Flow-Dependent Activation of Maxi K+ Channels in Apical Membrane of Rabbit Connecting Tubule

The Ca²⁺-activated maxi K⁺ channel was found in the apical membrane of everted rabbit connecting tubule (CNT) with a patch-clamp technique. The mean number of open channels (NP _o ) was markedly increased from 0.007 ± 0.004 to 0.189 ± 0.039 (n= 7) by stretching the patch membrane in a cell-attached configuration. This activation was suggested to be coupled with the stretch-activation of Ca²⁺-permeable cation channels, because the maxi K⁺ channel was not stretch-activated in both the cell-attached configuration using Ca²⁺-free pipette and in the inside-out one in the presence of 10 mm EGTA in the cytoplasmic side. The maxi K⁺ channel was completely blocked by extracellular 1 μm charybdotoxin (CTX), but was not by cytoplasmic 33 μm arachidonic acid (AA). On the other hand, the low-conductance K⁺ channel, which was also found in the same membrane, was completely inhibited by 11 μm AA, but not by 1 μm CTX. The apical K⁺ conductance in the CNT was estimated by the deflection of transepithelial voltage (ΔV _t ) when luminal K⁺ concentration was increased from 5 to 15 mEq. When the tubule was perfused with hydraulic pressure of 0.5 KPa, the ΔV _t was only −0.7 ± 0.4 mV. However, an increase in luminal fluid flow by increasing perfusion pressure to 1.5 KPa markedly enhanced ΔV _t to −9.4 ± 0.9 mV. Luminal application of 1 μm CTX reduced the ΔV _t to −1.3 ± 0.6 mV significantly in 6 tubules, whereas no significant change of ΔV _t was recorded by applying 33 μm AA into the lumen of 5 tubules (ΔV _t =−7.2 ± 0.5 mV in control vs.ΔV _t =−6.7 ± 0.6 mV in AA). These results suggest that the Ca²⁺-activated maxi K⁺ channel is responsible for flow-dependent K⁺ secretion by coupling with the stretch-activated Ca²⁺-permeable cation channel in the rabbit CNT. Received: 21 August 1997/Revised: 20 March 1998     

The Sensitivity of the Human Kv1.3 (hKv1.3) Lymphocyte K+ Channel to Regulation by PKA and PKC is Partially Lost in HEK 293 Host Cells

cDNA encoding the full-length hKv1.3 lymphocyte channel and a C-terminal truncated (Δ459-523) form that lacks the putative PKA Ser⁴⁶⁸ phosphorylation site were stably transfected in human embryonic kidney (HEK) 293 cells. Immunostaining of the transfected cells revealed a distribution at the plasma membrane that was uniform in the case of the full-length channel whereas clustering was observed in the case of the truncated channel. Some staining within the cell cytoplasm was found in both instances, suggesting an active process of biosynthesis. Analyses of the K⁺ current by the patch-clamp technique in the whole cell configuration showed that depolarizing steps to 40 mV from a holding potential (HP) of −80 mV elicited an outward current of 2 to 10 nA. The current threshold was positive to −40 mV and the current amplitude increased in a voltage-dependent manner. The parameters of activation were −5.7 and −9.9 mV (slope factor) and −35 mV (half activation, V _0.5) in the case of the full-length and truncated channels, respectively. The characteristics of the inactivation were 14.2 and 24.6 mV (slope factor) and −17.3 and −39.0 mV (V _0.5) for the full-length and truncated channels, respectively. The activation time constant of the full-length channel for potentials ranging from −30 to 40 mV decreased from 18 to 12 msec whereas the inactivation time constant decreased from 6600 msec at −30 mV to 1800 msec at 40 mV. The unit current amplitude measured in cells bathing in 140 mm KCl was 1.3 ± 0.1 pA at 40 mV, the unit conductance, 34.5 pS and the zero current voltage, 0 mV. Both forms of the channels were inhibited by TEA, 4-AP, Ni²⁺ and charybdotoxin. In contrast to the native (Jurkat) lymphocyte Kv1.3 channel that is fully inhibited by PKA and PKC, the addition of TPA resulted in 34.6 ± 7.3% and 38.7 ± 9.4% inhibition of the full-length and the truncated channels, respectively. 8-BrcAMP induced a 39.4 ± 5.4% inhibition of the full-length channel but had no effect (8.6 ± 8.3%) on the truncated channel. Cell dialysis with alkaline phosphatase had no effects, suggesting that the decreased sensitivity of the transfected channels to PKA and PKC was not due to an already phosphorylated channel. Patch extract experiments suggested that the hKv1.3 channel was partially sensitive to PKA and PKC. Cotransfecting the Kvβ1.2 subunit resulted in a decrease in the value of the time constant of inactivation of the full-length channel but did not modify its sensitivity to PKA and PKC. The cotransfected Kvβ2 subunit had no effects. Our results indicate that the hKv1.3 lymphocyte channel retains its electrophysiological characteristics when transfected in the Kvβ-negative HEK 293 cell line but its sensitivity to modulation by PKA and PKC is significantly reduced. Received: 18 June 1997/Revised: 7 October 1997     

Membrane Potassium Channels and Human Bladder Tumor Cells. I. Electrical Properties

These experiments were conducted to determine the membrane K⁺ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K⁺ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch clamp recordings. Cell depolarization resulted in activation of a Ca²⁺-dependent outward K⁺ current, 0.57 ± 0.13 nS/pF at −70 mV holding potential and 3.10 ± 0.15 nS/pF at 30 mV holding potential. Corresponding patch clamp measurements demonstrated a Ca²⁺-activated, voltage-dependent K⁺ channel (K_Ca) of 214 ± 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current and the K_Ca activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K⁺ channel, 21 ± 1 pS at positive transmembrane potential (V _m ) and 145 ± 13 pS at negative V _m . Glibenclamide (50 μm), Ba²⁺ (1 mm) and quinine (100 μm) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K⁺ channels in inside/out patches in a dose-dependent manner, and the IC₅₀= 46 μm. The identity of this K⁺ channel with an ATP-sensitive K⁺ channel (K_ATP) was confirmed by its inhibition with ATP (2 mm) and by its activation with diazoxide (100 μm). We conclude that plasma membranes of HTB-9 cells contain the K_Ca and a lower conductance K⁺ channel with properties consistent with a sulfonylurea receptor-linked K_ATP. Received: 12 June 1997/Revised: 21 October 1997     

Channel Activation Voltage Alone Is Directly Altered in an Isoform-specific Manner by Na<Subscript>v1.4</Subscript> and Na<Subscript>v1.5</Subscript> Cytoplasmic Linkers

The isoform-specific direct role of cytoplasmic loops in the gating of two voltage-gated sodium channel isoforms, the human cardiac channel (Na_v1.5; hH1) and the human adult skeletal muscle channel (Na_v1.4; hSkM1), was investigated. Comparison of biophysical characteristics was made among hSkM1, hH1, and several hSkM1/hH1 chimeras in which the putative cytoplasmic loops that join domain I to II (loop A) and domain II to III (loop B) from one isoform replaced one or both of the analogous loops from the other isoform. For all parameters measured, hSkM1 and hH1 behavior were significantly different. Comparison of hSkM1 and hH1 biophysical characteristics with the function of their respective chimeras indicate that only the half-activation voltage (V_a) is directly and differently altered by the species of cytoplasmic loop such that a channel consisting of one or both hSkM1 loops activates at smaller depolarizations, while a larger depolarization is required for activation of a channel containing one or both of the analogous hH1 loops. When either cardiac channel loop A or B is attached to hSkM1, a 6–7 mV depolarizing shift in V_a is measured, increasing to a nearly 20 mV depolarization when both cardiac-channel loops are attached. The addition of either skeletal muscle-channel loop to hH1 causes a 7 mV hyperpolarization in V_a, which increases to about 10 mV for the double loop chimera. There is no significant difference in either steady-state inactivation or in the recovery from inactivation data between hSkM1 and its chimeras and between hH1 and its chimeras. Data indicate that the cytoplasmic loops contribute directly to the magnitude of the window current, suggesting that channels containing skeletal muscle loops have three times the peak persistent channel activity compared to channels containing the cardiac loops. An electrostatic mechanism, in which surface charge differences among these loops might alter differently the voltage sensed by the gating mechanism of the channel, can not account for the observed isoform-specific effects of these loops only on channel activation voltage. In summary, although the DI-DII and DII-DIII loop structures among isoforms are not well conserved, these data indicate that only one gating parameter, V_a is affected directly and in an isoform-specific manner by these divergent loop structures, creating loop-specific window currents and percentages of persistently active channels at physiological voltages that will likely impact the excitability of the cell.     

State-Dependent Inhibition of Inactivation-Deficient CaV1.2 and CaV2.3 Channels By Mibefradil

The structural determinants of mibefradil inhibition were analyzed using wild-type and inactivation-modified Ca_V1.2 (α1C) and Ca_V2.3 (α1E) channels. Mibefradil inhibition of peak Ba²⁺ currents was dose- and voltage-dependent. An increase of holding potentials from −80 to −100 mV significantly shifted dose-response curves toward higher mibefradil concentrations, namely from a concentration of 108 ± 21 μm (n= 7) to 288 ± 17 μm (n= 3) for inhibition of half of the Ca_v1.2 currents (IC ₅₀) and from IC ₅₀= 8 ± 2 μm (n= 9) to 33 ± 7 μm (n= 4) for Ca_V2.3 currents. In the presence of mibefradil, Ca_V1.2 and Ca_V2.3 experienced significant use-dependent inhibition (0.1 to 1 Hz) and slower recovery from inactivation suggesting mibefradil could promote transition(s) to an absorbing inactivated state. In order to investigate the relationship between inactivation and drug sensitivity, mibefradil inhibition was studied in inactivation-altered Ca_V1.2 and Ca_V2.3 mutants. Mibefradil significantly delayed the onset of channel recovery from inactivation in CEEE (Repeat I + part of the I–II linker from Ca_V1.2 in the Ca_V2.3 host channel), in EC(AID)EEE (part of the I–II linker from Ca_V1.2 in the Ca_V2.3 host channel) as well as in Ca_V1.2 E462R, and Ca_V2.3 R378E (point mutation in the β-subunit binding motif) channels. Mibefradil inhibited the faster inactivating chimera EC(IS1-6)EEE with an IC ₅₀= 7 ± 1 μm (n= 3), whereas the slower inactivating chimeras EC(AID)EEE and CEEE were, respectively, inhibited with IC ₅₀= 41 ± 5 μm (n= 4) and IC ₅₀= 68 ± 9 μm (n= 5). Dose-response curves were superimposable for the faster EC(IS1-6)EEE and Ca_V2.3, whereas intermediate-inactivating channel kinetics (CEEE, Ca_V1.2 E462R, and Ca_V1.2 E462K) were inhibited by similar concentrations of mibefradil with IC ₅₀≈ 55–75 μm. The slower Ca_V1.2 wild-type and Ca_V1.2 Q473K channels responded to higher doses of mibefradil with IC ₅₀≈ 100–120 μm. Mibefradil was also found to significantly speed up the inactivation kinetics of slower channels (Ca_V1.2, CEEE) with little effect on the inactivation kinetics of faster-inactivating channels (Ca_V2.3). A open-channel block model for mibefradil interaction with high-voltage-activated Ca²⁺ channels is discussed and shown to qualitatively account for our observations. Hence, our data agree reasonably well with a ``receptor guarded mechanism' where fast inactivation kinetics efficiently trap mibefradil into the channel. Received: 14 March 2001/Revised: 25 June 2001     

Large Conductance Ca2+-Activated K+ Channels in Human Meningioma Cells

Cells from ten human meningiomas were electrophysiologically characterized in both living tissue slices and primary cultures. In whole cells, depolarization to voltages higher than +80 mV evoked a large K⁺ outward current, which could be blocked by iberiotoxin (100 nm) and TEA (half blocking concentration IC₅₀= 5.3 mm). Raising the internal Ca²⁺ from 10 nm to 2 mm shifted the voltage of half-maximum activation (V _1/2) of the K⁺ current from +106 to +4 mV. Respective inside-out patch recordings showed a voltage- and Ca²⁺-activated (BK _Ca ) K⁺ channel with a conductance of 296 pS (130 mm K⁺ at both sides of the patch). V _1/2 of single-channel currents was +6, −12, −46, and −68 mV in the presence of 1, 10, 100, and 1000 μm Ca²⁺, respectively, at the internal face of the patch. In cell-attached patches the open probability (P _o ) of BK _Ca channels was nearly zero at potentials below +80 mV, matching the activation threshold for whole-cell K⁺ currents with 10 nm Ca²⁺ in the pipette. Application of 20 μm cytochalasin D increased P _o of BK _Ca channels in cell-attached patches within minutes. These data suggest that the activation of BK _Ca channels in meningioma cells does not only depend on voltage and internal Ca²⁺ but is also controlled by the cytoskeleton. Received 18 June 1999/Revised: 18 January 2000     

Effect of Calcium and Calcium Channel Blockers on Transient Outward Current of F76 and D1 Neuronal Soma Membranes in the Subesophageal Ganglia of Helix aspersa

Twin-electrode voltage-clamp techniques were used to study the effect of calcium and calcium channel blockers on the transient outward current in isolated F76 and D1 neurones of Helix aspersa subesophageal ganglia in vitro (soma only preparation with no cell processes). On lowering extracellular Ca²⁺ concentration from 10 to 2 mm or removing extracellular calcium from the bathing medium, the threshold for this current shifted in a negative direction by 11.5 and 20 mV, respectively. On the other hand, increasing the extracellular Ca²⁺ concentration from 10 to 20 and to 40 mm shifted the steady-state inactivation curves in positive directions on the voltage axis by 7 and 15 mV, respectively. Upon application of calcium channel blockers, Co²⁺, La³⁺, Ni²⁺ and Cd²⁺, transient potassium current amplitude was reduced in a voltage-dependent manner, being more effective at voltages close to the threshold. The current was elicited even at a holding potential of −34 mV. The specific calcium channel blockers, amiloride and nifedipine did not shift the activation and steady-state inactivation curves and did not reduce the transient outward current amplitude. It was concluded that the transient outward current is not dependent on intracellular Ca²⁺ but that it is modulated by Ca²⁺ and di- and trivalent ions extracellularly. The effects of these ions are very unlikely to be due to a surface charge effect because the addition of La³⁺ (200 μm) completely reverses the shift in a hyperpolarizing direction when the extracellular Ca²⁺ concentration was reduced from 10 to 1 mm and additionally shifts the kinetics further still in a depolarizing direction. The responses seen here are consistent with a specific effect of di- and trivalent ions on the transient outward current channels leading to a modification of gating. Received: 30 March 1999/Revised: 5 October 1999     

Nitric Oxide Activates or Inhibits Skeletal Muscle Ryanodine Receptors Depending on Its Concentration, Membrane Potential and Ligand Binding

We show that rabbit skeletal RyR channels in lipid bilayers can be activated or inhibited by NO, in a manner that depends on donor concentration, membrane potential and the presence of channel agonists. 10 μm S-nitroso-N-acetyl-penicillamine (SNAP) increased RyR activity at −40 mV within 15 sec of addition to the cis chamber, with a 2-fold increase in frequency of channel opening (F _o ). 10 μm SNAP did not alter activity at +40 mV and did not further activate RyRs previously activated by 2 mm cis ATP at +40 or −40 mV. In contrast to the increase in F _o with 10 μm SNAP, 1 mm SNAP caused a 2-fold reduction in F _o but a 1.5-fold increase in mean open time (T _o ) at −40 mV in the absence of ATP. 1 mm SNAP or 0.5 mm sodium nitroprusside (SNP) induced ∼3-fold reductions in F _o and T _o at +40 or −40 mV when channels were activated by 2 mm cis ATP or in channels activated by 6.5 μm peptide A at −40 mV (peptide A corresponds to part of the II–III loop of the skeletal dihydropyridine receptor). Both SNAP-induced activation and SNAP/SNP-induced inhibition were reversed by 2 mm dithiothreitol. The results suggest that S-Nitrosylation or oxidation of at least three classes of protein thiols by NO each produced characteristic changes in RyR activity. We propose that, in vivo, initial release of NO activates RyRs, but stronger release increases [NO] and inhibits RyR activity and contraction. Received: 27 August 1999/Revised: 25 October 1999     

Electrophysiological Study of Chimeric Sodium Channels from Heart and Skeletal Muscle

The α-subunit cDNAs encoding voltage-sensitive sodium channels of human heart (hH1) and rat skeletal muscle (rSkM1) have been expressed in the tsA201 mammalian cell line, in which inactivation properties appear to be normal in contrast to Xenopus oocytes. A series of rSkM1/hH1 chimeric sodium channels has been evaluated to identify the domains of the α-subunits that are responsible for a set of electrophysiological differences between hH1 and rSkM1, namely, midpoints and slope factors of steady-state activation and inactivation, inactivation kinetics and recovery from inactivation kinetics and their voltage-dependence. The phenotype of chimeric channels in which each hH1 domain was successively introduced into a rSkM1 α-subunit framework confirmed the following conclusions. (i) The D4 and or/C-ter. are responsible for the slow inactivation of hH1 sodium channels. (ii) Concerning the other differences between rSkM1 and hH1: steady-state activation and inactivation, kinetics of recovery from inactivation, the phenotypes are determined probably by more than one domain of the α-subunit. Received: 20 January 1998/Revised: 19 March 1998     

A Patch-Clamp Study of Ion Channels in Protoplasts Prepared from the Marine Alga Valonia utricularis

The giant marine alga Valonia utricularis is a classical model system for studying the electrophysiology and water relations of plant cells by using microelectrode and pressure probe techniques. The recent finding that protoplasts can be prepared from the giant ``mother cells' (Wang, J., Sukhorukov, V.L., Djuzenova, C.S., Zimmermann, U., Müller, T., Fuhr, G., 1997, Protoplasma 196:123–134) allowed the use of the patch-clamp technique to examine ion channel activity in the plasmalemma of this species. Outside-out and cell-attached experiments displayed three different types of voltage-gated Cl<sup>−</sup> channels (VAC1, VAC2, VAC3, Valonia Anion Channel 1,2,3), one voltage-gated K<sup>+</sup> channel (VKC1, Valonia K <sup>+</sup> Channel 1) as well as stretch-activated channels. In symmetrical 150 mm Cl<sup>−</sup> media, VAC1 was most frequently observed and had a single channel conductance of 36 ± 7 pS (n= 4) in the outside-out and 33 ± 5 pS (n= 10) in the cell-attached configuration. The reversal potential of the corresponding current-voltage curves was within 0 ± 4 mV (n= 4, outside-out) and 9 ± 7 mV (n= 10, cell-attached) close to the Nernst potential of Cl<sup>−</sup> and shifted towards more negative values when cell-attached experiments were performed in asymmetrical 50:150 mm Cl<sup>−</sup> media (bath/pipette; E <sub>Cl−</sub> −20 ± 7 mV (n= 4); Nernst potential −28 mV). Consistent with a selectivity for Cl<sup>−</sup>, VAC1 was inhibited by 100 μM DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid). VAC1 was activated by a hyperpolarization of the patch. Boltzmann fits of the channel activity under symmetrical 150 mm Cl<sup>−</sup> conditions yielded a midpoint potential of −12 ± 5 mV (n= 4, outside-out) and −3 ± 6 mV (n= 9, cell-attached) and corresponding apparent minimum gating charges of 15 ± 3 (n= 4) and 18 ± 5 (n= 9). The midpoint potential shifted to more negative values in the presence of a Cl<sup>−</sup> gradient. VAC2 was activated by voltages more negative than E <sub>Cl−</sub> and was always observed together with VAC1, but less frequently. It showed a ``flickering' gating. The single channel conductance was 99 ± 10 pS (n= 6). VAC3 was activated by membrane depolarization and frequently exhibited several subconductance states. The single channel conductance of the main conductance state was 36 ± 5 pS (n= 5). VKC1 was also activated by positive clamped voltages. Up to three conductance states occurred whereby the main conductance state had a single channel conductance of 124 ± 27 pS (n= 6). In the light of the above results it seems to be likely that VAC1 contributes mainly to the Cl⁻ conductance of the plasmalemma of the turgescent ``mother cells' and that this channel (as well as VAC2) can operate in the physiological membrane potential range. The physiological significance of VAC3 and VKC1 is unknown, but may be related (as the stretch-activated channels) to processes involved in turgor regulation. Received: 24 June 1999/Revised: 2 September 1999     

Protection from Cell Death by mcl-1 Is Mediated by Membrane Hyperpolarization Induced By K+ Channel Activation

Mcl-1, a member of the Bcl-2 family, has been identified as an inhibitor of apoptosis induced by anticancer agents and radiation in myeloblastic leukemia cells. The molecular mechanism underlying this phenomenon, however, is not yet understood. In the present study, we report that hyperpolarization of the membrane potential is required for prevention of mcl-1 mediated cell death in murine myeloblastic FDC-P1 cells. In cells transfected with mcl-1, the membrane potential, measured by the whole-cell patch clamp, was hyperpolarized more than −30 mV compared with control cells. The membrane potential was repolarized by increased extracellular K⁺ concentration (56 mV per 10-fold change in K⁺ concentration). Using the cell-attached patch-clamp technique, K⁺ channel activity was 1.7 times higher in mcl-1 transfected cells (NP _o = 22.7 ± 3.3%) than control cells (NP _o = 13.2 ± 1.9%). Viabilities of control and mcl-1 transfected cells after treatment with the cytotoxin etoposide (20 μg/ml), were 37.9 ± 3.9% and 78.2 ± 2.0%, respectively. Suppression of K⁺ channel activity by 4-aminopyridine (4-AP) before etoposide treatment significantly reduced the viability of mcl-1 transfected cells to 49.0 ± 4.6%. These results indicate that as part of the prevention of cell death, mcl-1 causes a hyperpolarization of membrane potential through activation of K⁺ channel activity. Received: 30 March 1999/Revised: 20 July 1999     

Proton Inhibition of Sodium Channels: Mechanism of Gating Shifts and Reduced Conductance

Extracellular acidosis affects both permeation and gating of the expressed rat skeletal muscle Na⁺ channel (μ1). Reduction of the extracellular pH produced a progressive decrease in the maximal whole-cell conductance and a depolarizing shift in the whole-cell current-voltage relationship. A smaller depolarizing shift in the steady-state inactivation curve was observed. The pK of the reduction of maximal conductance was 6.1 over the pH range studied. An upper limit estimate of the pK of the shift of the half-activation voltage was 6.1. The relative reduction in the maximal whole-cell conductance did not change with higher [Na⁺] _o . The conductance of single fenvalerate-modified Na⁺ channels was reduced by extracellular protons. Although the single-channel conductance increased with higher [Na⁺] _o , the maximal conductances at pH 7.6, 7.0 and 6.0 did not converge at [Na⁺] _o up to 280 mm, inconsistent with a simple electrostatic effect. A model incorporating both Na⁺ and H⁺ binding in the pore and cation binding to a Gouy-Chapman surface charge provided a robust fit to the single-channel conductance data with an estimated surface charge density of 1e⁻/439?². Neither surface charge nor proton block alone suffices to explain the effects of extracellular acidosis on Na⁺ channel permeation; both effects play major roles in mediating the response to extracellular pH. Received: 14 May 1996/Revised: 19 September 1996     

Characteristics of Single, Large-Conductance Calcium-Dependent Potassium Channels (BKCa) from Smooth Muscle Cells Isolated from the Rabbit Mesenteric Artery

Smooth muscle cells isolated from the secondary and tertiary branches of the rabbit mesenteric artery contain large Ca²⁺-dependent channels. In excised patches with symmetrical (140 mm) K⁺ solutions, these channels had an average slope conductance of 235 ± 3 pS, and reversed in direction at −6.1 ± 0.4 mV. The channel showed K⁺ selectivity and its open probability (P _o ) was voltage-dependent. Iberiotoxin (50 nm) reversibly decreased P _o , whereas tetraethylammonium (TEA, at 1 mm) reduced the unitary current amplitude. Apamin (200 nm) had no effect. The channel displayed sublevels around 1/3 and 1/2 of the mainstate level. The effect of [Ca²⁺] on P _o was studied and data fitted to Boltzmann relationships. In 0.1, 0.3, 1.0 and 10 μm Ca²⁺, V _1/2 was 77.1 ± 5.3 (n= 18), 71.2 ± 4.8 (n= 16), 47.3 ± 10.1 (n= 11) and −14.9 ± 10.1 mV (n= 6), respectively. Values of k obtained in 1 and 10 μm [Ca²⁺] were significantly larger than that observed in 0.1 μm [Ca²⁺]. With 30 μm NS 1619 (a BK_Ca channel activator), V _1/2 values were shifted by 39 mV to the left (hyperpolarizing direction) and k values were not affected. TEA applied intracellularly, reduced the unitary current amplitude with a K _d of 59 mm. In summary, BK_Ca channels show a particularly weak sensitivity to intracellular TEA and they also display large variation in V _1/2 and k. These findings suggest the possibility that different types (isoforms) of BK_Ca channels may exist in this vascular tissue. Received: 22 December 1997/Revised: 27 March 1998     

Modulation of I A Potassium Current in Adrenal Cortical Cells by a Series of Ten Lanthanide Elements

The modulation of I _A K⁺ current by ten trivalent lanthanide (Ln³⁺) cations spanning the series with ionic radii ranging from 0.99 ? to 1.14 ? was characterized by the whole-cell patch clamp technique in bovine adrenal zona fasciculata (AZF) cells. Each of the ten Ln³⁺s reduced I _A amplitude measured at +20 mV in a concentration-dependent manner. Smaller Ln³⁺s were the most potent and half-maximally effective concentrations (EC₅₀s) varied inversely with ionic radius for the larger elements. Estimation of EC₅₀s yielded the following potency sequence: Lu³⁺ (EC₅₀= 3.0 μm) ≈ Yb³⁺ (EC₅₀= 2.7 μm) > Er³⁺ (EC₅₀= 3.7 μm) ≥ Dy³⁺ (EC₅₀= 4.7 μm) > Gd³⁺ (EC₅₀= 6.7 μm) ≈ Sm³⁺ (EC₅₀= 6.9 μm) > Nd³⁺ (EC₅₀= 11.2 μm) > Pr³⁺ (EC₅₀= 22.3 μm) > Ce³⁺ (EC₅₀= 28.0 μm) > La³⁺ (EC₅₀= 33.7 μm). Ln³⁺s altered selected voltage-dependent gating and kinetic parameters of I _A with a potency and order of effectiveness that paralleled the reduction of I _A amplitude. Ln³⁺s markedly slowed activation kinetics and shifted the voltage-dependence of I _A gating such that activation and steady-state inactivation occurred at more depolarized potentials. In contrast, Ln³⁺s did not measurably alter inactivation or deactivation kinetics and only slightly slowed kinetics of inactivated channels returning to the closed state. Replacement of external Ca²⁺ with Mg²⁺ had no effect on the concentration-dependent inhibition of I _A by Ln³⁺s. In contrast to their action on I _A K⁺ current, Ln³⁺s inhibited T-type Ca²⁺ currents in AZF cells without slowing activation kinetics. These results indicate that Ln³⁺ modulate I _A K⁺ channels through binding to a site on I _A channels located within the electric field but which is not specific for Ca²⁺. They are consistent with a model where Ln³⁺ binding to negative charges on the gating apparatus alters the voltage-dependence and kinetics of channel opening. Ln³⁺s modulate transient K⁺ and Ca²⁺ currents by two fundamentally different mechanisms. Received: 21 January 1997/Revised: 3 April 1998     

Large-conductance Calcium-activated Potassium Channels of Cultured Rat Melanotrophs

A large conductance, Ca²⁺-activated K⁺ channel of the BK type was examined in cultured pituitary melanotrophs obtained from adult male rats. In cell-attached recordings the slope conductance for the BK channel was ≈190 pS and the probability (P _o ) of finding the channel in the open state at the resting membrane potential was low (<<0.1). Channels in inside-out patches and in symmetrical 150 mm K⁺ had a conductance of ≈260 pS. The lower conductance in the cell-attached recordings is provisionally attributed to an intracellular K⁺ concentration of ≈113 mm. The permeability sequence, relative to K⁺, was K⁺ > Rb⁺ (0.87) > NH⁺ ₄ (0.17) > Cs⁺≥ Na⁺ (≤0.02). The slope conductance for Rb⁺ was much less than for K⁺. Neither Na⁺ nor Cs⁺ carried measurable currents and 150 mm internal Cs⁺ caused a flickery block of the channel. Internal tetraethylammonium ions (TEA⁺) produced a fast block for which the dissociation constant at 0 mV (K _D (0 mV)) was 50 mm. The K _D (0 mV) for external TEA⁺ was much lower, 0.25 mm, and the blocking reaction was slower as evidenced by flickery open channel currents. With both internal and external TEA⁺ the blocking reaction was bimolecular and weakly voltage dependent. External charybdotoxin (40 nm) caused a large and reversible decrease of P _o . The P _o was increased by depolarization and/or by increasing the concentration of internal Ca²⁺. In 0.1 μm Ca²⁺ the half-maximal P _o occurred at ≈100 mV; increasing Ca²⁺ to 1 μm shifted the voltage for the half-maximal P _o to −75 mV. The Ca²⁺ dependence of the gating was approximated by a fourth power relationship suggesting the presence of four Ca²⁺ binding sites on the BK channel. Received: 23 October/Revised: 15 December 1995     

Glycosylation Influences Gating and pH Sensitivity of I sK

The KvLQT1 and minK subunits that coassemble to form I _sK channels, contain potential N-glycosylation sites. To examine the role of glycosylation in channel function, a Chinese hamster ovary cell line deficient in glycosylation (Lec-1) and its parental cell line (Pro-5) were transiently transfected with human KvLQT1 (hKvLQT1) cDNA, alone and in combination with the rat (rminK) or human minK (hminK) cDNA. Functional KvLQT1 and I _sK currents were expressed in both cell lines, although amplitudes were larger in Pro-5 than Lec-1 cells transfected with hKvLQT1 and hKvLQT1/hminK. For I _sK , but not KvLQT1, the voltage-dependence of activation was shifted to more positive voltages and the activation kinetics were slower in the Lec-1 compared to the Pro-5 cells. The effect of extracellular acidification on recombinant KvLQT1 and I _sK currents was investigated in Pro-5 and Lec-1 cells. Changing external pH (pH _o ) from 7.4 to 6.0 significantly decreased the amplitude and increased the half-activation voltage (V _1/2) of KvLQT1 currents in Pro-5 and Lec-1 cells. In Pro-5 cells, decreasing pH _o reduced I _sK amplitude without increasing V _1/2, whether rminK or hminK was coexpressed with hKvLQT. In contrast, changing pH _o from 7.4 to 6.0 did not significantly change I _sK amplitude in Lec-1 cells. Thus, oligosaccharides attached to the minK subunit affect not only the gating properties, but also the pH sensitivity of I _sK . Received: 12 November 1999/Revised: 31 May 2000     

Characterization of the High-Affinity Verapamil Binding Site in a Plant Plasma Membrane Ca2+-selective Channel

Despite biochemical evidence for the existence of high-affinity phenylalkylamine receptors in higher plants, their effects on channel activity have only been demonstrated at relatively high concentrations. We have performed a quantitative single-channel analysis of the changes induced by extracellular verapamil in the rca channel [a wheat root plasma membrane Ca²⁺-selective channel (Pi?eros & Tester, 1995. Planta 195:478–488)]. Concentrations as low as 0.5 μm verapamil induced a blockade of the inward current, with no evident reduction of the single-channel current amplitude. Blockade by verapamil was concentration and voltage dependent. Preliminary analysis suggested the blockade was due to a reduction in the maximum open state probability rather than a change in V_0.5. Further analysis of the association and dissociation rate constants revealed a binding site located 56 to 59% down the voltage drop from the extracellular face of the channel, with a K _d (0) of 24 to 26 μm. This results in a K _d at −100 mV of 2 μm. Methoxyverapamil had qualitatively the same effects. This intra-pore binding site can be accessed directly from the extracellular side of the rca channel, but apparently not from the cytosolic side. Received: 15 August 1996/Revised: 23 December 1996     

A 59 Amino Acid Insertion Increases Ca2+ Sensitivity of rbslo1, a Ca2+-Activated K+ Channel in Renal Epithelia

We previously cloned a MaxiK channel α-subunit isoform, rbslo1, from rabbit kidney with an amino acid sequence highly homologous to mslo but with a 59 amino acid insertion between S8 and S9 (Morita et al., 1997. Am. J. Physiol. 273:F615–F624). rbslo1 activation properties differed substantially from mslo with much greater Ca²⁺ sensitivity, half-activation potential of −49 mV in 1 μm Ca²⁺. We now report single-channel analysis of rbslo1 and delA, a construct produced by removal of the 59 amino acid insertion at site A. delA is identical to mslo from upstream of S1 to downstream of S10 with the exception of 8 amino acids. Slope of the steady-state Boltzmann voltage activation curve was 8.1 mV per e-fold change in probability of opening for both rbslo1 and delA. The apparent [Ca²⁺] _i properties in delA were more like mslo but the voltage-activation properties remained distinctly rbslo1. Ca²⁺ affinity decreased and transmembrane voltage effects on apparent Ca²⁺ affinity increased in delA. The differences between rbslo1 and other cloned channels appear to be localized at insertion site A with both the insertion sequence and amino acid substitutions near site A being important. The steeper activation slope makes the channel more responsive to small changes in transmembrane voltage while the insertion sequence makes the channel functional at physiological low levels of [Ca²⁺] _i . Received: 23 August 1999     

Charged Residues in Surface-located Loops Influence Voltage Gating of Porin from Haemophilus influenzae Type b

Porin of Haemophilus influenzae type b (341 amino acids; M _r 37782) determines the permeability of the outer membrane to low molecular mass compounds. Purified Hib porin was subjected to chemical modification of lysine residues by succinic anhydride. Electrospray ionization mass spectrometry identified up to 12 modifications per porin molecule. Tryptic digestion of modified Hib porin followed by reverse phase chromatography and matrix assisted laser desorption ionization time-of-flight mass spectrometry mapped the succinylation sites. Most modified lysines are positioned in surface-located loops, numbers 1 and 4 to 7. Succinylated porin was reconstituted into planar lipid bilayers, and biophysical properties were analyzed and compared to Hib porin: there was an increased average single channel conductance compared to Hib porin (1.24+/−0.41 vs. 0.85+/−0.40 nanosiemens). The voltage-gating activity of succinylated porin differed considerably from that of Hib porin. The threshold voltage for gating was decreased from 75 to 40 mV. At 80 mV, steady-state conductance for succinylated porin was 50–55% of the instantaneous conductance. Hib porin at 80 mV showed a decrease to 89–91% of the instantaneous current levels. We propose that surface-located lysine residues are determinants of voltage gating for porin of Haemophilus influenzae type b. Received: 11 August 2000/Revised: 8 September 2000     

Expression of Cyclic Nucleotide-Gated Cation Channels in Airway Epithelial Cells

Using the whole-cell patch-clamp technique, the selectivity and pharmacology of 8-Br-cGMP-stimulated currents in the human alveolar cell line A549 was compared to 8-Br-cGMP-stimulated currents in HK293 cells transfected with hαCNC1. Whole cell currents stimulated by 8-Br-cGMP in HK293 cells transfected with hαCNC1 or A549 cells are carried by inward sodium and outward potassium with nearly the same selectivity. The whole-cell inward currents that are stimulated by 8-Br-cGMP in HK293 cells transfected with hαCNC1 are inhibited by l-cis-diltiazem with an IC₅₀ of 154 μm, by 2′,4′-dichlorobenzamil with an IC₅₀ of 50 μm and by amiloride with an IC₅₀ of 133 μm. The whole-cell inward currents in A549 cells that are stimulated by 8-Br-cGMP, are inhibited by l-cis-diltiazem with an IC₅₀ of 87 μm, by 2′4′-dichlorobenzamil with an IC₅₀ of 38 μm and by amiloride with an IC₅₀ of 32 μm suggesting that these airway cells contain cyclic nucleotide-gated cation channels. RT-PCR data suggest that mRNA of both αCNC1 and βCNC subunits are present in A549 cells and the presence of the βCNC subunit, may as previously reported, increase the affinity of these channel blockers compared to the hαCNC1 subunit alone. The mRNA of two other isoforms of this channel, CNC2 and CNC3, are also expressed in the A549 cell line. This study documents the IC₅₀ of externally applied channel blockers that can be used for in vitro or in vivo experiments to document sodium absorption via cyclic nucleotide-gated cation channels in airway cells. Received: 24 February/Revised: 28 May 1999