Inwardly rectifying K(+) channels in esophageal smooth muscle

The whole cell patch-clamp technique was used to investigate whether there were inwardly rectifying K(+) (K(ir)) channels in the longitudinal muscle of cat esophagus. Inward currents were observable on membrane hyperpolarization negative to the K(+) equilibrium potential (E(k)) in freshly isolated esophageal longitudinal muscle cells. The current-voltage relationship exhibited strong inward rectification with a reversal potential (E(rev)) of -76.5 mV. Elevation of external K(+) increased the inward current amplitude and positively shifted its E(rev) after the E(k), suggesting that potassium ions carry this current. External Ba(2+) and Cs(+) inhibited this inward current, with hyperpolarization remarkably increasing the inhibition. The IC(50) for Ba(2+) and Cs(+) at -60 mV was 2.9 and 1.6 mM, respectively. Furthermore, external Ba(2+) of 10 microM moderately depolarized the resting membrane potential of the longitudinal muscle cells by 6.3 mV while inhibiting the inward rectification. We conclude that K(ir) channels are present in the longitudinal muscle of cat esophagus, where they contribute to its resting membrane potential.     

Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium

Inward-rectifier K channel: using macroscopic voltage clamp and single- channel patch clamp techniques we have identified the K+ channel responsible for potassium recycling across basolateral membranes (BLM) of principal cells in intact epithelia isolated from frog skin. The spontaneously active K+ channel is an inward rectifier (Kir) and is the major component of macroscopic conductance of intact cells. The current- voltage relationship of BLM in intact cells of isolated epithelia, mounted in miniature Ussing chambers (bathed on apical and basolateral sides in normal amphibian Ringer solution), showed pronounced inward rectification which was K(+)-dependent and inhibited by Ba2+, H+, and quinidine. A 15-pS Kir channel was the only type of K(+)-selective channel found in BLM in cell-attached membrane patches bathed in physiological solutions. Although the channel behaves as an inward rectifier, it conducts outward current (K+ exit from the cell) with a very high open probability (Po = 0.74-1.0) at membrane potentials less negative than the Nernst potential for K+. The Kir channel was transformed to a pure inward rectifier (no outward current) in cell- attached membranes when the patch pipette contained 120 mM KCl Ringer solution (normal NaCl Ringer in bath). Inward rectification is caused by Mg2+ block of outward current and the single-channel current-voltage relation was linear when Mg2+ was removed from the cytosolic side. Whole-cell current-voltage relations of isolated principal cells were also inwardly rectified. Power density spectra of ensemble current noise could be fit by a single Lorentzian function, which displayed a K dependence indicative of spontaneously fluctuating Kir channels. Conclusions: under physiological ionic gradients, a 15-pS inward- rectifier K+ channel generates the resting BLM conductance in principal cells and recycles potassium in parallel with the Na+/K+ ATPase pump.     

Ca2+-activated K+ channels in murine endothelial cells: block by intracellular calcium and magnesium

The intermediate (IK(Ca)) and small (SK(Ca)) conductance Ca(2+)-sensitive K(+) channels in endothelial cells (ECs) modulate vascular diameter through regulation of EC membrane potential. However, contribution of IK(Ca) and SK(Ca) channels to membrane current and potential in native endothelial cells remains unclear. In freshly isolated endothelial cells from mouse aorta dialyzed with 3 microM free [Ca(2+)](i) and 1 mM free [Mg(2+)](i), membrane currents reversed at the potassium equilibrium potential and exhibited an inward rectification at positive membrane potentials. Blockers of large-conductance, Ca(2+)-sensitive potassium (BK(Ca)) and strong inward rectifier potassium (K(ir)) channels did not affect the membrane current. However, blockers of IK(Ca) channels, charybdotoxin (ChTX), and of SK(Ca) channels, apamin (Ap), significantly reduced the whole-cell current. Although IK(Ca) and SK(Ca) channels are intrinsically voltage independent, ChTX- and Ap-sensitive currents decreased steeply with membrane potential depolarization. Removal of intracellular Mg(2+) significantly increased these currents. Moreover, concomitant reduction of the [Ca(2+)](i) to 1 microM caused an additional increase in ChTX- and Ap-sensitive currents so that the currents exhibited theoretical outward rectification. Block of IK(Ca) and SK(Ca) channels caused a significant endothelial membrane potential depolarization (approximately 11 mV) and decrease in [Ca(2+)](i) in mesenteric arteries in the absence of an agonist. These results indicate that [Ca(2+)](i) can both activate and block IK(Ca) and SK(Ca) channels in endothelial cells, and that these channels regulate the resting membrane potential and intracellular calcium in native endothelium.     

Resting and activation-dependent ion channels in human mast cells

The mechanism of mediator secretion from mast cells in disease is likely to include modulation of ion channel activity. Several distinct Ca(2+), K(+), and Cl(-) conductances have been identified in rodent mast cells, but there are no data on human mast cells. We have used the whole-cell variant of the patch clamp technique to characterize for the first time macroscopic ion currents in purified human lung mast cells and human peripheral blood-derived mast cells at rest and following IgE-dependent activation. The majority of both mast cell types were electrically silent at rest with a resting membrane potential of around 0 mV. Following IgE-dependent activation, >90% of human peripheral blood-derived mast cells responded within 2 min with the development of a Ca(2+)-activated K(+) current exhibiting weak inward rectification, which polarized the cells to around -40 mV and a smaller outwardly rectifying Ca(2+)-independent Cl(-) conductance. Human lung mast cells showed more heterogeneity in their response to anti-IgE, with Ca(2+)-activated K(+) currents and Ca(2+)-independent Cl(-) currents developing in approximately 50% of cells. In both cell types, the K(+) current was blocked reversibly by charybdotoxin, which along with its electrophysiological properties suggests it is carried by a channel similar to the intermediate conductance Ca(2+)-activated K(+) channel. Charybdotoxin did not consistently attenuate histamine or leukotriene C(4) release, indicating that the Ca(2+)-activated K(+) current may enhance, but is not essential for, the release of these mediators.     

Characteristics of Kcnn4 channels in the apical membranes of an intestinal epithelial cell line

Intermediate-conductance K(+) (Kcnn4) channels in the apical and basolateral membranes of epithelial cells play important roles in agonist-induced fluid secretion in intestine and colon. Basolateral Kcnn4 channels have been well characterized in situ using patch-clamp methods, but the investigation of Kcnn4 channels in apical membranes in situ has been hampered by a layer of mucus that prevents seal formation. In the present study, we used patch-clamp methods to characterize Kcnn4 channels in the apical membrane of IEC-18 cells, a cell line derived from rat small intestine. A monolayer of IEC-18 cells grown on a permeable support is devoid of mucus, and tight junctions enable selective access to the apical membrane. In inside-out patches, Ca(2+)-dependent K(+) channels observed with iberiotoxin (a Kcnma1/large-conductance, Ca(2+)-activated K(+) channel blocker) and apamin (a Kcnn1-3/small-conductance, Ca(2+)-activated K(+) channel blocker) present in the pipette solution exhibited a single-channel conductance of 31 pS with inward rectification. The currents were reversibly blocked by TRAM-34 (a Kcnn4 blocker) with an IC(50) of 8.7 ± 2.0 μM. The channels were not observed when charybdotoxin, a peptide inhibitor of Kcnn4 channels, was added to the pipette solution. TRAM-34 was less potent in inhibiting Kcnn4 channels in patches from apical membranes than in patches from basolateral membranes, which was consistent with a preferential expression of Kcnn4c and Kcnn4b isoforms in apical and basolateral membranes, respectively. The expression of both isoforms in IEC-18 cells was confirmed by RT-PCR and Western blot analyses. This is the first characterization of Kcnn4 channels in the apical membrane of intestinal epithelial cells.     

Functional roles of charged amino acid residues on the wall of the cytoplasmic pore of Kir2.1

It is known that rectification of currents through the inward rectifier K(+) channel (Kir) is mainly due to blockade of the outward current by cytoplasmic Mg(2+) and polyamines. Analyses of the crystal structure of the cytoplasmic region of Kir2.1 have revealed the presence of both negatively (E224, D255, D259, and E299) and positively (R228 and R260) charged residues on the wall of the cytoplasmic pore of Kir2.1, but the detail is not known about the contribution of these charged residues, the positive charges in particular, to the inward rectification. We therefore analyzed the functional significance of these charged amino acids using single/double point mutants in order to better understand the structure-based mechanism underlying inward rectification of Kir2.1 currents. As a first step, we used two-electrode voltage clamp to examine inward rectification in systematically prepared mutants in which one or two negatively or positively charged amino acids were neutralized by substitution. We found that the intensity of the inward rectification tended to be determined by the net negative charge within the cytoplasmic pore. We then used inside-out excised patch clamp recording to analyze the effect of the mutations on blockade by intracellular blockers and on K(+) permeation. We observed that a decrease in the net negative charge within the cytoplasmic pore reduced both the susceptibility of the channel to blockade by Mg(2+) or spermine and the voltage dependence of the blockade. It also reduced K(+) permeation; i.e., it decreased single channel conductance, increased open-channel noise, and strengthened the intrinsic inward rectification in the total absence of cytoplasmic blockers. Taken together, these data suggest that the negatively charged cytoplasmic pore of Kir electrostatically gathers cations such as Mg(2+), spermine, and K(+) so that the transmembrane pore is sufficiently filled with K(+) ions, which enables strong voltage-dependent blockade with adequate outward K(+) conductance.     

Clotrimazole-sensitive K+ currents regulate pacemaker activity in interstitial cells of Cajal

Interstitial cells of Cajal (ICC) are pacemaker cells for gut peristaltic motor activity. Compared with cardiac pacemaker cells, little is known about mechanisms that regulate ICC excitability. The objective of the present study was to investigate a potential role for clotrimazole (CTL)-sensitive K currents (I(CTL)) in the regulation of ICC excitability and pacemaker activity. ICC were studied in situ and in short-term culture by using the whole cell patch-clamp configuration. In situ, ICC exhibited spontaneous transient inward currents followed by transient outward currents. CTL blocked outward currents, thereby increasing the net inward currents, and depolarized ICC, thereby establishing CTL-sensitive channels as regulators of ICC pacemaker activity. In short-term culture, a I(CTL) was identified that showed increased conductance when depolarized from the resting membrane potential to 0 mV and subsequent inward rectification at further depolarized potentials. The I(CTL) markedly increased with increasing intracellular calcium and was insensitive to the ether-à-go-go-related K channel blocker E-4031 and the large-conductance calcium-activated K channel blocker iberiotoxin. I(CTL) contributed 3-9 nS to the whole cell conductance at 0 mV membrane potential under physiological conditions; it was fast activating (tau = 88 ms), showed little time-dependent inactivation, and exhibited a deactivation time constant of 38 ms. The nitric oxide donor sodium nitroprusside (SNP) increased I(CTL). Single-channel activity, activated by calcium and SNP, was inhibited by CTL, with a single-channel conductance of approximately 38 pS. In summary, ICC generate a I(CTL) on depolarization through an intermediate-conductance calcium-activated K channel that regulates pacemaker activity and ICC excitability.     

Sodium-potassium-ATPase electrogenicity in cerebral precapillary arterioles

Electrogenicity of the Na(+)/K(+) pump has the capability to generate a large negative membrane potential independently of ion-channel current. The high background membrane resistance of arterioles may make them susceptible to such an effect. Pump current was detected by patch-clamp recording from smooth muscle cells in fragments of arterioles (diameter 24-58 microm) isolated from pial membrane of rabbit cerebral cortex. The current was 20 pA at -60 mV, and the extrapolated zero current potential was -160 mV. Two methods of estimating the effect of pump electrogenicity on resting potential indicated an average contribution of -35 mV. In 20% of the recordings, block of inward rectifier K(+) channels by 10-100 microM Ba(2+) led to a small depolarization, but hyperpolarization was a more common response. Ba(2+) also inhibited depolarization evoked by 20 mM K(+). In arterioles within intact pial membrane, Ba(2+) failed to evoke constriction but inhibited K(+)-induced constriction. The data suggest that cerebral arterioles are vulnerable to the hyperpolarizing effect of the Na(+)/K(+) pump, excessive effects of which are prevented by depolarizing inward rectifier K(+) current     

Two K+ channel types, muscarinic agonist-activated and inwardly rectifying, in a Cl- secretory epithelium: the avian salt gland

Patches of membrane on cells isolated from the nasal salt gland of the domestic duck typically contained two types of K+ channel. One was a large-conductance ("maxi") K+ channel which was activated by intracellular calcium and/or depolarizing membrane voltages, and the other was a smaller-conductance K+ channel which exhibited at least two conductance levels and displayed pronounced inward rectification. Barium blocked both channels, but tetraethylammonium chloride and quinidine selectively blocked the larger K+ channel. The large K+ channel did not appear to open under resting conditions but could be activated by application of the muscarinic agonist, carbachol. The smaller channels were open under resting conditions but the gating was not affected by carbachol. Both of these channels reside in the basolateral membranes of the Cl- secretory cells but they appear to play different roles in the life of the cell.     

Properties of a potassium channel in the basolateral membrane of renal proximal convoluted tubule and the effect of cyclosporine on it

We studied the potassium channel in the basolateral membrane of the rat proximal convoluted tubule as affected by cyclosporine A. Proximal convoluted tubules were dissected from the rat kidney under a stereoscopic microscope, without a preliminary enzyme treatment. The standard configuration for single-channel tight seal patch-clamp technique was used to record channel currents. A small conductance, stretch-sensitive potassium channel could be observed spontaneously in most of the cell-attached patches as the gigaohm seal was formed. In the inside-out configuration, channel activity was diminished. The K(+) channel appeared to be an inward rectifier. The limiting inward slope conductance was 28.3+/-1.7 pS (Vp was between 40 mV and 80 mV, n=6) and the outward chord conductance was 5.6+/-0.3 pS (Vp was between -40 and -60 mV, n=5). The open dwell time constants of the potassium channel were 0.524 ms and 5.087 ms, while the closed dwell time constants were 1.029 ms and 16.500 ms. The opening probability of the channel decreased when the extracellular fluid was acidified. Cyclosporine A had no significant effect on the potassium channel of the proximal tubular cell in the basolateral membrane at concentrations of 10 and 50 microg/ml, while at 100 microg/ml, it decreased the opening probability.     

Structural basis of inward rectification: cytoplasmic pore of the G protein-gated inward rectifier GIRK1 at 1.8 A resolution

Inward rectifier K(+) channels govern the resting membrane voltage in many cells. Regulation of these ion channels via G protein-coupled receptor signaling underlies the control of heart rate and the actions of neurotransmitters in the central nervous system. We have determined the protein structure formed by the intracellular N- and C termini of the G protein-gated inward rectifier K(+) channel GIRK1 at 1.8 A resolution. A cytoplasmic pore, conserved among inward rectifier K(+) channels, extends the ion pathway to 60 A, nearly twice the length of a canonical transmembrane K(+) channel. The cytoplasmic pore is lined by acidic and hydrophobic amino acids, creating a favorable environment for polyamines, which block the pore. These results explain in structural and chemical terms the basis of inward rectification, and they also have implications for G protein regulation of GIRK channels.     

Role of hyperpolarization-activated currents for the intrinsic dynamics of isolated retinal neurons

The intrinsic dynamics of bipolar cells and rod photoreceptors isolated from tiger salamanders were studied by a patch-clamp technique combined with estimation of effective impulse responses across a range of mean membrane voltages. An increase in external K(+) reduces the gain and speeds the response in bipolar cells near and below resting potential. High external K(+) enhances the inward rectification of membrane potential, an effect mediated by a fast, hyperpolarization-activated, inwardly rectifying potassium current (K(IR)). External Cs(+) suppresses the inward-rectifying effect of external K(+). The reversal potential of the current, estimated by a novel method from a family of impulse responses below resting potential, indicates a channel that is permeable predominantly to K(+). Its permeability to Na(+), estimated from Goldman-Hodgkin-Katz voltage equation, was negligible. Whereas the activation of the delayed-rectifier K(+) current causes bandpass behavior (i.e., undershoots in the impulse responses) in bipolar cells, activation of the K(IR) current does not. In contrast, a slow hyperpolarization-activated current (I(h)) in rod photoreceptors leads to pronounced, slow undershoots near resting potential. Differences in the kinetics and ion selectivity of hyperpolarization-activated currents in bipolar cells (K(IR)) and in rod photoreceptors (I(h)) confer different dynamical behavior onto the two types of neurons.     

Expression of intermediate-conductance, Ca2+-activated K+ channel (KCNN4) in H441 human distal airway epithelial cells

Electrophysiological studies of H441 human distal airway epithelial cells showed that thapsigargin caused a Ca(2+)-dependent increase in membrane conductance (G(Tot)) and hyperpolarization of membrane potential (V(m)). These effects reflected a rapid rise in cellular K(+) conductance (G(K)) and a slow fall in amiloride-sensitive Na(+) conductance (G(Na)). The increase in G(Tot) was antagonized by Ba(2+), a nonselective K(+) channel blocker, and abolished by clotrimazole, a KCNN4 inhibitor, but unaffected by other selective K(+) channel blockers. Moreover, 1-ethyl-2-benzimidazolinone (1-EBIO), which is known to activate KCNN4, increased G(K) with no effect on G(Na). RT-PCR-based analyses confirmed expression of mRNA encoding KCNN4 and suggested that two related K(+) channels (KCNN1 and KCNMA1) were absent. Subsequent studies showed that 1-EBIO stimulates Na(+) transport in polarized monolayers without affecting intracellular Ca(2+) concentration ([Ca(2+)](i)), suggesting that the activity of KCNN4 might influence the rate of Na(+) absorption by contributing to G(K). Transient expression of KCNN4 cloned from H441 cells conferred a Ca(2+)- and 1-EBIO-sensitive K(+) conductance on Chinese hamster ovary cells, but this channel was inactive when [Ca(2+)](i) was <0.2 microM. Subsequent studies of amiloride-treated H441 cells showed that clotrimazole had no effect on V(m) despite clear depolarizations in response to increased extracellular K(+) concentration ([K(+)](o)). These findings thus indicate that KCNN4 does not contribute to V(m) in unstimulated cells. The present data thus establish that H441 cells express KCNN4 and highlight the importance of G(K) to the control of Na(+) absorption, but, because KCNN4 is quiescent in resting cells, this channel cannot contribute to resting G(K) or influence basal Na(+) absorption.     

Properties of a tonically active, sodium-permeable current in mouse urinary bladder smooth muscle

Urinary bladder smooth muscle (UBSM) elicits depolarizing action potentials, which underlie contractile events of the urinary bladder. The resting membrane potential of UBSM is approximately -40 mV and is critical for action potential generation, with hyperpolarization reducing action potential frequency. We hypothesized that a tonic, depolarizing conductance was present in UBSM, functioning to maintain the membrane potential significantly positive to the equilibrium potential for K(+) (E(K); -85 mV) and thereby facilitate action potentials. Under conditions eliminating the contribution of K(+) and voltage-dependent Ca(2+) channels, and with a clear separation of cation- and Cl(-)-selective conductances, we identified a novel background conductance (I(cat)) in mouse UBSM cells. I(cat) was mediated predominantly by the influx of Na(+), although a small inward Ca(2+) current was detectable with Ca(2+) as the sole cation in the bathing solution. Extracellular Ca(2+), Mg(2+), and Gd(3+) blocked I(cat) in a voltage-dependent manner, with K(i) values at -40 mV of 115, 133, and 1.3 microM, respectively. Although UBSM I(cat) is extensively blocked by physiological extracellular Ca(2+) and Mg(2+), a tonic, depolarizing I(cat) was detected at -40 mV. In addition, inhibition of I(cat) demonstrated a hyperpolarization of the UBSM membrane potential and decreased the amplitude of phasic contractions of isolated UBSM strips. We suggest that I(cat) contributes tonically to the depolarization of the UBSM resting membrane potential, facilitating action potential generation and thereby a maintenance of urinary bladder tone.     

Inhibition, by an amphiphilic substance, niflumic acid, of the inward rectification of the crustacean muscle fiber

Agents such as TEA+ or CS+ ions, these last ions instead of K+ ions in poor K extracellular solution, known to reduce or abolish the inwardly rectifying channel in many preparations produced no effect in crayfish muscle membrane By contrast, poor Cl extracellular solution (Cl- ions were replaced by CH3OSO3- ions) blocked the inward current activated by hyperpolarizing pulses and produced an increase of the resting potential. Niflumic acid is a agent which inhibited the inward going rectification of the crayfish muscle membrane. Apparent dissociation constant of niflumic acid with membrane sites was equal to about 6 X 10(-8) M; this value corresponds to that given by Cousin & Motais (1979) concerning translocation of Cl- ions in the membrane of red cells. Activation of the inward going rectification in the crayfish membrane is responsible of an inward current carried by Cl- ions.     

Charges in the Cytoplasmic Pore Control Intrinsic Inward Rectification and Single-Channel Properties in Kir1.1 and Kir2.1 Channels

An E224G mutation of the Kir2.1 channel generates intrinsic inward rectification and single-channel fluctuations in the absence of intracellular blockers. In this study, we showed that positively charged residues H226, R228 and R260, near site 224, regulated the intrinsic inward rectification and single-channel properties of the E224G mutant. By carrying out systematic mutations, we found that the charge effect on the intrinsic inward rectification and single-channel conductance is consistent with a long-range electrostatic mechanism. A Kir1.1 channel where the site equivalent to E224 in the Kir2.1 channel is a glycine residue does not show inward rectification or single-channel fluctuations. The G223K and N259R mutations of the Kir1.1 channel induced intrinsic inward rectification and reduced the single-channel conductance but did not generate large open-channel fluctuations. Substituting the cytoplasmic pore of the E224G mutant into the Kir1.1 channel induced open-channel fluctuations and intrinsic inward rectification. The single-channel conductance of the E224G mutant showed inward rectification. Also, a voltage-dependent gating mechanism decreased open probability during depolarization and contributed to the intrinsic inward rectification in the E224G mutant. In addition to an electrostatic effect, a close interaction of K⁺ with channel pore may be required for generating open-channel fluctuations in the E224G mutant.     

Electrophysiological properties of isolated rat liver cells

The electrophysiological properties of isolated rat liver cells were studied using the patch clamp method in whole-cell configuration. The membrane potential in isolated hepatocytes was -42 +/- 7 mV (n = 20). The input resistance (Rin) and the time constant (tau m) were 51 +/- 17 M (the range of 34 to 180 M omega) (n = 20) and 4.2 +/- 1.0 msec (the range of 3 to 16.5 ms) (n = 20). Assuming that the specific membrane capacitance is 1 microF/cm2, the membrane resistance and membrane capacitance were 42. +/- 9.0 K omega cm2 and 87 +/- 27 pF. These values indicate that isolated rat hepatocytes are not abnormally permeable or leaky. The current-voltage relationship was linear with no rectification. The depolarizing pulse from the resting potential did not induce fast or slow inward currents even when norepinephrine or high Ca2 (3.6 mM) were applied. This indicates that there is no voltage-sensitive Ca2+ channel in the isolated hepatocytes.     

Anomalous permeabilities of the egg cell membrane of a starfish in K(+)-Tl(+) mixtures

The electrical properties of “inward” rectifying egg cell membranes of the starfish mediastera aequalis have been studied in the presence of K(+)-Tl(+) mixtures. When the ratio of the external concentrations of these ions is changed while their sum is kept constant, both the conductance and the zero-current membrane potential go through a minimum, showing clear discrepancies from theoretical results based on conventional electrodiffusion models (E.g., Goldman’s equation). By contrast, when the ration of the two concentrations is fixed and their sum varied, the potential follows an ideal Nernst slope, consistent with Goldman’s equation. The membrane conductance which, according to previous studies on similar membranes, is to be viewed as a function of the displacement of the membrane potential from its resting value δV, shows marked differences between the cases in which K(+) or Tl(+) are the predominant ions: when K(+) is the predominant permeant ion in solution, the addition of small amounts of Tl(+) inhibits the current, while corresponding blocking effects of K(+) on the current are not observed when Tl(+) is the predominant permeant ion. Also, the time course of the conductance during voltage clamp is different in the two cases, being much faster in Tl(+) than in K(+) solution for comparable values of δV. Most of the above features are accounted for by a model in which it is assumed that the ionic channels have external binding sites for cations and that their permeability properties depend on the species of the cation bound (K(+)or Tl(+) in the present experiments).     

Regulation of ion conductance in frog skin by isoproterenol

The effect of isoproterenol on apical and basolateral membrane conductance in principal cells of short-circuited frog skin was analyzed using microelectrodes. Isoproterenol (10(-6) mol/l) increased the apical membrane conductance in addition to stimulating Cl- conductive pathways outside the principal cells. The effect on apical Na+ channels explains the increase in amiloride sensitive short-circuit current. Basolateral membrane conductance increased only slightly. Steady-state I/V relationships of the basolateral membrane indicate that the inward rectification of basolateral membrane K+ channels was not altered.