Sodium Blocking Induced by a Point Mutation at the C-Terminal End of the Pore Helix of the KAT1 Channel

A plant hyperpolarization-activating K⁺ channel, KAT1, is highly selective for K⁺ over Na⁺ and is little affected by external Na⁺, which is crucial to take up K⁺ effectively in a Na⁺-containing environment. It has been shown that a mutation at the location (Thr256) preceding the selectivity signature sequence dramatically enhanced the sensitivity of the KAT1 channel to external Na⁺. We report here electrophysiological experiments for the mechanism of action of external Na⁺ on KAT1 channels. The Thr256 residue was substituted with either glutamine (Q) or glutamate (E). The wild-type channel was insensitive to external Na⁺. However, the activity of both mutant channels was significantly depressed by Na⁺ with apparent dissociation constants of 6.7 mm and 11.3 mm for T256Q and T256E, respectively. The instantaneous current-voltage relationships revealed distinct blocking mechanisms for these mutants. For T256Q a typical voltage-dependent fast blocking was shown. On the other hand, the blocking for the T256E mutant was voltage-independent at low Na⁺ concentrations and became voltage-dependent at higher concentrations. At extreme hyperpolarization the blocking was relieved significantly. These data strongly suggest that the mutation at the end of the pore helix rearranged the selectivity filter and allows Na⁺ to penetrate into the pore. Received: 16 October 2000/Revised: 20 February 2001     

Increased Resistance to Extracellular Cation Block by Mutation of the Pore Domain of the Arabidopsis Inward-rectifying K+ Channel KAT1

Inward-rectifying potassium channels in plant cells provide important mechanisms for low-affinity K⁺ uptake and membrane potential control in specific cell types, including guard cells, pulvinus cells, aleurone cells and root hair cells. K⁺ channel blockers are potent tools for studying the physiological functions and structural properties of K⁺ channels. In the present study the structural and biophysical mechanisms of Cs⁺ and TEA⁺ block of a cloned Arabidopsis inward-rectifying K⁺ channel (KAT1) were analyzed. Effects of the channel blockers Cs⁺ and TEA⁺ were characterized both extracellularly and intracellularly. Both external Cs⁺ and TEA⁺ block KAT1 currents. A mutant of KAT1 (``m2KAT1'; H267T, E269V) was produced by site-directed mutagenesis of two amino acid residues in the C-terminal portion of the putative pore (P) domain. This mutant channel was blocked less by external Cs⁺ and TEA⁺ than the wild-type K⁺ channel. Internal TEA⁺ and Cs⁺ did not significantly block either m2KAT1 or KAT1 channels. Other properties, such as cation selectivity, voltage-dependence and proton activation did not show large changes between m2KAT1 and KAT1, demonstrating the specificity of the introduced mutations. These data suggest that the amino acid positions mutated in the inward-rectifying K⁺ channel, KAT1, are accessible to external blockers and may be located on the external side of the membrane, as has been suggested for outward-rectifying K⁺ channels. Received: 31 July 1995/Revised: 5 January 1996     

The Impermeant Ion Methylammonium Blocks K+ and NH+ 4 Currents through KAT1 Channel Differently: Evidence for Ion Interaction in Channel Permeation

The permeation properties of KAT1, an inward rectifying potassium channel from plant cells, were investigated with different ions in the external medium. With either K⁺, NH⁺ ₄ or methylammonium (MA) in the external solution, the channel, expressed in Xenopus oocytes, appeared permeable to K⁺ and, to a lesser extent, to NH⁺ ₄ but not to the slightly bigger, methylated analogue of NH⁺ ₄, MA. Substituting NH⁺ ₄ for K⁺ shifted the voltage dependency of channel activation further negative and hastened activation kinetics. This suggests that channel operation depends on the transported substrate. In mixed solution (50 mm K⁺, 50 mm MA) MA inhibited K⁺ current in a voltage-independent manner. The maximum block did not exceed 50% of the K⁺ current. In contrast, when NH⁺ ₄ was the permeant ion (50 mm NH⁺ ₄, 50 mm MA) MA caused a voltage-dependent, slowly developing open channel block, achieving complete inhibition at very negative voltages. The latter block could be partially overcome by the addition of K⁺ in the external solution. The data support a model in which ions, after entering the channel pore, compete with different affinities for binding sites on their permeation pathway. Received: 6 October 1997/Revised: 28 January 1998     

Potassium Uptake Through the TOK1 K+ Channel in the Budding Yeast

The current through TOK1 (YKC1), the outward-rectifying K⁺ channel in Saccharomyces cerevisiae, was amplified by expressing TOK1 from a plasmid driven by a strong constitutive promoter. TOK1 so hyper-expressed could overcome the K⁺ auxotrophy of a mutant missing the two K⁺ transporters, TRK1 and TRK2. This trk1Δtrk2Δ double mutant hyperexpressing the TOK1 transgene had a higher internal K⁺ content than one expressing the empty plasmid. We examined protoplasts of these TOK1-hyperexpressing cells under a patch clamp. Besides the expected K⁺ outward current activating at membrane potential (V _m ) above the K⁺ equilibrium potential (E _K+ ), a small inward current was consistently observed when the V _m was slightly below E _K+ . The inward and the outward currents are similar in their activation rates, deactivation rates, ion specificities and Ba²⁺ inhibition, indicating that they flow through the same channel. Thus, the yeast outwardly rectifying K⁺ channel can take up K⁺ into yeast cells, at least under certain conditions. Received: 1 October 1998/Revised: 9 December 1998     

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     

Cysteine Mapping in the Ion Selectivity and Toxin Binding Region of the Cardiac Na+ Channel Pore

Aqueous exposure of critical residues in the selectivity region of voltage gated Na⁺ channels was studied by cysteine-scanning mutagenesis at three positions in each of the SS2 segments of domains III (D3) and IV (D4) of the human heart Na⁺ channel. Ionic currents were modified by charged cysteine-specific methanethiosulfonate (MTS) reagents, (2-aminoethyl)methanethiosulfonate (MTSEA⁺) and (2-sulfonatoethyl)methanethiosulfonate (MTSES⁻) in all six of the Cys-substituted channels, including Trp → Cys substitutions at homologous positions in D3 and D4 that were predicted in secondary structure models to have buried side chains. Furthermore, in the absence of MTS modification, each of the Cys mutants showed a reduction in tetrodotoxin (TTX) block by a factor >10². Cysteine substitution without MTS modification abolished the alkali metal ion selectivity in K1418C (D3), but not in A1720C (the corresponding position in D4) suggesting that the lysine but not the alanine side chains contribute to selectivity even though both were exposed. Neither position responded to MTSES⁻ suggesting that these residues occupy either a size- or charge-restricted region of the pore. By contrast, MTSES⁻ markedly increased, and MTSEA⁺ markedly decreased conductance of D1713C (D4) suggesting that the acidic side chain of Asp¹⁷¹³ acts electrostatically in an unrestricted region. These results suggest that Lys¹⁴¹⁸ lies in a restricted region favorable to cations, whereas Asp¹⁷¹³ is at a more peripheral location in the Na⁺ channel pore. Received: 8 May 1996/Revised: 15 August 1996     

Evidence for Negative Charge in the Conduction Pathway of the Cardiac Ryanodine Receptor Channel Provided by the Interaction of K+ Channel N-type Inactivation Peptides

We have investigated the interaction of two peptides (ShB — net charge +3 and ShB:E12KD13K — net charge +7) derived from the NH₂-terminal domain of the Shaker K⁺ channel with purified, ryanodine-modified, cardiac Ca²⁺-release channels (RyR). Both peptides produced well resolved blocking events from the cytosolic face of the channel. At a holding potential of +60 mV the relationship between the probability of block and peptide concentration was described by a single-site binding scheme with 50% saturation occurring at 5.92 ± 1.06 μm for ShB and 0.59 ± 0.14 nm for ShB:E12KD13K. The association rates of both peptides varied with concentration (4.0 ± 0.4 sec⁻¹μm ⁻¹ for ShB and 2000 ± 200 sec⁻¹μm ⁻¹ for ShB:E12KD13K); dissociation rates were independent of concentration. The interaction of both peptides was influenced by applied potential with the bulk of the voltage-dependence residing in K_off. The effectiveness of the inactivation peptides as blockers of RyR is enhanced by an increase in net positive charge. As is the case with inactivation and block of K⁺ channels, this is mediated by a large increase in K_on. These observations are consistent with the proposal that the conduction pathway of RyR contains negatively charged sites which will contribute to the ion handling properties of this channel. Received: 15 December 1997/Revised: 13 March 1998     

Characterization of a Chloride-Selective Channel from Rough Endoplasmic Reticulum Membranes of Rat Hepatocytes: Evidence for a Block by Phosphate

We have characterized the conduction and blocking properties of a chloride channel from rough endoplasmic reticulum membranes of rat hepatocytes after incorporation into a planar lipid bilayer. Our experiments revealed the existence of a channel with a mean conductance of 164 ± 5 pS in symmetrical 200 mm KCl solutions. We determined that the channel was ten times more permeable for Cl⁻ than for K⁺, calculated from the reversal potential using the Goldman-Hodgkin-Katz equation. The channel was voltage dependent, with an open probability value ranging from 0.9 at −20 mV to 0.4 at +60 mV. In addition to its fully open state, the channel could also enter a flickering state, which appeared to involve rapid transitions to zero current level. Our results showed a decrease of the channel mean open time combined with an increase of the channel mean closed time at positive potentials. An analysis of the dwell time distributions for the open and closed intervals led to the conclusion that the observed fluctuation pattern was compatible with a kinetic scheme containing a single open state and a minimum of three closed states. The permeability sequence for test halides determined from reversal potentials was Br⁻ > Cl⁻ > I⁻≈ F⁻. The voltage dependence of the open probability was modified by the presence of halides in trans with a sequence reflecting the permeability sequence, suggesting that permeant anions such as Br⁻ and Cl⁻ have access to an internal site capable of controlling channel gating. Adding NPPB to the cis chamber inhibited the channel activity by increasing fast flickering and generating long silent periods, whereas channel activity was not affected by 50 μm DNDS in trans. The channel was reversibly inhibited by adding phosphate to the trans chamber. The inhibitory effect of phosphate was voltage-dependent and could be reversed by addition of Cl⁻. Our results suggest that channel block involves the interaction of HPO²⁻ ₄ with a site located at 70% of the membrane span. Received: 10 January 1997/Revised: 29 May 1997     

Effects of ATP-sensitive Potassium Channel Regulators on Chloride Channels in the Sarcoplasmic Reticulum Vesicles from Rabbit Skeletal Muscle

The lipid bilayer technique was used to examine the effects of the ATP-sensitive K⁺ channel inhibitor (glibenclamide) and openers (diazoxide, minoxidil and cromakalim) and Cl⁻ channel activators (GABA and diazepam) on two types of chloride channels in the sarcoplasmic reticulum (SR) from rabbit skeletal muscle. Neither diazepam at 100 μm nor GABA at 150 μm had any significant effect on the conductance and kinetics of the 75 pS small chloride (SCl) channel. Unlike the 150 pS channel, the SCl channel is sensitive to cytoplasmic glibenclamide with K _i ∼ 30 μm. Glibenclamide induced reversible decline in the values of current (maximal current amplitude, I _max and average mean current, I′) and kinetic parameters (frequency of opening F _o , probability of the channel being open P _o and mean open time, T _o , of the SCl channel. Glibenclamide increased mean closed time, T _c , and was a more potent blocker from the cytoplasmic side (cis) than from the luminal side (trans) of the channel. Diazoxide increased I′, P _o , and T _o in the absence of ATP and Mg²⁺ but it had no effect on I _max and also failed to activate or remove the glibenclamide- and ATP-induced inhibition of the SCl channel. Minoxidil induced a transient increase in I′ followed by an inhibition of I _max, whereas cromakalim reduced P _o and I′ by increasing channel transitions to the closed state and reducing T _o without affecting I _max. The presence of diazoxide, minoxidil or cromakalim on the cytoplasmic side of the channel did not prevent [ATP] _cis or [glibenclamide] _cis from blocking the channel. The data suggest that the action(s) of these drugs are not due to their effects on the phosphorylation of the channel protein. The glibenclamide- and cromakalim-induced effects on the SCl channel are mediated via a ``flicker' type block mechanism. Modulation of the SCl channel by [diazoxide] _cis and [glibenclamide] _cis highlights the therapeutic potential of these drugs in regulating the Ca²⁺-counter current through this channel. Received: 2 September 1997/Revised: 20 March 1998     

Temperature-Dependent Expression of a Squid Kv1 Channel in Sf9 Cells and Functional Comparison with the Native Delayed Rectifier

SqKv1A is a cDNA that encodes a Kv1 (Shaker-type) α-subunit expressed only in the giant axon and the parental giant fiber lobe (GFL) neurons of the squid stellate ganglion. We incorporated SqKv1A into a recombinant baculovirus for expression in the insect Sf9 cell line. Whole-cell patch-clamp recordings reveal that very few cells display functional potassium current (I _K) if cultured at the standard postinfection temperature of 27°C. At 18°C, less SqKv1A protein is produced than at 27°C, but cells with I _K currents are much more numerous and can survive for at least 20 days postinfection (vs. ∼5 days at 27°C). Activation and deactivation kinetics of SqKv1A in Sf9 cells are slower (∼3- and 10-fold, respectively) than those of native channels in GFL neurons, but have similar voltage dependencies. The two cell types show only subtle differences in steady-state voltage-dependence of conductance and inactivation. Rates of I _K inactivation in 20 mm external K are identical in the two cell types, but the sensitivity of inactivation to external tetraethylammonium (TEA) and K ions differ: inactivation of SqKv1A in Sf9 cells is slowed by external TEA and K ions, whereas inactivation of GFL I _K is largely insensitive. Functional differences are discussed in terms of factors that may be specific to cell-type, including the presence of presently unidentified Kv1 subunits in GFL neurons that might form heteromultimers with SqKv1A.     

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     

Effects of Cationic Substitutions on Delayed Rectifier Current in Type I Vestibular Hair Cells

The resting potassium current (I _KI ) in gerbil dissociated type I vestibular hair cells has been characterized under various ionic conditions in whole cell voltage-clamp. When all K⁺ in the patch electrode solution was replaced with Na⁺, (Na⁺) _in or Cs⁺, (Cs⁺) _in , large inward currents were evoked in response to voltage steps between −90 and −50 mV. Activation of these currents could be described by a Hodgkin-Huxley-type kinetic scheme, the order of best fit increasing with depolarization. Above ∼−40 mV currents became outward and inactivated with a monoexponential time course. Membrane resistance was inversely correlated with external K⁺ concentration. With (Na⁺) _in , currents were eliminated when K⁺ was removed from the external solution or following extracellular perfusion of 4-aminopyridine, indicating that currents flowed through I _KI channels. Also, reduction of K⁺ entry through manipulation of membrane potential reduced the magnitude of the outward current. Under symmetrical Cs⁺, 0 K⁺ conditions I _KI is highly permeable to Cs⁺. However, inward currents were reduced when small amounts of external K⁺ were added. Higher concentrations of K⁺ resulted in larger currents indicating an anomalous mole fraction effect in mixtures of external Cs⁺ and K⁺. Received: 23 June 1999/Revised: 27 September 1999     

Enhanced Closed-state Inactivation in a Mutant Shaker K+ Channel

Many mutations that shift the voltage dependence of activation in Shaker channels cause a parallel shift of inactivation. The I2 mutation (L382I in the Shaker B sequence) is an exception, causing a 45 mV activation shift with only a 9 mV shift of inactivation midpoint relative to the wildtype (WT) channel. We compare the behavior of WT and I2 Shaker 29-4 channels in macropatch recordings from Xenopus oocytes. The behavior of WT channels can be described by both simple and detailed kinetic models which assume that inactivation proceeds only from the open state. The behavior of I2 channels requires that they inactivate from closed states as well, a property characteristic of voltage-gated sodium channels. A detailed ``multiple-state inactivation' model is presented that describes both activation and inactivation of I2 channels. The results are consistent with the view that residue L382 is associated with the receptor for the inactivation particles in Shaker channels. Received: 16 December 1996/Revised: 5 February 1997     

Burst Kinetics of Co-expressed Kir6.2/SUR1 Clones: Comparison of Recombinant with Native ATP-sensitive K+ Channel Behavior

Co-expression of clones encoding Kir6.2, a K⁺ inward rectifier, and SUR1, a sulfonylurea receptor, reconstitutes elementary features of ATP-sensitive K⁺ (K_ATP) channels. However, the precise kinetic properties of Kir6.2/SUR1 clones remain unknown. Herein, intraburst kinetics of Kir6.2/SUR1 channel activity, heterologously co-expressed in COS cells, displayed mean closed times from 0.7 ± 0.1 to 0.4 ± 0.03 msec, and from 0.4 ± 0.1 to 2.0 ± 0.2 msec, and mean open times from 1.9 ± 0.4 to 4.5 ± 0.8 msec, and from 12.1 ± 2.4 to 5.0 ± 0.2 msec between −100 and −20 mV, and +20 to +80 mV, respectively. Burst duration for Kir6.2/SUR1 activity was 17.9 ± 1.8 msec with 5.6 ± 1.5 closings per burst. Burst kinetics of the Kir6.2/SUR1 activity could be fitted by a four-state kinetic model defining transitions between one open and three closed states with forward and backward rate constants of 1905 ± 77 and 322 ± 27 sec⁻¹ for intraburst, 61.8 ± 6.6 and 23.9 ± 5.8 sec⁻¹ for interburst, 12.4 ± 6.0 and 13.6 ± 2.9 sec⁻¹ for intercluster events, respectively. Intraburst kinetic properties of Kir6.2/SUR1 clones were essentially indistinguishable from pancreatic or cardiac K_ATP channel phenotypes, indicating that intraburst kinetics per se were insufficient to classify recombinant Kir6.2/SUR1 amongst native K_ATP channels. Yet, burst kinetic behavior of Kir6.2/SUR1 although similar to pancreatic, was different from that of cardiac K_ATP channels. Thus, expression of Kir6.2/SUR1 proteins away from the pancreatic micro-environment, confers the burst kinetic identity of pancreatic, but not cardiac K_ATP channels. This study reports the kinetic properties of Kir6.2/SUR1 clones which could serve in the further characterization of novel K_ATP channel clones. Received: 12 March 1997/Revised: 5 May 1997     

Anion-selectivity of the Swelling-activated Osmolyte Channel in Eel Erythrocytes

Osmotic swelling of fish erythrocytes activates a broad-specificity permeation pathway that mediates the volume-regulatory efflux of taurine and other intracellular osmolytes. This pathway is blocked by inhibitors of the erythrocyte band 3 anion exchanger, raising the possibility that band 3 is involved in the volume-regulatory response. In this study of eel erythrocytes, a quantitative comparison of the pharmacology of swelling-activated taurine transport with that of band 3-mediated SO²⁻ ₄ transport showed there to be significant differences between them. N-ethylmaleimide and quinine were effective inhibitors of swelling-activated taurine transport but caused little, if any, inhibition of band 3. Conversely, DIDS was a more potent inhibitor of band 3-mediated SO²⁻ ₄ flux than of swelling-activated taurine transport. In cells in isotonic medium, pretreated then co-incubated with 0.1 mm DIDS, the band 3-mediated transport of SO²⁻ ₄ and Cl⁻ was reduced to a low level. Exposure of these cells to a hypotonic medium containing 0.1 mm DIDS was followed by the activation of a Cl⁻ permeation pathway showing the same inhibitor sensitivity as swelling-activated taurine transport. The data are consistent with swelling-activated transport of taurine and Cl⁻ being via a common pathway. A comparison of the swelling-activated transport rates for taurine and Cl⁻ with those for several other solutes was consistent with the hypothesis that this pathway is an anion-selective channel, similar to those that mediate the volume-regulatory efflux of Cl⁻ and organic osmolytes from mammalian cells. Received: 7 July 1995/Revised: 2 September 1995     

The Actions of Calmodulin Antagonists W-7 and TFP and of Calcium on the Gating Kinetics of the Calcium-activated Large Conductance Potassium Channel of the Chara Protoplasmic Drop: A Substate-sensitive Analysis

The effects of the calmodulin antagonists W-7 and trifluoperazine have been measured on the Ca²⁺-activated potassium channel in the membrane surrounding protoplasmic drops expressed from internodal cells of charophyte plants. The large-conductance (170 pS), voltage- and Ca²⁺-dependent gating, and prominent conductance substrate of this channel shows a strong kinetic resemblance to those of the Maxi-K channel from animal cells. This is the first study of the action of calmodulin antagonists which measures their effects on the most populated substates as well as the closed and main open states of Maxi-K channels. The substate analysis provides new evidence for different modes of action of- and different bindings sites for these calmodulin antagonists. Neither antagonist produces the simple closure of the channel reported previously as its effect on the Maxi-K channel, though both do induce flicker-block, reducing the mean current to near zero at high concentrations following an inverted Michaelis-Menten curve. W-7 reduces residence time in the fully open state, thus raising, in the same proportions, the probabilities of finding the channel in the closed state or a pre-existing substate. Its binding to the channel is voltage- and calcium-dependent. In contrast, trifluoperazine reduces residence in the open state and promotes an apparently new substate which overlaps the closed state at −50 mV but is distinguishable from it at voltages more negative than −100 mV. This substate may represent times that trifluoperazine is bound to the channel. Both antagonists have effects clearly distinguishable from that of withdrawing calcium from the channel, which does not affect open state residence time but increases closed state residence time. Thus neither antagonist reverses the activating effect of Ca²⁻. This is good kinetic evidence against the view that the channel is activated by Ca²⁺-calmodulin and that the effect of a calmodulin antagonist is to reverse this process by making Ca²⁻-calmodulin less available. Received: 26 August 1996/Revised: 7 October 1996     

The Antifungal Imidazole Clotrimazole and its Major In Vivo Metabolite are Potent Blockers of the Calcium-Activated Potassium Channel in Murine Erythroleukemia Cells

Clotrimazole (CLT), a member of the antifungal imidazole family of compounds, has been found to inhibit both calcium (Ca²⁺)-activated ⁸⁶Rb and potassium (K) fluxes of human red cells and to inhibit red cell binding of ¹²⁵I-charybdotoxin (ChTX) [11]. We have now used patch-clamp techniques to demonstrate reversible inhibition of whole cell K_Ca2+ currents in murine erythroleukemia (MEL) cells by submicromolar concentrations of CLT. Inhibition was equivalent whether currents were elicited by bath application of the Ca²⁺ ionophore A23187 or by dialyzing cells with a pipette solution containing micromolar concentrations of free Ca²⁺. The extent of inhibition of whole cell MEL K_Ca2+ currents was voltage-dependent, decreasing with increasing test potential. We also determined the single channel basis of the CLT inhibition in MEL cells by demonstrating the inhibition of a calcium-activated, ChTX-sensitive K channel by CLT in outside-out patches. The channel was also blocked by the des-imidazolyl metabolite of CLT, 2-chlorophenyl-bisphenyl-methanol (MET II) [15], thus demonstrating that the imidazole ring is not required for the inhibitory action of CLT. Single K_Ca2+ channels were also evident in inside-out patches of MEL cells. Block of K current by CLT was not unique to MEL cells. CLT also inhibited a component of the whole cell K current in PC12 cells. Channel specificity of block by CLT was determined by examining its effects on other types of voltage-sensitive currents. CLT block showed the following rank order of potency: K currents in PC12 cells > Ca²⁺ currents in PC12 cells ≫ Na currents in sympathetic neurons. These results demonstrate that direct inhibition of single K_Ca2+ by CLT can be dissociated from inhibition of cytochrome P-450 in MEL cells. Received: 10 September 1996/Revised: 12 December 1996     

SITS Blockade Induces Multiple Subconductance States in a Large Conductance Chloride Channel

The effect of the chloride channel blocker 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS) on the gating and amplitude of an endothelial chloride channel was explored using the outside-out configuration of the patch-clamp technique. Under control conditions the channel displayed two main gating modes: shut and fully open. Transitions to equally spaced subconductance states were rarely observed (less than 10 events/minute). At low concentrations (<45 μm), SITS increased the number of transitions to the three subconductance states in a concentration-dependent manner, while reducing the number of transitions to the fully open state. This effect was maintained after removing SITS from the bath solution, suggesting that the modifications in the channel induced by SITS were irreversible. All four conducting states had similar current-voltage relationships. At higher concentrations (>45 μm), SITS reduced the amplitude of all conducting states (three subconductances and fully open). This effect was fully reversible upon SITS removal from the bath solution. A half-inhibitory concentration (IC₅₀) of 55.6 ± 2.7 μm (+60 mV) and 66.7 ± 2.2 (−60 mV) was obtained from the fitting to a Langmuir function. All these results are compatible with the existence of two SITS binding sites in the chloride channel: one of high affinity responsible for the increment in the number of transitions to subconductance states, and one low affinity binding site involved in the reduction of the amplitude of all conducting states. Received: 29 October 1998/Revised: 16 February 1999