Deleting 448EAVK in the first intracellular loop dramatically decreases apparent Ca2+ affinity. In contrast, mutating the adjacent amino acids 444EEEE abolishes intrinsic voltage dependence without altering the apparent Ca2+affinity. Voltage-dependent gating of Ano1 measured in the presence of intracellular Ca2+ was facilitated by anions with high permeability or by an increase in [Cl−]e. Our data show that the transition between closed and open states is governed by Ca2+ in a voltage-dependent manner and suggest that anions allosterically modulate Ca2+-binding affinity. This mechanism provides a unified explanation of CaCC channel gating by voltage and ligand that has long been enigmatic.

Loss of Hyperpolarization-activated Cl− Current in Salivary Acinar Cells from Clcn2 Knockout Mice

ClC-2 is localized to the apical membranes of secretory epithelia where it has been hypothesized to play a role in fluid secretion. Although ClC-2 is clearly the inwardly rectifying anion channel in several tissues, the molecular identity of the hyperpolarization-activated Cl− current in other organs, including the salivary gland, is currently unknown. To determine the nature of the hyperpolarization-activated Cl− current and to examine the role of ClC-2 in salivary gland function, a mouse line containing a targeted disruption of theClcn2 gene was generated. The resulting homozygousClcn2 −/− mice lacked detectable hyperpolarization-activated chloride currents in parotid acinar cells and, as described previously, displayed postnatal degeneration of the retina and testis. The magnitude and biophysical characteristics of the volume- and calcium-activated chloride currents in these cells were unaffected by the absence of ClC-2. Although ClC-2 appears to contribute to fluid secretion in some cell types, both the initial and sustained salivary flow rates were normal inClcn2 −/− mice following in vivostimulation with pilocarpine, a cholinergic agonist. In addition, the electrolytes and protein contents of the mature secretions were normal. Because ClC-2 has been postulated to contribute to cell volume control, we also examined regulatory volume decrease following cell swelling. However, parotid acinar cells from Clcn2 −/−mice recovered volume with similar efficiency to wild-type littermates. These data demonstrate that ClC-2 is the hyperpolarization-activated Cl− channel in salivary acinar cells but is not essential for maximum chloride flux during stimulated secretion of saliva or acinar cell volume regulation.

Secretion and cell volume regulation by salivary acinar cells from mice lacking expression of the Clcn3 Cl− channel gene

Salivary gland acinar cells shrink when Cl− currents are activated following cell swelling induced by exposure to a hypotonic solution or in response to calcium‐mobilizing agonists. The molecular identity of the Cl− channel(s) in salivary cells involved in these processes is unknown, although ClC‐3 has been implicated in several tissues as a cell‐volume‐sensitive Cl− channel. We found that cells isolated from mice with targeted disruption of the Clcn3 gene undergo regulatory volume decrease in a fashion similar to cells from wild‐type littermates. Consistent with a normal regulatory volume decrease response, the magnitude and the kinetics of the swell‐activated Cl− currents in cells from ClC‐3‐deficient mice were equivalent to those from wild‐type mice. It has also been suggested that ClC‐3 is activated by Ca2+‐calmodulin‐dependent protein kinase II; however, the magnitude of the Ca2+‐dependent Cl− current was unchanged in the Clcn3−/‐ animals. In addition, we observed that ClC‐3 appeared to be highly expressed in the smooth muscle cells of glandular blood vessels, suggesting a potential role for this channel in saliva production by regulating blood flow, yet the volume and ionic compositions of in vivo stimulated saliva from wild‐type and null mutant animals were comparable. Finally, in some cells ClC‐3 is an intracellular channel that is thought to be involved in vesicular acidification and secretion. Nevertheless, the protein content of saliva was unchanged in Clcn3−/‐ mice. Our results demonstrate that the ClC‐3 Cl− channel is not a major regulator of acinar cell volume, nor is it essential for determining the secretion rate and composition of saliva.

Cytosolic Ca2+ and Ca2+‐activated Cl− current dynamics: insights from two functionally distinct mouse exocrine cells

The dynamics of Ca2+ release and Ca2+‐activated Cl− currents in two related, but functionally distinct exocrine cells, were studied to gain insight into how the molecular specialization of Ca2+ signalling machinery are utilized to produce different physiological endpoints: in this case, fluid or exocytotic secretion. Digital imaging and patch‐clamp methods were used to monitor the temporal and spatial properties of changes in cytosolic Ca2+ concentration ([Ca2+]c) and Cl− currents following the controlled photolytic release of caged‐InsP3 or caged‐Ca2+. In parotid and pancreatic acinar cells, changes in [Ca2+]c and activation of a Ca2+‐activated Cl− current occurred with close temporal coincidence. In parotid, a rapid global Ca2+ signal was invariably induced, even with low‐level photolytic release of threshold amounts of InsP3. In pancreas, threshold stimulation generated an apically delimited [Ca2+]c signal, while a stronger stimulus induced a global [Ca2+]c signal which exhibited characteristics of a propagating wave. InsP3 was more effective in parotid, where [Ca2+]c signals initiated with shorter latency and exhibited a faster time‐to‐peak than in pancreas. The increased potency of InsP3 in parotid probably results from a four‐fold higher number of InsP3 receptors as measured by radiolabelled InsP3 binding and western blot analysis. The Ca2+ sensitivity of the Cl− channels in parotid and pancreas was determined from the [Ca2+]‐current relationship measured during a dynamic ‘Ca2+ ramp’ produced by the continuous, low‐level photolysis of caged‐Ca2+. In addition to a greater number of InsP3 receptors, the Cl− current density of parotid acinar cells was more than four‐fold greater than that of pancreatic cells. Whereas activation of the current was tightly coupled to increases in Ca2+ in both cell types, local Ca2+ clearance was found to contribute substantially to the deactivation of the current in parotid. These data reveal specializations of common modules of Ca2+‐release machinery and subsequent effector activation that are specifically suited to the distinct functional roles of these two related cell types.

Three distinct chloride channels control anion movements in rat parotid acinar cells.

1. We used the whole‐cell configuration of the patch clamp technique to examine the different macroscopic Cl‐ currents present in single rat parotid acinar cells. 2. Cell swelling produced by negative osmotic pressure (hypotonic bath solutions) induced a large outwardly rectifying Cl‐ current with little or no time and voltage dependence. In contrast, an increase in intracellular [Ca2+] induced by ionomycin activated Cl‐ currents with very different properties. Ca(2+)‐activated Cl‐ currents showed outward rectification, relatively slow activation kinetics and marked voltage dependence. These results are consistent with the existence of two different outwardly rectifying Cl‐ channels in rat parotid cells. 3. In conditions designed to eliminate the activation of these two Cl‐ currents, a third type of current was observed. This third current was activated in a time‐dependent manner by hyperpolarized potentials and was about equally permeant to Cl‐, I‐ and Br‐. 4. The properties of the hyperpolarization‐activated current were similar to those of the cloned ClC‐2 channel. Polymerase chain reaction‐based methods and ribonuclease protection analyses indicated the presence in parotid gland of mRNA homologous to ClC‐2. 5. Individual parotid acinar cells expressed all three types of Cl‐ channels. Each type of channel may contribute to Cl‐ efflux in distinct stages of the secretion process depending on the intracellular [Ca2+], cell volume and membrane potential.

COMPARISON OF VOLTAGE-ACTIVATED CL- CHANNELS IN RAT PAROTID ACINAR CELLS WITH CLC-2 IN A MAMMALIAN EXPRESSION SYSTEM

Abstract. Rat parotid acinar cells express Cl− currents that are activated in a time-dependent manner by hyperpolarized potentials. ClC-2, a member of the ClC gene family, codes for a voltage-gated, inward rectifying anion channel when expressed in Xenopus oocytes. In the present study, we found that cDNA derived from individual parotid acinar cells contained sequence identical to that reported for ClC-2 in rat brain and heart. A polyclonal antibody generated against the N-terminal cytoplasmic domain of ClC-2 recognized an approximately 100 kD protein on western blots of both brain and parotid gland. ClC-2 expressed in oocytes has different kinetics from the currents found in parotid acinar cells. Since the ClC-2 channel was cloned from and its transcripts are expressed in mammalian tissue, we compared the channel properties of acinar cells to a mammalian expression system. We expressed ClC-2 channels in human embryonic kidney cells, HEK 293, using recombinant ClC-2 DNA and ClC-2 DNA fused with DNA coding for jellyfish green fluorescent protein (GFP). Confocal microscopy revealed that the expressed ClC-2-GFP chimera protein localized to the plasma membrane. Whole cell Cl− currents from HEK 293 cells expressing ClC-2-GFP were similar, if not identical, to the Cl− currents recorded from cells transfected with ClC-2 cDNA (no GFP). The voltage-dependence and kinetics of ClC-2 channels expressed in HEK 293 cells were quite similar to those in acinar cells. Channels in parotid acinar and HEK 293 cells activated at more positive membrane potentials and with a faster time course than the channels expressed in Xenopus oocytes. In summary, we found that ClC-2 message and protein are expressed in salivary cells and that the properties of voltage-activated, inward rectifying Cl− channels in acinar cells are similar to those generated by the ClC-2-GFP construct expressed in HEK 293 cells. The properties of the ClC-2 anion channel seem to be dependent on the type of cell background in which it is expressed.

Interaction of Ba2+ with the Pores of the Cloned Inward Rectifier K+ Channels Kir2.1 Expressed in Xenopus Oocytes

Interactions of Ba2+ with K+ and molecules contributing to inward rectification were studied in the cloned inward rectifier K+ channels, Kir2.1. Extracellular Ba2+ blocked Kir2.1 channels with first-order kinetics in a Vm-dependent manner. At Vm more negative than -120 mV, the Kd-Vm relationship became less steep and the dissociation rate constants were larger, suggesting Ba2+ dissociation into the extracellular space. Both depolarization and increasing [K+]i accelerated the recovery from extracellular Ba2+ blockade. Intracellular K+ appears to relieve Ba2+ blockade by competitively slowing the Ba2+ entrance rate, instead of increasing its exit rate by knocking off action. Intracellular spermine (100 microM) reduced, whereas 1 mM [Mg2+]i only slightly reduced, the ability of intracellular K+ to repulse Ba2+ from the channel pore. Intracellular Ba2+ also blocked outward IKir2.1 in a voltage-dependent fashion. At Vm >/= +40 mV, where intrinsic inactivation is prominent, intracellular Ba2+ accelerated the inactivation rate of the outward IKir2.1 in a Vm-independent manner, suggesting interaction of Ba2+ with the intrinsic gate of Kir2.1 channels.

Functional interactions between P2X4 and P2X7 receptors from mouse salivary epithelia

Mouse parotid acinar cells express P2X4 and P2X7 receptors (mP2X4R and mP2X7R) whose physiological function remains undetermined. Here we show that mP2X4R expressed in HEK‐293 cells do not allow the passage of tetraethylammonium (TEA+) and promote little, if any, ethidium bromide (EtBr) uptake when stimulated with ATP or BzATP. In contrast, mP2X7R generates slowly decaying TEA+ current, sustained Na+ current and promotes robust EtBr uptake. However, ATP‐activated TEA+ current from acinar cells was unlike that generated by mP2X7R or mP2X4R. Functional interactions between mP2X4R and mP2X7R were investigated in HEK cells co‐transfected with different mP2X4 : mP2X7 cDNA ratios and using solutions containing either TEA+ or Na+ ions. Co‐expressed channels generated a TEA+ current that displayed faster decay during ATP stimulation than mP2X7R alone. Moreover, cells transfected with a 2 : 1 cDNA ratio displayed decaying kinetics similar to those observed in acinar cells. Concentration–response curves in Na+‐containing solutions were constructed for heterologously expressed mP2X4R, mP2X7R and mP2X4R:mP2X7R co‐expressions as well as acinar cells. The EC50 values determined were 11, 220, 434 and 442 μm, respectively. Na+ currents generated by expressing mP2X4R or mP2X7R alone were potentiated by ivermectin (IVM). In contrast, IVM potentiation in acinar cells and HEK cells co‐expressing P2X4 and P2X7 (1 : 1 or 2 : 1 cDNA ratios) was seen only when the ATP concentration was lowered from 5 to 0.03 mm. Taken together our observations indicate a functional interaction between murine P2X7 and P2X4 receptors. Such interaction might occur in acinar cells to shape the response to extracellular ATP in salivary epithelia.

Differences in regulation of Ca2+-activated Cl− channels in colonic and parotid secretory cells

We investigated the regulation of Ca2+-activated Cl- channels in cells from the human colonic cell line T84 and acinar cells from rat parotid glands. The participation of multifunctional Ca2+- and calmodulin-dependent protein kinase (CaM kinase) II in the activation of these channels was studied using selective inhibitors of calmodulin and CaM kinase II. Ca2+-dependent Cl- currents were recorded using the whole cell patch-clamp technique. Direct inhibition of CaM kinase II by 40 μM peptide 281-302 or by 10 μM KN-62, another CaM kinase inhibitor, did not block the Cl- current in parotid acinar cells, whereas in T84 cells KN-62 markedly inhibited the Ca2+-dependent Cl- current. We also used the calmodulin-binding domain peptide 290-309 (0.5 μM), which competitively inhibits the activation of CaM kinase II. This peptide reduced the Cl- current in T84 cells by ∼70% but was without effect on the channels in parotid acinar cells. We conclude that the Ca2+-dependent Cl- channels in T84 cells are activated by CaM kinase II but that the channels in parotid acinar cells must be regulated by a fundamentally different Ca2+-dependent mechanism that does not utilize CaM kinase II or any calmodulin-dependent process.

Conformation-dependent regulation of inward rectifier chloride channel gating by extracellular protons

We have investigated the gating properties of the inward rectifier chloride channel (Clir) from mouse parotid acinar cells by external protons (H+o) using the whole‐cell patch‐clamp technique. Increasing the pHo from 7.4 to 8.0 decreased the magnitude of Clir current by shifting the open probability to more negative membrane potentials with little modification of the activation kinetics. The action of elevated pH was independent of the conformational state of the channel. The effects of low pH on Clir channels were dependent upon the conformational state of the channel. That is, application of pH 5.5 to closed channels essentially prevented channel opening. In contrast, application of pH 5.5 to open channels actually increased the current. These results are consistent with the existence of two independent protonatable sites: (1) a site with a pK near 7.3, the titration of which shifts the voltage dependence of channel gating; and (2) a site with pK = 6.0. External H+ binds to this latter site (with a stoichiometry of two) only when the channels are closed and prevent channel opening. Finally, block of channels by Zn2+ and Cd2+ was inhibited by low pH media. We propose that mouse parotid Clir current has a bimodal dependence on the extracellular proton concentration with maximum activity near pH 6.5: high pH decreases channel current by shifting the open probability to more negative membrane potentials and low pH also decreases the current but through a proton‐dependent stabilization of the channel closed state.