Ted Begenisich

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.

Sodium channel permeation in squid axons. I: Reversal potential experiments.

1. Na channel reversal potentials were studied in perfused voltage clamped squid giant axons. The concentration dependence of ion selectivity was determined with both external and internal changes in Na and ammonium concentrations. 2. A tenfold change in the internal ammonium activity results in a 42 mV shift in the reversal potential, rather than the 56 mV shift expected from the Goldman, Hodgkin, Katz equation for a constant PNa/PNH4 ratio. However, changing [Na]o tenfold at constant internal [NH4] gives approximately the expected 56 mV shift. Therefore, the apparent channel selectivity depends upon the internal ammonium concentration but not the external Na concentration. 3. With ammonium outside and Na inside, the calculated permeability ratio is nearly constant, regardless of the permeant ion concentration. 4. Internal Cs ions can alter the Na/K permeability ratio. 5. The results are considered in terms of a three‐barrier, two‐site ionic permeation model.

How many conductance states do potassium channels have

al-ternativeis difficulttoruleout,however,becauseionic movementsthroughmembranechannelsarenotyetwellenoughunderstoodto permitdefinitive predictions aboutthenoiseassociated with thepermeation process. Ifit turns outthat thedominantnoisesourcein these experiments is the transport process rather than channel gating, ourmeasurementsgiveanupperlimitoftheaveragesinglechannelconductance.Finally, it must be emphasized that, even if the dominant source for fluctuationsin ourexperiments is potassiumchannelgating, our results do not necessarily implythatpotassiumchannelsareall identicalorthattheyhaveonlytwoconductancestates.

Regulation of chloride channels in secretory epithelia.

Abstract. 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.

Sodium Flux Ratio in Voltage-clamped Squid Giant Axons

The sodium flux ratio across the axolemma of internally perfused, voltage-clamped giant axons of Loligo pealei has been measured at various membrane potentials. The flux ratio exponent obtained from these measurements was about unity and independent of membrane voltage over the 50 mV range from about -20 to l mV. These results, combined with previous measurements of ion permeation through sodium channels, show that the sodium channel behaves like a multi-ion pore with two ion binding sites that are rarely simultaneously occupied by sodium.

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.

Sodium channel permeation in squid axons. II: Non-independence and current-voltage relations.

1. The concentration and voltage dependence of current through the Na channels of squid giant axons was studied. The permeant cations Na, K and ammonium were used. 2. The Na channel current at a fixed voltage saturates as the internal permeant ion concentration is increased. The half‐saturation activities at 50 mV were found to be 623, 268, 161 mM for Na, NH4, and K, respectively. These Km values for Na and K appear to be voltage‐dependent. 3. Instantaneous current‐voltage relations were determined with two different internal Na concentrations and with Na outside and K inside. 4. A three‐barrier, two‐site ion permeation model previously used in describing Na channel reversal potentials is shown here to account for the Na channel currents as well.

Adrenergic modulation of the delayed rectifier potassium channel in calf cardiac Purkinje fibers

We have investigated the modulation of the delayed rectifier potassium channel in calf cardiac Purkinje fibers by the neurohormone norepinephrine. We find that 0.5 microM norepinephrine increases this K channel current by a factor of 2.7. A maximal increase of about four was found for concentrations of 1 microM and above. Norepinephrine produced a small (less than 5 mV) and variable shift of the K channel reversal potential toward more negative values. The kinetics of the potassium channel are well described by a two-exponential process, both in the absence and presence of norepinephrine. However, norepinephrine substantially decreases the slower time constant with no significant effect on the fast time constant. Potassium channel activation curves in the presence of norepinephrine are very similar to control curves except at large positive potentials. A simple sequential three-state model for this channel can reproduce these data both with and without norepinephrine. The logarithms of the rate constants derived from this model are quadratic functions of voltage, suggesting the involvement of electric field-induced dipoles in the gating of this channel. Most of the kinetic effects of norepinephrine appear to be on a single rate constant.

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.

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.