Jan Eggermont

Volume-activated Cl− channels

1. An increase in cell volume activates, in most mammalian cells, a Cl- current, ICl,vol. This current is involved in a variety of cellular functions, such as the maintenance of a constant cell volume, pH regulation, and control of membrane potential. It might also play a role in the regulation of cell proliferation and in the processes that control transition from proliferation to differentiation. This review focuses on various aspects of this current, including its biophysical characterisation and its functional role for various cell processes. 2. Volume-activated Cl- channels show all outward rectification. Iodide is more permeable than chloride. In some cell types, ICl,vol inactivates at positive potentials. Single channel conductance can be divided mainly into two groups: small (< 5 pS) and medium conductance channels (around 50 pS). 3. The pharmacology and modulation of these channels are reviewed in detail, and suggest the existence of an heterogeneous family of multiple volume-activated Cl- channels. 4. Molecular candidates for this channel (i.e. ClC-2, a member of the ClC-family of voltage-dependent Cl- channels, the mdr-1 encoded P-glycoprotein, the nucleotide-sensitive pICln protein and phospholemman) will be discussed.

Regulation of a swelling‐activated chloride current in bovine endothelium by protein tyrosine phosphorylation and G proteins

1 The role of protein tyrosine phosphorylation and of G proteins in the activation of a swelling‐activated Cl− current (ICl,swell) in calf pulmonary artery endothelial (CPAE) cells was studied using the whole‐cell patch clamp technique. ICl,swell was activated by reducing the extracellular osmolality by either 12.5 % (mild hypotonicity) or 25 % (strong hypotonicity). 2 The protein tyrosine kinase (PTK) inhibitors tyrphostin B46, tyrphostin A25 and genistein inhibited ICl,swell with IC50 values of, respectively, 9.2 ± 0.2, 61.4 ± 1.7 and 62.9 ± 1.3μM. Tyrphostin A1, a tyrphostin analogue with little effect on PTK activity, and daidzein, an inactive genistein analogue, were without effect on ICl,swell. 3 The protein tyrosine phosphatase (PTP) inhibitors Na3VO4 (200 μM) and dephostatin (20 μM) potentiated ICl,swell activated by mild hypotonicity by 47 ± 9 and 69 ± 15 %, respectively. 4 Intracellular perfusion with GTPγS (100 μM) transiently activated a Cl− current with an identical biophysical and pharmacological profile to ICl,swell. This current was inhibited by the tested PTK inhibitors and potentiated by the PTP inhibitors. Hypertonicity‐induced cell shrinkage completely inhibited the GTPγS‐activated Cl− current. 5 Intracellular perfusion with GDPβS (1 mM) caused a time‐dependent inhibition of ICl,swell, which was more pronounced when the current was activated by mild hypotonicity. 6 Our results demonstrate that the activity of endothelial swelling‐activated Cl− channels is dependent on tyrosine phosphorylation and suggest that G proteins regulate the sensitivity to cell swelling.

Properties of heterologously expressed hTRP3 channels in bovine pulmonary artery endothelial cells

1 We combined patch clamp and fura‐2 fluorescence methods to characterize human TRP3 (hTRP3) channels heterologously expressed in cultured bovine pulmonary artery endothelial (CPAE) cells, which do not express the bovine trp3 isoform (btrp3) but express btrp1 and btrp4. 2 ATP, bradykinin and intracellular InsP3 activated a non‐selective cation current (IhTRP3) in htrp3‐transfected CPAE cells but not in non‐transfected wild‐type cells. During agonist stimulation, the sustained rise in [Ca2+]i was significantly higher in htrp3‐transfected cells than in control CPAE cells. 3 The permeability for monovalent cations was PNa > PCs≈PK >> PNMDG and the ratio PCa/PNa was 1·62 ± 0·27 (n= 11). Removal of extracellular Ca2+ enhanced the amplitude of the agonist‐activated IhTRP3 as well as that of the basal current The trivalent cations La3+ and Gd3+ were potent blockers of IhTRP3 (the IC50 for La3+ was 24·4 ± 0·7 μM). 4 The single‐channel conductance of the channels activated by ATP, assessed by noise analysis, was 23 pS. 5 Thapsigargin and 2,5‐di‐tert‐butyl‐1,4‐benzohydroquinone (BHQ), inhibitors of the organellar Ca2+‐ATPase, failed to activate IhTRP3. U‐73122, a phospholipase C blocker, inhibited IhTRP3 that had been activated by ATP and bradykinin. Thimerosal, an InsP3 receptor‐sensitizing compound, enhanced IhTRP3, but calmidazolium, a calmodulin antagonist, did not affect IhTRP3. 6 It is concluded that hTRP3 forms non‐selective plasmalemmal cation channels that function as a pathway for agonist‐induced Ca2+ influx.

Differential expression of volume-regulated anion channels during cell cycle progression of human cervical cancer cells

1 This study investigated the volume‐regulated anion channel (VRAC) of human cervical cancer SiHa cells under various culture conditions, testing the hypothesis that the progression of the cell cycle is accompanied by differential expression of VRAC activity. 2 Exponentially growing SiHa cells expressed VRACs, as indicated by the presence of large outwardly rectifying currents activated by hypotonic stress with the anion permeability sequence I− > Br− > Cl−. VRACs were potently inhibited by tamoxifen with an IC50 of 4.6 μm. 3 Fluorescence‐activated cell sorting (FACS) experiments showed that 59 ± 0.5, 5 ± 0.5 and 36 ± 1.1% of unsynchronized, exponentially growing cervical cancer SiHa cells were in G0/G1, S and G2/M stage, respectively. Treatment with aphidicolin (5 μm) arrested 88 ± 1.4% of cells at the G0/G1 stage. 4 Arrest of cell growth in the G0/G1 phase was accompanied by a significant decrease of VRAC activity. The normalized hypotonicity‐induced current decreased from 48 ± 5.2 pA pF−1 at +100 mV in unsynchronized cells to 15 ± 2.6 pA pF−1 at +100 mV in aphidicolin‐treated cells. After removal of aphidicolin, culturing in medium containing 10% fetal calf serum triggered a rapid re‐entry into the cell cycle and a concomitant recovery of VRAC density. 5 Pharmacological blockade of VRACs by tamoxifen or NPPB caused proliferating cervical cancer cells to arrest in the G0/G1 stage, suggesting that activity of this channel is critical for G1/S checkpoint progression. 6 This study provides new information on the functional significance of VRACs in the cell cycle clock of human cervical cancer cells.

Role of Rho and Rho kinase in the activation of volume‐regulated anion channels in bovine endothelial cells

1 We have studied the modulation of volume‐regulated anion channels (VRACs) by the small GTPase Rho and by one of its targets, Rho kinase, in calf pulmonary artery endothelial (CPAE) cells. 2 RT‐PCR and immunoblot analysis showed that both RhoA and Rho kinase are expressed in CPAE cells. 3 I Cl,swell, the chloride current through VRACs, was activated by challenging CPAE cells with a 25 % hypotonic extracellular solution (HTS) or by intracellular perfusion with a pipette solution containing 100 μM GTPγS. 4 Pretreatment of CPAE cells with the Clostridium C2IN‐C3 fusion toxin, which inactivates Rho by ADP ribosylation, significantly impaired the activation of ICl,swell in response to the HTS. The current density at +100 mV was 49 ± 13 pA pF−1 (n= 17) in pretreated cells compared with 172 ± 17 pA pF−1 (n= 21) in control cells. 5 The volume‐independent activation of ICl,swell by intracellular perfusion with GTPγS was also impaired in C2IN‐C3‐pretreated cells (31 ± 7 pA pF−1, n= 11) compared with non‐treated cells (132 ± 21 pA pF−1, n= 15). 6 Activation of ICl,swell was pertussis toxin (PTX) insensitive. 7 Y‐27632, a blocker of Rho kinase, inhibited ICl,swell and delayed its activation. 8 Inhibition of Rho and of Rho kinase by the above‐described treatments did not affect the extent of cell swelling in response to HTS. 9 These experiments provide strong evidence that the Rho‐Rho kinase pathway is involved in the VRAC activation cascade.

Activation of volume‐regulated chloride currents by reduction of intracellular ionic strength in bovine endothelial cells

1 We have studied the effects of intracellular ionic strength (Γi) on the swelling‐activated whole‐cell Cl− current (ICl,swell) in cultured calf pulmonary artery endothelial cells (CPAE cells). 2 Reducing Γi from 155 to 95 mM at constant osmolarity and Cl− concentration activates an outwardly rectifying current that is mainly carried by Cl− ions and inactivates at positive potentials. The amplitude of the current is larger at more reduced levels of Γi. 3 The permeability ratio for the anions I−, Br−, Cl− and gluconate (PI: PBr: PCl: Pgluc) was 1.35 : 1.03 : 1 : 0.17. 3 Blockers of the swelling‐activated Cl− current in CPAE cells also inhibit the current which is activated by a reduction in Γi with an IC50 of 1.1 μM for tamoxifen, 1.3 μM for mibefradil, and 35 μM for quinidine. 4 The protein tyrosine kinase inhibitors tyrphostin B46 (50 μM) and genistein (100 μM), which inhibit ICl,swell in CPAE cells, also inhibited the Γi‐induced current by 92.9 ± 2.4% (n= 3) and 41.2 ± 5.0% (n= 4), respectively. 5 Hypertonic extracellular solutions rapidly and reversibly antagonized the Γi‐activated current, whereas increasing Γi from 155 to 195 mM precluded activation of ICl,swell by hypotonic shock. 6 It is concluded that a reduction of Γi activates an anion current that is identical to that activated by cell swelling. Changes in intracellular ionic strength may shift the volume set point for activation of ICl, swell.

Volume-activated Cl- currents in different mammalian non-excitable cell types.

The existence and properties of volume-activated Cl− currents were studied in 15 different cell types (endothelium: human umbilical vein, human aorta, bovine pulmonary artery; fibroblasts: Swiss 3T3, L, C3H 10T1/2 and COS-1; epithelium: KB3, HeLa and A6; blood cells: RBL-2H3 and Jurkat; endothelioma cells derived from both subcutaneous and thymic hemangiomas; skin: IGR1 melanoma). Volume-activated Cl− currents with common characteristics, i.e. small conductance, outward rectification, higher permeability for iodide than for chloride and sensitivity to block by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) could be elicited in all cells. The block of this current by tamoxifen and dideoxyforskolin is different for the various cell types, as well as the time course and the amplitude of the responses induced by repetitive applications of hypotonicity. Volume-activated Cl− channels with similar biophysical properties are therefore widespread among mammalian cells. This may reflect either a single Cl− channel that is ubiquitously expressed or a family of functionally related Cl− channels with cell specific expression patterns.

Effects of cyclic nucleotide dependent protein kinases on the endoplasmic reticulum Ca2+ pump of bovine pulmonary artery

This paper describes the stimulation by cyclic nucleotide dependent protein kinases on the Ca2+ uptake by isolated endoplasmic reticulum (ER) vesicles from the bovine main pulmonary artery. This ER fraction has previously been shown to be highly enriched in phospholamban, a protein kinase substrate that has been well characterized in cardiac sarcoplasmic reticulum (SR), where its phosphorylation is accompanied by an increased rate of Ca2+ uptake. As previously observed for the phosphorylation of phospholamban, the stimulation of the rate of Ca uptake was as high with cGMP dependent protein kinase as with cAMP dependent protein kinase. The effect of phosphorylation of the ER membranes from smooth muscle on the Ca2+ uptake was smaller than that seen in cardiac SR, and it was only observed if albumin was included during the isolation of the membranes. This relatively small effect is probably not due to a lower ratio of phospholamban to Ca2(+)-transport enzyme in the ER membranes as compared to cardiac SR. Several alternative explanations are discussed.

Caveolin-1 modulates the activity of the volume-regulated chloride channel.

1 Caveolae are small invaginations of the plasma membrane that have recently been implicated in signal transduction. In the present study, we have investigated whether caveolins, the principal protein of caveolae, also modulate volume‐regulated anion channels (VRACs). 2 I Cl,swell, the cell swelling‐induced chloride current through VRACs, was studied in three caveolin‐1‐deficient cell lines: Caco‐2, MCF‐7 and T47D. 3 Electrophysiological measurements showed that ICl,swell was very small in these cells and that transient expression of caveolin‐1 restored ICl,swell. The caveolin‐1 effect was isoform specific: caveolin‐1β but not caveolin‐1α upregulated VRACs. This correlated with a different subcellular distribution of caveolin‐1α (perinuclear location) from caveolin‐1β (perinuclear and peripheral). 4 To explain the modulation of ICl,swell by caveolin‐1 we propose that caveolin increases the availability of VRACs in the plasma membrane or, alternatively, that it plays a crucial role in the signal transduction cascade of VRACs.

Use of a bicistronic GFP-expression vector to characterise ion channels after transfection in mammalian cells

Abstract Transient transfection of ion channels into mammalian cells is a useful method with which to study ion channel properties. However, a general problem in transient transfection procedures is how to select cells that express the transfected cDNA. We have constructed a bicistronic vector, pCINeo/IRES-GFP, which utilises a red-shifted variant of Green Fluorescent Protein as an in vivo cell marker. Incorporation of an ion channel cDNA into the bicistronic unit allows coupled expression of the ion channel and Green Fluorescent Protein. After transient transfection of COS cells with pCINeo/IRES-GFP containing a rat delayed rectifier K+ channel cDNA (RCK1, Kv1.1), all green cells (n = 32) expressed the RCK1 channel as identified by the well known kinetics, K+ selectivity and pharmacology of Kv1.1. In contrast, non-fluorescent cells (n = 24) were negative with respect to RCK1 expression. It is concluded that the bicistronic pCINeo/IRES-GFP vector provides an efficient and non-invasive way of identifying cells which express ion channels after transfection. This novel method should greatly facilitate functional studies of ion channels transfected into mammalian cells.