The Journal of Membrane Biology

Begenisich, T. ; Melvin, J.E.

doi: 10.1007/s002329900372pmid: 9592072

Fluid and electrolyte secretion from secretory epithelia is a highly regulated process. Chloride channel activity at the apical membrane determines the rate and direction of salt and water secretion. Multiple classes of Cl− channels with distinct gating mechanisms are involved in moving ions and water. Secretory agonists that induce intracellular increases in two second messenger systems, cAMP and [Ca2+] i , are generally associated with secretion. However, changes in cell volume and the membrane potential may also play a role in regulating fluid and electrolyte secretion in some tissues. In this review we discuss the regulation of the different types of Cl− channels found in secretory epithelia.

Park, K. ; Arreola, J. ; Begenisich, T. ; Melvin, J.E.

doi: 10.1007/s002329900373pmid: 9592073

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.

Pedersen, S.F. ; Prenen, J. ; Droogmans, G. ; Hoffmann, E.K. ; Nilius, B.

doi: 10.1007/s002329900374pmid: 9592074

A Ca2+-activated (I Cl,Ca) and a swelling-activated anion current (I Cl,vol) were investigated in Ehrlich ascites tumor cells using the whole cell patch clamp technique. Large, outwardly rectifying currents were activated by an increase in the free intracellular calcium concentration ([Ca2+] i ), or by hypotonic exposure of the cells, respectively. The reversal potential of both currents was dependent on the extracellular Cl− concentration. I Cl,Ca current density increased with increasing [Ca2+] i , and this current was abolished by lowering [Ca2+] i to <1 nm using 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA). In contrast, activation of I Cl,vol did not require an increase in [Ca2+] i . The kinetics of I Cl,Ca and I Cl,vol were different: at depolarized potentials, I Cl,Ca as activated in a [Ca2+] i - and voltage-dependent manner, while at hyperpolarized potentials, the current was deactivated. In contrast, I Cl,vol exhibited time- and voltage-dependent deactivation at depolarized potentials and reactivation at hyperpolarized potentials. The deactivation of I Cl,vol was dependent on the extracellular Mg2+ concentration. The anion permeability sequence for both currents was I − > Cl− > gluconate. I Cl,Ca was inhibited by niflumic acid (100 μm), 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 μm) and 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS, 100 μm), niflumic acid being the most potent inhibitor. In contrast, I Cl,vol was unaffected by niflumic acid (100 μm), but abolished by tamoxifen (10 μm). Thus, in Ehrlich cells, separate chloride currents, I Cl,Ca and I Cl,vol, are activated by an increase in [Ca2+] i and by cell swelling, respectively.

Kertser, S. ; Bobryshev, A. ; Voitenko, S. ; Gmiro, V. ; Brovtsyna, N. ; Skok, V.

doi: 10.1007/s002329900375pmid: 9592075

Acetylcholine-induced membrane currents and excitatory postsynaptic currents (EPSCs) were recorded from the neurons of rat superior cervical ganglion (SCG) using the whole-cell patch clamp and the two-electrode voltage clamp techniques, correspondingly. The EPSC decay was bi-exponential, with fast and slow components characterized by time constants 5.5 ± 0.5 msec and 20.4 ± 1.2 msec (mean ±sem; n= 23), respectively. Blocking of these currents by a series of newly synthesized bis-cationic ammonium compounds, the pentamethonium and pentaethonium derivatives, was analyzed. Blocking effects were due to a block of nicotinic acetylcholine receptor (nAChR) open channel, with mean blocker binding rate constants for the fast component three to five times higher than those for the slow component. Dimensions of a nAChR ionic channel were deduced from a relationship between blocking activity of the compounds and the size of the projections of their three-dimensional molecular models on the neuronal membrane plane. The results suggest that there are two populations of nAChRs in rat SCG neurons; while these polulations differ in the rate constants of the binding by the blocker to their open channels, they exhibit similar channel diameter, 11.8 Å, at the level at which the blockers bind to the channel.

Edwards, J.C. ; Tulk, B. ; Schlesinger, P.H.

doi: 10.1007/s002329900376pmid: 9592076

. p64 is a protein identified as a chloride channel by biochemical purification from kidney microsomes. We expressed p64 in HeLa cells using a recombinant vaccinia virus/T7 RNA polymerase driven system. Total cell membranes were prepared from infected/transfected cells and fused to a planar lipid bilayer. A novel chloride channel activity was found in cells expressing p64 and not in control cells. The p64-associated activity shows strong anion over cation selectivity. Single channels show prominent outward rectification with single channel conductance at positive potentials of 42 pS. The chloride channel activity is activated by treatment of the membranes with alkaline phosphatase and inhibited by DNDS and by TS-TM calix(4)arene. Whole membrane anion permeability was determined by a chloride efflux assay, revealing that membranes from cells expressing p64 showed a small but highly significant increase in chloride permeability, consistent with expression of a novel chloride channel activity.

, A. ; , Rakowska; Danker, T. ; Schneider, S.W. ; Oberleithner, H.

doi: 10.1007/s002329900377pmid: 9592077

Bidirectional transport of molecules between nucleus and cytoplasm through the nuclear pore complexes (NPCs) spanning the nuclear envelope plays a fundamental role in cell function and metabolism. Nuclear import of macromolecules is a two-step process involving initial recognition of targeting signals, docking to the pore and energy-driven translocation. ATP depletion inhibits the translocation step. The mechanism of translocation itself and the conformational changes of the NPC components that occur during macromolecular transport, are still unclear. The present study investigates the effect of ATP on nuclear pore conformation in isolated nuclear envelopes from Xenopus laevis oocytes using the atomic force microscope. All experiments were conducted in a saline solution mimicking the cytosol using unfixed nuclear envelopes. ATP (1 mm) was added during the scanning procedure and the resultant conformational changes of the NPCs were directly monitored. Images of the same nuclear pores recorded before and during ATP exposure revealed dramatic conformational changes of NPCs subsequent to the addition of ATP. The height of the pores protruding from the cytoplasmic surface of the nuclear envelope visibly increased while the diameter of the pore opening decreased. The observed changes occurred within minutes and were transient. The slow-hydrolyzing ATP analogue, ATP-γ-S, in equimolar concentrations did not exert any effects. The ATP-induced shape change could represent a nuclear pore ``contraction.''

Fernández, R. ; Malnic, G.

doi: 10.1007/s002329900378pmid: 9592078

MDCK cells display several acid-base transport systems found in intercalated cells, such as Na+-H+ exchange, H+–K+ ATPase and Cl−/HCO− 3 exchange. In this work we studied the functional activity of a vacuolar H+-ATPase in MDCK cells and its chloride dependence. We measured intracellular pH (pHi) in monolayers grown on glass cover slips utilizing the pH sensitive probe BCECF. To analyze the functional activity of the H+ transporters we observed the intracellular alkalinization in response to an acute acid load due to a 20 mm NH+ 4 pulse, and calculated the initial rate of pHi recovery (dpHi/dt). The cells have a basal pHi of 7.17 ± 0.01 (n= 23) and control dpHi/dt of 0.121 ± 0.006 (n= 23) pHi units/min. This pHi recovery rate is markedly decreased when Na+ was removed, to 0.069 ± 0.004 (n= 16). It was further reduced to 0.042 ± 0.005 (n= 12) when concanamycin 4.6 × 10−8 m (a specific inhibitor of the vacuolar H+-ATPase) was added to the zero Na+ solution. When using a solution with zero Na+, low K+ (0.5 mm) plus concanamycin, pHi recovery fell again, significantly, to 0.023 ± 0.006 (n= 14) as expected in the presence of a H+–K+-ATPase. This result was confirmed by the use of 5 × 10−5 m Schering 28080. The Na+ independent pHi recovery was significantly reduced from 0.069 ± 0.004 to 0.042 ± 0.004 (n= 12) when NPPB 10−5 m (a specific blocker of Cl− channels in renal tubules) was utilized. When the cells were preincubated in 0 Cl−/normal Na+ solution for 8 min. before the ammonium pulse, the pHi recovery fell from 0.069 ± 0.004 to 0.041 ± 0.007 (n= 12) in a Na+ and Cl− free solution. From these results we conclude that: (i) MDCK cells have two Na+-independent mechanisms of pHi recovery, a concanamycin sensitive H+-ATPase and a K+ dependent, Schering 28080 sensitive H+–K+ ATPase; and, (ii) pHi recovery in Na+-free medium depends on the presence of a chloride current which can be blocked by NPPB and impaired by preincubation in Cl−–free medium. This finding supports a role for chloride in the function of the H+ ATPase, which might be electrical shunting or a biochemical interaction.

Cai, S. ; Garneau, L. ; Sauvé, R.

doi: 10.1007/s002329900379pmid: 9592079

The pharmacological profile of a voltage-independent Ca2+-activated potassium channel of intermediate conductance (IK(Ca2+)) present in bovine aortic endothelial cells (BAEC) was investigated in a series of inside-out and outside-out patch-clamp experiments. Channel inhibition was observed in response to external application of ChTX with a half inhibition concentration of 3.3 ± 0.3 nm (n= 4). This channel was insensitive to IbTX, but channel block was detected following external application of MgTX and StK leading to the rank order toxin potency ChTX > StK > MgTX >>IbTX. A reduction of the channel unitary current amplitude was also measured in the presence of external TEA, with half reduction occurring at 23 ± 3 mm TEA (n= 3). The effect of TEA was voltage insensitive, an indication that TEA may bind to a site located on external side of the pore region of this channel. Similarly, the addition of d-TC to the external medium caused a reduction of the channel unitary current amplitude with half reduction at 4.4 ± 0.3 mm (n= 4). In contrast, application of d-TC to the bathing medium in inside-out experiments led to the appearance of long silent periods, typical of a slow blocking process. Finally, the IK(Ca2+) in BAEC was found to be inhibited by NS1619, an activator of the Ca2+-activated potassium channel of large conductance (Maxi K(Ca2+)), with a half inhibition value of 11 ± 0.8 μm (n= 4). These results provide evidence for a pharmacological profile distinct from that reported for the Maxi K(Ca2+) channel, with some features attributed to the voltage-gated KV1.2 potassium channel.