Osmotic shrinkage of human cervical cancer cells induces an extracellular Cl- -dependent nonselective cation channel,which requires p38 MAPK

This study is to integrate a functional role of nonselective cation (NSC) channels into a model of volume regulation on osmotic shrinkage for human cervical cancer cells. Application of a hypertonic solution (400 mosm kg(-1)) induced cell shrinkage, which was accompanied by a 7-fold increase of inward currents at -80 mV from -4.1 +/- 0.4 pA pF(-1) to -29 +/- 1.1 pA pF(-1) (n = 36, p < 0.001). There is a good correlation of channel activity and cell volume changes. Replacement of bath Na(+) by K(+), Cs(+), Li(+), or Rb(+) did not affect the stimulated inward current significantly, but replacement by Ca(2+), Ba(2+), or the impermeable cation N-methyl-d-glucamine abolished the inward current; this demonstrates that the shrinkage-induced currents discriminate poorly between monovalent cations but are not carried by divalent cations. Replacement of extracellular Cl(-) by gluconate abolished the shrinkage-induced currents in a concentration-dependent manner without changing the reversal potential. Gadolinium (Gd(3+)) inhibited the stimulated current, whereas bumetanide and amiloride had no inhibitory effect. Cell shrinkage triggered mitogen-activated protein (MAP) kinase cascades leading to the activation of MAP/extracellular signal-regulated kinase 1/2 (ERK1/2) kinase (MEK1/2), and p38 kinase. Interference with p38 MAPK by either the specific inhibitor (SB202190), or a dominant-negative mutant profoundly suppressed the activation of the shrinkage-induced NSC channels. In contrast, the regulatory mechanism of shrinkage-induced NSC channels was independent of the volume-responsive MEK1/2 signaling pathway. More importantly, the cell volume response to hypertonicity was inhibited significantly in p38 dominant-negative mutant or by SB202190. Therefore, p38 MAPK is critically involved in the activation of a shrinkage-induced NSC channel, which plays an important role in the volume regulation of human cervical cancer cells.     

A non-selective cation channel in the apical membrane of cultured A6 kidney cells.

The patch-voltage clamp technique was used to investigate the characteristics of a non-selective cation channel (NSCC) identified in the apical membrane of cultured A6 toad kidney cells. The NSCC was present in cell-attached and inside-out membrane patches. The characteristics of this NSCC are as follows: (a) linear current-voltage relationship with a channel conductance of 21 +/- 2 pS; (b) a low selectivity between Na+ and K+ (1.5:1); (c) a high selectivity of Na+ to Cl- (greater than 45:1); (d) this channel has a single open state and two closed states; (e) the open-time constant and the second closed-time constant of this channel are voltage dependent; and (f) this NSCC is insensitive to amiloride (10(-7) M). We conclude that the NSCC resembles previously described non-selective cation channels. The NSCC of the apical membrane of A6 cells may aid in the movement of Na+ and K+ in response to varying ionic concentrations across the apical membrane.     

Maxi-channels recorded in situ from ICC and pericytes associated with the mouse myenteric plexus

Ion channels are fundamental to gastrointestinal pacemaking by interstitial cells of Cajal (ICC). Previously, we have recorded a high-conductance chloride channel (HCCC) from ICC, both in culture and in situ, associated with the myenteric plexus. The biophysical properties of the HCCC (conductance, subconductances, voltage- and time-dependent inactivation) suggest it is a member of a class called the maxi-anion channels. In this study we further investigated the properties of the HCCC in situ. Our main finding was that the HCCC is not strictly a chloride channel but has a relative sodium-chloride permeability (P(Na/Cl)) of 0.76 to 1.64 (depending on the method of measurement). Therefore, we have renamed the HCCC the "maxi-channel." A maxi-channel was also expressed by pericytes associated with the vasculature near the myenteric plexus. This had a lower P(Na/Cl) (0.33 to 0.49, depending on the method of measurement) but similar conductance (326 ± 7 vs. 316 ± 24 pS for ICC). This is the first report of cation permeability equaling anion permeability in a maxi-anion channel. As such, the properties of the maxi-channels described in this article may have implications for the maxi-anion channel field, as well as for studies of their role in ICC and pericytes.     

Ion channels in human endothelial cells.

Ion channels were studied in human endothelial cells from umbilical cord by the patch clamp technique in the cell attached mode. Four different types of ion channels were recorded: i) potassium channel current that rectifies at positive potentials in symmetrical potassium solutions (inward rectifier); ii) low-conductance non-selective cation channel with a permeability ratio K:Na:Ca = 1:0.9:0.2; iii) high-conductance cation-selective channel that is about 100 times more permeable for calcium than for sodium or potassium; iv) high-conductance potassium channel with a permeability ratio K:Na = 1:0.05. The extrapolated reversal potential of the inwardly rectifying current was near to the potassium equilibrium potential. The slope conductance decreased from 27 pS in isotonic KCl solution to 7 pS with 5.4 mmol/l KCl and 140 mmol/l NaCl in the pipette but 140 mmol/l KCl in the bath. The low-conductance non-selective cation channel showed a single-channel conductance of 26 pS with 140 mmol/l Na outside, 28 pS with 140 mmol/l K outside, and rectified in inward direction in the presence of Ca (60 mmol/l Ca, 70 mmol/l Na, 2.7 mmol/l K in the pipette) at negative potentials. The current could be observed with either chloride or aspartate as anion. The high-conductance non-selective channel did not discriminate between Na and K. The single-channel conductance was about 50 pS. The extrapolated reversal potential was more positive than +40 mV (140 K or 140 Na with 5 Ca outside). Both the 26 and 50 pS channel showed a run-down, and they rapidly disappeared in excised patches. The high-conductance potassium channel with a single-channel conductance of 170 pS was observed only rarely. It reversed near the expected potassium equilibrium potential. The 26 pS channel could be stimulated with histamine and thrombin from outside in the cell-attached mode. Both the 26 pS as well as the 50 pS channel can mediate calcium flux into the endothelial cell.     

Ion channels in mammalian proximal renal tubules.

In the plasma membranes of mammalian proximal renal tubules single ion channels were investigated mainly in isolated tubules perfused on one side, in isolated nonperfused (collapsed) tubules and in primary cell cultures. With these techniques, the following results were obtained: in the luminal membrane of isolated one-sided perfused tubules of rabbit and mouse S3 segments, K(+)-selective channels with single-channel conductance (g) of 33 pS and 63 pS, respectively, were recorded. In primary cultures of rabbit S1 segments, a small-conductance (42 pS) as well as a large-conductance (200 pS) K+ channel were observed. The latter was Ca2(+)- and voltage-sensitive. In cultured cells a Ca2(+)-activated, nonselective cation channel with g = 25 pS was also recorded. On the other hand, an amiloride-sensitive channel with g = 12 pS, which was highly selective for Na+ over K+, was observed in the isolated perfused S3 segment. In the basolateral membrane of isolated perfused S3 segments, two types of K+ channels with g = 46 pS and 36 pS, respectively, were observed. The latter channel was not dependent on cytosolic Ca2+ in cell-excised patches. A K+ channel with g = 54 pS was recorded in isolated nonperfused S1 segments. This channel showed inward rectification and was more active at depolarizing potentials. In isolated perfused S3 segments, in addition to the K+ channels also a nonselective cation channel with g = 28 pS was observed. This channel was highly dependent on cytosolic Ca2+ in cell-free patches. It can be concluded that the K+ channels both in the luminal and contraluminal cell membrane are involved in the generation of the cell potential. Na+ channels in the luminal membrane may participate in Na+ reabsorption, whereas the function of a basolateral cation channel remains unclear. Recently, single anion-selective channels were recorded in membranes of endocytotic vesicles, isolated from rat proximal tubules. Vesicles were enlarged by the dehydration/rehydration method and investigated with the patch clamp technique. The Cl- channel had a conductance of 73 pS, the current-voltage curve was linear and the channel inactivated at high negative clamp potentials. It is suggested that this channel is responsible for charge neutrality during active H+ uptake into the endosomes.     

Hypertonic activation of a non-selective cation conductance in HeLa cells and its contribution to cell volume regulation

In whole-cell recordings on single HeLa cells, the hypertonic activation of a cation conductance with a selectivity ratio P(Na):P(Li):P(K):P(Cs):P(NMDG):P(Ca):P(Cl) of 1.00:0.86:0.84:0.56:0.10:0.07:0.15 was observed. This (non-selective) cation conductance was reduced to 59 and 30% of maximal stimulation by Gd(3+) and flufenamate, respectively, but it was insensitive to amiloride (with each compound applied at 100 microm/l). As was determined by the Coulter counter technique, the cation conductance was the main mechanism of regulatory volume increase (RVI) in HeLa cells. Whereas a significant contribution of Na(+)/H(+) antiport was also detectable, Na(+)-K(+)-2Cl(-) symport most likely did not contribute to RVI.     

Cation selective channel in fetal alveolar type II epithelium.

A cation selective channel was identified in the apical membrane of fetal rat (Wistar) alveolar type II epithelium using the patch clamp technique. The single channel conductance was 23 +/- 1.2 pS (n = 16) with symmetrical NaCl (140 mM) solution in the bath and pipette. The channel was highly permeable to Na+ and K+ (PNa/PK = 0.9) but essentially impermeant to chloride and gluconate. Membrane potential did not influence open state probability when measured in a high Ca2+ (1.5 mM) bath. The channel reversibly inactivated when the bath was exchanged with a Ca(2+)-free (less than 10(-9) M) solution. The Na+ channel blocker amiloride (10(-6) M) applied to the extracellular side of the membrane reduced P(open) relative to control patches; P(control) = 0.57 +/- 0.11 (n = 5), P(amiloride) = 0.09 +/- 0.07 (n = 4, p less than 0.01), however, amiloride did not significantly influence channel conductance (g); g(control) 19 +/- 0.9 pS (n = 5), 18 +/- 3.0 pS (n = 4). More than one current level was observed in 42% (16/38) of active patches; multiple current levels (ranging from 2 to 6) were of equal amplitude suggesting the presence of multiple channels or subconductance states. Channel activity was also evident in cell attached patches. Since monolayers of these cells absorb Na+ via an amiloride sensitive transport mechanism we speculate that this amiloride sensitive cation selective channel is a potential apical pathway for electrogenic Na+ transport in the alveolar region of the lung.     

AICAR decreases the activity of two distinct amiloride-sensitive Na+-permeable channels in H441 human lung epithelial cell monolayers

Transepithelial transport of Na(+) across the lung epithelium via amiloride-sensitive Na(+) channels (ENaC) regulates fluid volume in the lung lumen. Activators of AMP-activated protein kinase (AMPK), the adenosine monophosphate mimetic AICAR, and the biguanide metformin decreased amiloride-sensitive apical Na(+) conductance (G(Na+)) in human H441 airway epithelial cell monolayers. Cell-attached patch-clamp recordings identified two distinct constitutively active cation channels in the apical membrane that were likely to contribute to G(Na+): a 5-pS highly Na(+) selective ENaC-like channel (HSC) and an 18-pS nonselective cation channel (NSC). Substituting NaCl with NMDG-Cl in the patch pipette solution shifted the reversal potentials of HSC and NSC, respectively, from +23 mV to -38 mV and 0 mV to -35 mV. Amiloride at 1 microM inhibited HSC activity and 56% of short-circuit current (I(sc)), whereas 10 microM amiloride partially reduced NSC activity and inhibited a further 30% of I(sc). Neither conductance was associated with CNG channels as there was no effect of 10 microM pimoside on I(sc), HSC, or NSC activity, and 8-bromo-cGMP (0.3-0.1 mM) did not induce or increase HSC or NSC activity. Pretreatment of H441 monolayers with 2 mM AICAR inhibited HSC/NSC activity by 90%, and this effect was reversed by the AMPK inhibitor Compound C. All three ENaC proteins were identified in the apical membrane of H441 monolayers, but no change in their abundance was detected after treatment with AICAR. In conclusion, activation of AMPK with AICAR in H441 cell monolayers is associated with inhibition of two distinct amiloride-sensitive Na(+)-permeable channels by a mechanism that likely reduces channel open probability.     

Synthetic mammalian C-type natriuretic peptide forms large cation channels

We report the first evidence that synthetic human C-type natriuretic peptide-22 and the OaC-type natriuretic peptide-39(18-39), a 22 amino acid fragment of the OaC-type natriuretic peptide-39 from platypus venom, can function directly by forming a novel voltage-gated weakly cation-selective channel in negatively charged artificial lipid bilayer membranes. The channel activity is characterized by a tendency for inactivation at negative voltages, e.g. -60 and -70 mV, whereas at positive voltages the channel is fully open. The channel has a maximal cord conductance of 546+/-23 pS (n = 16) and shows weak outward rectification. The sequence and the permeability ratios were P(K)+: P(Cs)+: P(Na)+: P(choline)+ 1:0.88:0.76:0.13, respectively. The addition of 50 mM TEA+ cis (a blocker of outwardly rectifying K+ channels), 20 mM Cs+ cis (a blocker of inwardly rectifying K+ channels) or 0.5 mM glibenclamide cis (a blocker of ATP-sensitive K+ channels) to the cis chamber did not affect the conductance or the kinetics of the OaC-type natriuretic peptide-39(18-39)-formed channels (n = 2-5). It is concluded that the weak cation selectivity, large conductance and high open probability as well as their voltage dependency are consistent with the ability of these peptides to cause that loss of compartmentation of the membrane, which is a characteristic feature of adverse conditions that cause C-type natriuretic peptide-related pathologies.     

Influence of extracellular monovalent cations on pore and gating properties of P2X7 receptor-operated single-channel currents

Using the patch-clamp method, we studied the influence of external alkali and organic monovalent cations on the single-channel properties of the adenosine triphosphate (ATP)-activated recombinant human P2X(7) receptor. The slope conductance of the hP2X(7) channel decreased and the reversal potential was shifted to more negative values as the ionic diameter of the organic test cations increased. From the relationship between single-channel conductance and the dimensions of the inward current carrier, the narrowest portion of the pore was estimated to have a mean diameter of approximately 8.5 A. Single-channel kinetics and permeation properties remained unchanged during receptor activation by up to 1 mM ATP(4-) for >1 min, arguing against a molecular correlate of pore dilation at the single P2X(7) channel level. Substitution of extracellular Na(+) by any other alkali or organic cation drastically increased the open probability of the channels by prolonging the mean open time. This effect seems to be mediated allosterically through an extracellular voltage-dependent Na(+) binding site with a K(d) of approximately 5 mM Na(+) at a membrane potential of -120 mV. The modulation of the ATP-induced hP2X(7) receptor gating by extracellular Na(+) could be well described by altering the rate constant from the open to the neighboring closed state in a C-C-C-O kinetic receptor model. We suggest that P2X(7) receptor-induced depolarization and associated K(+)-efflux may reduce Na(+) occupancy of the regulatory Na(+) binding site and thus increase the efficacy of ATP(4-) in a feed-forward manner in P2X(7) receptor-expressing cells.     

Single-channel recordings from two types of amiloride-sensitive epithelial Na+ channels

We report here the first evidence in intact epithelial cells of unit conductance events from amiloride-sensitive Na+ channels. The events were observed when patch-clamp recordings were made from the apical surface of cultured epithelial kidney cells (A6). Two types of channels were observed: one with a high selectivity to Na+ and one with relatively low selectivity. The characteristics of the low-selectivity channel are as follows: single-channel conductance ranged between 7 and 10 pS (mean = 8.4 +/- 1.3), the current-voltage (I-V) relationship displayed little if any nonlinearity over a range of +/- 80 mV (with respect to the patch pipette) and the channel Na+/K+ selectivity was approximately 3-4:1. Amiloride, a cationic blocker of the channel, reduced channel mean open time and increased channel mean closed times as the voltage of the cell interior was made more negative. Amiloride induced channel flickering at increased negative potentials (intracellular potential with respect to the patch) but did not alter the single-channel conductance or the I-V relationship from that observed in control patches. The characteristics of the high-selectivity channel are: a single-channel conductance of 1-3 pS (mean = 2.8 +/- 1.2), the current-voltage relationship is markedly nonlinear with a Na+/K+ selectivity greater than 20:1. The mean open and closed times for the two types of channels are quite different, the high-selectivity channel being open only about 10% of the time while the low-selectivity channel is open about 30% of the time.     

Functional properties of endogenous receptor- and store-operated calcium influx channels in HEK293 cells

Activation of phospholipase C (PLC)-mediated signaling pathways in non-excitable cells causes the release of calcium (Ca2+) from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores and activation of Ca2+ influx via plasma membrane Ca2+ channels. The properties and molecular identity of plasma membrane Ca2+ influx channels in non-excitable cells is a focus of intense investigation. In the previous studies we used patch clamp electrophysiology to describe the properties of Ca2+ influx channels in human carcinoma A431 cell lines. Now we extend our studies to human embryonic kidney HEK293 cells. By using a combination of Ca2+ imaging and whole cell and single channel patch clamp recordings we discovered that: 1) HEK293 cells contain four types of plasma membrane Ca2+ influx channels: I(CRAC), Imin, Imax, and I(NS); 2) I(CRAC) channels are highly Ca2+-selective (P(Ca/Cs)>1000) and I(CRAC) single channel conductance is too small for single channel analysis; 3) Imin channels in HEK293 cells display functional properties identical to Imin channels in A431 cells, with single channel conductance of 1.2 pS for divalent cations, 10 pS for monovalent cations, and divalent cation selectivity P(Ba/K)=20; 4) Imin channels in HEK293 cells are activated by InsP3 and inhibited by phosphatidylinositol 4,5-bisphosphate, but store-independent; 5) when compared with Imin, Imax channels have higher conductance for divalent (17 pS) and monovalent (33 pS) cations, but less selective for divalent cations (P(Ba/K)=4), 6) Imax channels in HEK293 cells can be activated by InsP3 or by Ca2+ store depletion; 7) I(NS) channels are non-selective (P(Ba/K)=0.4) and display a single channel conductance of 5 pS; and 8) I(NS) channels are not gated by InsP3 but activated by depletion of intracellular Ca2+ stores. Our findings provide novel information about endogenous Ca2+ channels supporting receptor-operated and store-operated Ca2+ influx pathways in HEK293 cells.     

The role of Pre-H2 domains of alpha- and delta-epithelial Na+ channels in ion permeation, conductance, and amiloride sensitivity

Epithelial Na(+) channels (ENaC) regulate salt and water re-absorption across the apical membrane of absorptive epithelia such as the kidney, colon, and lung. Structure-function studies have suggested that the second transmembrane domain (M2) and the adjacent pre- and post-M2 regions are involved in channel pore formation, cation selectivity, and amiloride sensitivity. Because Na(+) selectivity, unitary Na(+) conductance (gamma(Na)), and amiloride sensitivity of delta-ENaC are strikingly different from those of alpha-ENaC, the hypothesis that the pre-H2 domain may contribute to these characterizations has been examined by swapping the pre-H2, H2, and both (pre-H2+H2) domains of delta- and alpha-ENaCs. Whole-cell and single channel results showed that the permeation ratio of Li(+) and Na(+) (P(Li)/P(Na)) for the swap alpha chimeras co-expressed with betagamma-ENaC in Xenopus oocytes decreased significantly. In contrast, the ratio of P(Li)/P(Na) for the swap delta constructs was not significantly altered. Single channel studies confirmed that swapping of the H2 and the pre-H2+H2 domains increased the gamma(Na) of alpha-ENaC but decreased the gamma(Na) of delta-ENaC. A significant increment in the apparent inhibitory dissociation constant for amiloride (K(i)(amil)) was observed in the alpha chimeras by swapping the pre-H2, H2, and pre-H2+H2 domains. In contrast, a striking decline of K(i)(amil) was obtained in the chimeric delta constructs with substitution of the H2 and pre-H2+H2 domains. Our results demonstrate that the pre-H2 domain, combined with the H2 domain, contributes to the P(Li)/P(Na) ratio, single channel Na(+) conductance, and amiloride sensitivity of alpha- and delta-ENaCs.     

Low conductance sodium channels in canine cardiac Purkinje cells.

Low conductance sodium (Na) channels have been observed in nerve, skeletal muscle, and cardiac cells. In cardiac tissues the higher amplitude, more commonly observed Na channel was first investigated in detail by Cachelin et al. (Cachelin, A.B., J.E. de Peyer, S. Kokubun, and H. Reuter, 1983, J. Physiol. (Lond.), 340:389-402). They also reported low amplitude Na channel events. We have studied this low conductance Na channel in single canine cardiac Purkinje cells using cell-attached patches. Patch pipette solutions contained either 140 or 280 mM NaCl, and cells were bathed in a solution of 150 mM KCl to bring their resting potential close to zero. In 140 mM Na+, during steps to -50 mV, the lower and higher openings had amplitudes of 0.57 +/- 0.2 and 1.2 +/- 0.2 pA (means +/- SD of Gaussian fits). In 280 mM Na+ at -50 mV, amplitudes were 0.72 +/- 0.2 and 1.55 +/- 0.2 pA. Over a substantial voltage range, the lower events had amplitudes of about one-third that of the higher events. The frequency of the low conductance openings varied in different patches from zero to 22% of total openings. Histograms of open durations and latencies at several voltages suggested no difference in kinetics between the two channel events. The behavior of the low conductance channels was more consistent with a second population of channels rather than a second open state.     

Poly-L-glutamine forms cation channels: relevance to the pathogenesis of the polyglutamine diseases

We report that long-chain poly-L-glutamine forms cation-selective channels when incorporated into artificial planar lipid bilayer membranes. The channel was permeable to alkali cations and H(+) ions and virtually impermeable to anions; the selectivity sequence based on the single-channel conductance was H(+) > Cs(+) > K(+) > Na(+). The cation channel was characterized by long-lived open states (often lasting for several minutes to tens of minutes) interrupted by brief closings. The appearance of the channel depended critically on the length of polyglutamine chains; ion channels were observed with 40-residue stretches, whereas no significant conductance changes were detected with 29-residue tracts. The channel-forming threshold length of poly-L-glutamine was thus between 29 and 40 residues. A molecular mechanics calculation suggests a mu-helix (. Biophys. J. 69:1130-1141) as a candidate molecular structure of the channel. The channel-forming nature of long-chain poly-L-glutamine may provide a clue to the elucidation of the pathogenetic mechanism of the polyglutamine diseases, a group of inherited neurodegenerative disorders including Huntington's disease.     

Divalent cation effects on acetylcholine-activated channels at the frog neuromuscular junction

The effects of the divalent cations Ca and Mg on the properties of ACh-activated channels at the frog neuromuscular junction were studied using a two-microelectrode voltage clamp. The divalent cation concentration was varied from 2 to 40 mM in solutions containing 50% normal Na. The reversal potential was determined by interpolation of the acetylcholine (ACh)-induced current versus voltage relationship. The single-channel conductance and the mean channel lifetime were calculated from fluctuation analysis of the ACh-induced end-plate current. Extracellular Na and/or divalent cations affected the reversal potential of endplate channels in a way that cannot be described by the Goldman-Hodgkin-Katz equation or by a simple two-barrier, one-binding site model of the channel if the assumption was made that permeability ratios were constant and not a function of ion concentrations. Increasing the divalent cation concentration decreased the single-channel conductance to approximately 10 pS in solutions with 50% Na and 40 mM divalent cation concentrations. The effect of the divalent cations Ca and Mg on the mean channel lifetime was complex and dependent on whether the divalent cation was Ca or Mg. The mean channel lifetime was not significantly changed in most solutions with increased Ca concentration, while it was slightly prolonged by increased Mg concentration.     

A Na+- and Cl- -activated K+ channel in the thick ascending limb of mouse kidney

This study investigates the presence and properties of Na+-activated K+ (K(Na)) channels in epithelial renal cells. Using real-time PCR on mouse microdissected nephron segments, we show that Slo2.2 mRNA, which encodes for the K(Na) channels of excitable cells, is expressed in the medullary and cortical thick ascending limbs of Henle's loop, but not in the other parts of the nephron. Patch-clamp analysis revealed the presence of a high conductance K+ channel in the basolateral membrane of both the medullary and cortical thick ascending limbs. This channel was highly K+ selective (P(K)/P(Na) approximately 20), its conductance ranged from 140 to 180 pS with subconductance levels, and its current/voltage relationship displayed intermediate, Na+-dependent, inward rectification. Internal Na+ and Cl- activated the channel with 50% effective concentrations (EC50) and Hill coefficients (nH) of 30 +/- 1 mM and 3.9 +/- 0.5 for internal Na+, and 35 +/- 10 mM and 1.3 +/- 0.25 for internal Cl-. Channel activity was unaltered by internal ATP (2 mM) and by internal pH, but clearly decreased when internal free Ca2+ concentration increased. This is the first demonstration of the presence in the epithelial cell membrane of a functional, Na+-activated, large-conductance K+ channel that closely resembles native K(Na) channels of excitable cells. This Slo2.2 type, Na+- and Cl--activated K+ channel is primarily located in the thick ascending limb, a major renal site of transcellular NaCl reabsorption.