Potassium channel openers act through an activation of ATP-sensitive K+ channels in guinea-pig cardiac myocytes

In a previous article (Escande et al. 1988a), we have shown that cromakalim (BRL 34915), a potassium channel opener (PCO), is a potent activator of ATP-sensitive K⁺ channels in cardiac cells. In the present article, the influence on K⁺ channels of two other potassium channel openers chemically unrelated to cromakalim, RP 49356 and pinacidil, has been investigated in patch-clamped isolated cardiac myocytes. In the whole-cell configuration, K⁺ currents were recorded in the presence of 50 M TTX and 3 M nitrendipine or 3 mM cobalt. Like cromakalim, RP 49356 or pinacidil activated a time-independent outward current at 33–35°C but not at 19–21°C, which showed little voltage-dependency in the potential range –60 to +60 mV. Its amplitude was a function of the agonist concentration, e.g. it was 2.1±0.4 nA at +60 mV with 30 M RP 49356 and 4.3±0.8 nA with 300 M. In control conditions, glibenclamide, a blocker of K⁺-ATP channels in pancreatic and heart cells, affected neither the inward rectifier,i _K1, nor the delayed K⁺ current,i _K. At 3 M, glibenclamide fully prevented the effects of 300 M RP 49356 or pinacidil. At lower concentrations, glibenclamide partially counteracted the activation by PCOs of a K⁺ current. In the cell-attached contiguration, externally applied RP 49356 or pinacidil caused opening of large channels which reversed around 0 mV in a high K⁺ external medium. In inside-out patches, both RP 49356 or pinacidil activated K⁺-ATP channels by increasing the time period for which the channels remained in the open state. It is concluded that, like cromakalim, RP 49356 and pinacidil are potent activators of K⁺-ATP channels in cardiac myocytes.     

ATP-sensitive K+ channels in rat ventricular myocytes are blocked and inactivated by internal divalent cations

K⁺ currents were recorded from ATP-sensitive channels in inside-out patches from isolated rat ventricular myocytes. In the absence of internal divalent cations the current voltage relationship could be described by constant-field assumptions with a permeability of 1.25×10^–13 cm²/s; outward currents saturated under a high driving force for K⁺ movement. Internal 0.1–5.0 mM Mg²⁺, 0.1 M Ca²⁺ and 10 mM Na⁺ each depressed the flux of K⁺ ions moving outwards through open channels. Internal 0.1–5.0 mM Mg²⁺, 0.1–1.0 M Ca²⁺ and 1–10 M Ba²⁺ and Sr²⁺ blocked K⁺ channel activity in a dose-and voltage-dependent manner. Run-down channels could be reactivated by Mg-ATP, but not by AMP-PNP, ATPS or Mg-free ATP which suggested that phosphorylation of the channels was involved in their activity. Ca²⁺ (>=1 M) and Sr²⁺ (1 mM) markedly inactivated K⁺ ATP channels, millimolar Ba²⁺ or Mg²⁺ were less effective. This suggested that the run down of the channels was a Ca²⁺-dependent dephosphorylation of the K⁺ channel protein.     

Positive cooperativity in activation of the cardiac muscarinic K+ channel by intracellular GTP

Concentration-dependent effects of intracellular GTP on activation of the muscarinic K⁺ channel were examined in inside-out patches of cardiac atrial myocytes. The pipette solution contained 0.1 M ACh. GTP (0.01–30 M) and 0.5 mM MgCl₂ were applied to the inside side of the patch membrane. K⁺ channels were activated with GTP concentration above 0.1 M. Channel activation reached a maximal value with 1–3 M GTP. It decreased at GTP concentrations larger than 3 M, probably due to desensitization. The dependence of the open probability of the channel on intracellular GTP showed a sigmoidal relationship with a Hill coefficient of around 3. A positive cooperative effect of intracellular GTP on the K⁺ channel may play an important role in amplifying the signal from the membrane receptor to the K⁺ channel.     

Pinacidil activates the ATP-sensitive K+ channel in inside-out and cell-attached patch membranes of guinea-pig ventricular myocytes

Patch-clamp techniques were used to study the effects of pinacidil on the adenosine-5-triphosphate (ATP)-sensitive K⁺ channel current in guinea-pig ventricular myocytes. In inside-out patches, the ATP-sensitive K⁺ channel current could be recorded at an internal ATP concentration of 0.5 mM or less and almost complete inhibition was achieved by raising the concentration to 2 mM. Application of pinacidil (10–30 M) in the presence of 2 mM ATP restored the current, whereas 5 mM ATP antagonized the effect of pinacidil. The conductance of the channel at symmetrical K⁺ concentrations of 140 mM was 75 pS with a slight inward rectification at voltages positive to +40 mV. There was no significant change in the conductance after application of pinacidil. In 0.5 mM ATP, at –80 mV, both the distributions of the open time and the life-time of bursts could be fitted by a single exponential. An increase in ATP concentration decreased the mean life-time of bursts, whereas pinacidil increased it with little increase in the mean open time. Closed time distributions of the channel were fitted by at least two exponentials, with a fast and a slow time constant. An increase in ATP concentration markedly increased the slow time constant associated with a decrease in the number of bursts, whereas the effect of pinacidil was opposite to that of increased ATP. These results indicate that pinacidil increases the open-state probability of the ATP-sensitive K⁺ channel. In cell-attached patches, application of pinacidil (100–200 M) to the extracellular solution reversibly induced the channel activity, which showed similar properties to those of the ATP-sensitive K⁺ channel recorded in cell-free patches.     

Interaction between external Na+ and mexiletine on Na+ channel in guinea-pig ventricular myocytes

To assess the modulation of Na⁺ channel block with local anaesthetics by the change of external Na⁺ concentration ([Na⁺]_o), we examined the block by mexiletine at different [Na⁺]_o using the whole-cell and the cell-attached configurations of the patch-clamp technique. Lowering [Na⁺]_o increased the degree of use dependent block of the whole-cell Na⁺ current. The external Na⁺ dependence of the Na⁺ current block was caused by the interaction of mexiletine with the activated Na⁺ channel, but not with the inactivated channel. In single-Na⁺ channel current recordings at a reduced [Na⁺]_o of 70 mM, mexiletine shortened the mean open time of the channels (1.32±0.06 ms in the control vs. 0.86±0.12 ms with the drug, P<0.05) without changes in the unitary current amplitude, whereas the drug did not affect mean open time at a [Na⁺]_o of 140 mM. Moreover, the open time distributions during drug exposure at the reduced [Na⁺]_o were better fitted to a double exponential than to a single exponential in four out of six experiments. These data suggest that mexiletine induces two conductive states: the native open state and a state representing the first step of open channel block. The transition from the former to the latter is dependent on [Na⁺]_o, suggesting an antagonistic interaction of external Na⁺ with mexiletine.     

Functional linkage of the cardiac ATP-sensitive K+ channel to the actin cytoskeleton

The role of the cytoskeleton in the rundown and reactivation of adenosine triphosphate (ATP) sensitive K⁺ channels (KATP channels) was examined by perturbing selectively the intracellular surface of inside-out membrane patches excised from guinea-pig ventricular myocytes. Actin filament-depolymerizing agents (cytochalasins and desoxyribonuclease I) accelerated channel rundown, while actin filament stabilizer (phalloidin) or phosphatidylinositol biphosphate (PIP₂; inhibitor of F-actin-severing proteins) inhibited spontaneous and/or Ca²⁺-induced rundown. When rundown was induced by cytochalasin D or by long exposure to high Ca²⁺, channel activity could not be restored by exposure to MgATP, but application of F-actin with MgATP could reinstitute channel activity. The processes of rundown and reactivation of cardiac K_ATP channels may thus be influenced by the assembly and disassembly of the actin cytoskeletal network, which provides a novel regulatory mechanism of this channel.     

Aromatic aldehydes and aromatic ketones open ATP-sensitive K+ channels in guinea-pig ventricular myocytes

Patch-clamp techniques were used to study the effects of three carbonyl compounds, 3,4-dihydroxy-benzaldehyde, 2,3-dihydroxybenzaldehyde, and 2,4-dihydroxy-acetophenone, on the adenosine-5-triphosphate(ATP)-sensitive K⁺ channel current (I _K.ATP) in guinea-pig ventricular myocytes. 3,4-Dihydroxybenzaldehyde (0.5–1 mM) shortened the action potential duration, and this effect was inhibited by application of a specific blocker of I _K.ATP, glibenclamide. The shortening of the action potential duration was shown to be caused by a time-independent outward current. In the cell-attached patch configuration, all three compounds activated a kind of single-channel current, which showed an inward rectification at positive potentials and which had a linear current/voltage relation at negative potentials, having a conductance of 90 pS. The current reversed at about 0 mV in symmetrical K⁺ concentrations on both sides of the membrane. In excised patches this current was blocked by internal application of ATP. Thus we identified this channel as I _K.ATP. The activation effects of two aromatic aldehydes were stronger than that of the aromatic ketone. The effect of these compounds on I _K.ATP was not reduced by addition of cysteine (10 mM). In inside-out patches, 3,4-dihydroxybenzaldehyde increased the activity of I _K.ATP, which had been blocked by 0.5 mM MgATP in the presence of 0.5 mM ADP, but the activation effect was variable and much weaker than that in the cell-attached configuration, and was completely eliminated in the absence of ADP. These results suggest that these compounds: (a) modulate I _K.ATP perhaps through an intracellular mechanism, (b) bind covalently to proteins to form a Schiff base which may by responsible for the effects, and (c) may require an ADP-dependent process.     

Short-term desensitization of muscarinic K+ channel current in isolated atrial myocytes and possible role of GTP-binding proteins

The short-term desensitization of the acetylcholine (ACh)-induced K⁺ channel current was examined in single atrial cells of guinea-pig heart. The tight-seal whole cell voltage clamp technique was used. The solution in the pipettes contained GTP or guanosine-5-O-(3-thiotriphosphate) (GTP-S, a non-hydrolyzable GTP analogue). In GTP-loaded cells, ACh evoked a specific K⁺ channel current via GTP-binding proteins (G) in a dose-dependent manner. The K⁺ current showed agonist-dependent desensitization similar to those reported in other cardiac tissues (Nilius 1983; Carmeliet and Mubagwa 1986). The cellular response to ACh was also desensitized by activation of P1-purinergic receptors with adenosine (Ado). In GTP-S-loaded cells, the K⁺ current was gradually induced even in the absence of agonists, probably due to direct activation of G proteins by GTP-S. In the early phase of the spontaneous current increase, ACh evoked a large current transiently. As the GTP-S-induced activation of the current progressed, the magnitude of the ACh-evoked current transient became smaller and finally negligible. Similar results were obtained when Ado was used as an agonist instead of ACh to induce the K⁺ current. Therefore, it is indicated that the agonistreceptor interaction may not be essential for the desensitization of ACh-induced K⁺ current in atrial myocytes.     

Sodium current block caused by group IIb cations in calf Purkinje fibres and in guinea-pig ventricular myocytes

The action of group IIb cations [Cadmium (Cd²⁺), Zinc (Zn²⁺), Mercury (Hg²⁺)] on the cardiac fast sodium current (I _Na) was investigated in calf Purkinje fibres and in ventricular cells isolated from guinea-pig hearts. In calf Purkinje fibres, I _Na was depressed by submillimolar concentrations of Zn²⁺ and Hg²⁺. With both cations, the current reduction occurred at all voltages in the range of current activation and the voltage dependence of peak current was unchanged. The degree of peak current inhibition depended on the cation concentration but not on voltage. The position of the inactivation curve on the voltage axis was unaltered at cation concentrations giving substantial current inhibition, and moved to the right only with concentration exceeding 1–1.5 mM. These effects can be interpreted as due to I _Na channel blockade. The action of Zn²⁺ and Hg²⁺ was similar to that described earlier of Cd²⁺ on Purkinje fibres (DiFrancesco et al. 1985b). I _Na was also inhibited by group IIb cations in isolated guinea-pig ventricular cells. Depression of I _Na by Cd²⁺, Zn²⁺ and Hg²⁺ was essentially voltage-independent, in agreement with its being caused by channel block. The dependence of I _Na block by Cd²⁺ upon external Na concentration [Na⁺ ₀] was investigated in ventricular myocytes. The fraction of I _Na block by 0.1 mM CdCl₂ was 0.50 at 140 mM, 0.81 at 70 mM and 0.83 at 35 mM [Na⁺]₀. A similar increase of block efficiency at low [Na⁺ ₀] was observed with 0.05 mM CdCl₂. In both the Purkinje fibre and the ventricular cell, the order of potency of I _Na block by group IIb cations was Hg²⁺> Zn²⁺> Cd²⁺. Manganese (Mn²⁺, 2–5 mM), an ion of group VIIa, also depressed the I _Na in Purkinje fibres and ventricular myocytes. This effect was however due mainly to a positive shift on the voltage dependence of current kinetics rather than to a reduction of the conductance of the channel (G _Na), and can be accounted for by an ion-screening action of Mn²⁺ on the external membrane surface. The block by group IIb cations is a typical property of cardiac Na⁺ channels and characterizes the cardiac as opposed to other types of Na⁺ channel. Offprint request to: D. DiFrancesco     

Role of intracellular Mg2+ in the activation of muscarinic K+ channel in cardiac atrial cell membrane

Effects of intracellular Mg²⁺ in the activation of a muscarinic K⁺ channel were examined in single atrial cells, using patch-recording techniques. In cell-attached patch recordings, acetylcholine (ACh) or adenosine (Ado), present in the pipette, activated a specific population of K⁺ channels. In inside-out patches, openings of the K⁺ channel by ACh or Ado diminished and did not resume until Mg²⁺ was added to the perfusate which contained GTP or GTP-S, a non-hydrolyzable GTP analogue. Channel openings caused by GTP faded by removing Mg²⁺, while GTP-S-induced openings persisted steadily even when both Mg²⁺ and GTP-S were removed. In contrast to the case of GTP-induced channel openings, the GTP-S-induced openings were not inhibited by the A protomer of pertussi toxin with NAD. From these observations, we concluded: 1) Intracellular Mg²⁺ is essential for GTP to activate the GTP-binding protein. 2) Deactivation of the N protein may be caused by hydrolysis of GTP to GDP. This process may not require Mg²⁺. 3) During the activation by GTP analogues, the N protein may be dissociated into its subunits.     

The Ca2+-sensitive K+-currents underlying the slow afterhyperpolarization of bullfrog sympathetic neurones

Ca²⁺-sensitive K⁺ currents involved in the slow afterhyperpolarization (a.h.p.) of an action potential of bullfrog sympathetic neurones were studied with a single-electrode voltage clamp method. The outward tail current (I_AH) generated after the end of a depolarizing command pulse (from the holding potential of –60 mV to 0 mV, 5–20 ms in duration), mimicking an action potential, was separated into at least two exponential components (I_AHf and I_AHs). They were identified as K⁺ currents, since their reversal potentials were close to the K⁺ equilibrium potential and they were sensitive to external K⁺. The time constant of I_AHf (t _f; 44 ms at –60 mV) was decreased by membrane hyperpolarization from –40 to –80 mV, while that of I_AHs (t _s; 213 ms) remained constant. Removal of external Ca²⁺ or addition of Cd²⁺ significantly decreased the I_AHs amplitude (A_s) andt _f without a change int _s and the I_AHf amplitude (A_f). On the other hand, increasing Ca²⁺ influx by applying repetitive command pulses enhanced both A_f and A_s with negligible effects ont _f andt _s, and produced a much slower component. Intracellular injection of EGTA reduced A_f with no effect ont _f, and increased A_s with a decreasedt _s. Both muscarine and (±)-tubocurarine, which reduced I_AHs, hardly affected I_AHf. These results indicate that a.h.p. is induced by the activation of two distinct Ca²⁺-dependent K⁺ channels, which differ in voltage sensitivity, Ca²⁺-dependence and pharmacology.     

Inhibition of voltage-dependent Na+ and K+ currents by forskolin in nodes of Ranvier

The effect of forskolin on voltage-activated Na⁺ and K⁺ currents in nodes of Ranvier from the toad, Bufo marinus, has been examined using the vaseline-gap voltageclamp technique. Peak Na⁺ currents (I _Na) were reduced by 35% and the rate of decline of Na⁺ current during continuous depolarization was accelerated following treatment with 450 M forskolin. However, the voltage-dependence of steady-state inactivation as well as the rate of recovery from fast inactivation remained unchanged. Upon repetitive depolarization at 1–10 Hz, a further inhibition of I _Na (60%) was observed. This use-dependent or phasic inhibition recovers slowly at -80 mV ( 13 s) and had a voltage-dependence like that of activation of the Na conductance. Near maximal steady-state phasic inhibition occurred with depolarizing pulse durations of only 4 ms, consistent with a direct involvement of the open Na⁺ channel in the blocking process. Inhibition of the delayed K⁺ current (I _K) was characterized by a concentration-dependent reduction in steady-state current amplitude (IC₅₀ 80 M) and a concentration-independent acceleration of current inactivation. A similar inhibition of I _K was obtained with 1,9-dideoxyforskolin, a homolog which does not activate adenylate cyclase (AC). The results suggest that the inhibition of I _K and perhaps I _Na follows directly from drug binding and is not a consequence of AC activation.     

A stretch-activated K+ channel in the basolateral membrane ofXenopus kidney proximal tubule cells

The present study examined whether a basolateral potassium ion (K⁺) channel is activated by membrane-stretching in the cell-attached patch. A K⁺ channel of conductance of 27.5 pS was most commonly observed in the basolateral membrane ofXenopus kidney proximal tubule cells. Channel activity increased with hyperpolarizing membrane potentials [at more positive pipette potentials (V _p)]. Open probability (P _o) was 0.03, 0.13, and 0.21 atV _p values of 0, 40, and 80 mV, respectively. Barium (0.1 mM) in the pipette reducedP _o by 79% at aV _p of 40 mV. Application of negative hydraulic pressure (−16 to −32 cm H₂O) to the pipette markedly activated outward currents (fromP _o=0.01 to 0.75) at aV _p of −80 mV, but not inward currents at aV _p of 80 mV. The size of the activated outward currents (from cell to pipette) did not change by replacing chloride with gluconate in the pipette. These results indicate that a stretch-activated K⁺ channel exists in the basolateral membrane of proximal tubule cells. It may play an important role as a K⁺ exit pathway when the cell membrane is stretched (for example, by cell swelling).     

Acetylcholine-mediated K+ channel activity in guinea-pig atrial cells is supported by nucleoside diphosphate kinase

We studied the role of nucleoside diphosphate kinase (NDPK) in acetylcholine-mediated muscarinic K⁺ channel activation in inside-out patches of guinea-pig atrial cells. NDPK-catalysed activation of the muscarinic K⁺ channels by adenosine triphosphate-Mg²⁺ (ATP-Mg²⁺) is not prevented by occupation of the muscarinic receptor [by acetylcholine (ACh) or atropine], nor by uncoupling of the receptor from the G protein by pertussis-toxin-catalysed adenosine diphosphate (ADP)-ribosylation of G_K. In the presence of ACh, addition of 0.1 mM guanosine triphosphate (GTP) after activation of the channels by 4 mM ATP alone resulted in a moderate increase of channel activity (in contrast to block in the absence of ACh): NDPK-mediated direct transphosphorylation is uncoupled by the G nucleotide but agonist-induced guanosine diphosphate (GDP)-to-GTP exchange takes over activation of the channels. Moreover, ACh-dependent channel stimulation was possible in inside-out patches while ATP and GDP were present in the bathing solution (in contrast to the complete absence of channel activation in the absence of ACh). This indicates that NDPK synthesises sufficient GTP to support channel activation by exchange. Hence, it is postulated that the main functional role of NDPK under physiological conditions is to provide a local supply of GTP (using GDP and ATP) in the immediate vicinity of the G protein, thereby maintaining a high local GTP/GDP ratio and ensuring adequate receptor-mediated regulation of muscarinic K⁺ channel activity.     

ATP4− and ATP·Mg inhibit the ATP-sensitive K+ channel of rat ventricular myocytes

K⁺ currents were recorded from ATP-sensitive channels in inside-out membrane patches excised from isolated rat ventricular myocytes. ATP-sensitive K⁺ channel inhibition could be evoked by ATP in the absence of magnesium where most ATP would be present as the free acid ATP^4–. Channel inhibition was enhanced when the same total concentration of ATP was applied in the presence of magnesium, where most ATP would be bound as ATP·Mg. Dose-response relationships for ATP-sensitive K⁺ channel inhibition evoked by ATP had a Hill coefficient of 2 andK _i of 17 and 30 M for ATP in the presence and absence of magnesium respectively. This was the obverse of the expected results if ATP^4– were to be the sole form of ATP to effect channel closure. ATP-sensitive K⁺ channel inhibition evoked by ATPS, AMP-PNP and AMP-PCP was also enhanced in the presence of magnesium. It is concluded that the ATP-sensitive K⁺ channel of rat ventricular myocytes binds and is closed by both the free-acid and divalent-cationbound forms of ATP.     

High-conductance K+ channel in apical membranes of principal cells cultured from rabbit renal cortical collecting duct anlagen

Using the patch clamp technique, one type of K⁺ channel was identified in the apical cell membrane of cultured principal cells of rabbit renal collecting ducts in the cell-attached or excised-patch configuration. The channel was highly selective for K⁺ over Na⁺ (typically 30-70-fold) and had a conductance of 180, SD±39 pS (n=6), referred to a situation of 140 mmolar K⁺-Ringer solution present on either surface of the patch membrane. Channel activity was completely blocked by Ba²⁺ (5 mmol/l) and partially inhibited by Na⁺. The latter was evidenced by a deviation from Goldman rectification at high cytoplasm-positive membrane potentials, which was observed when Na⁺ competed with K⁺ for channel entrace from the cytoplasmic surface. Channel open probability depended strongly on membrane voltage and cytoplasmic Ca²⁺ concentration. Open-close kinetics exhibited double exponential behaviour, with a strong voltage dependence of the slow open time constant. Infrequently also a substate conductance level was identified. The voltage and calcium dependence suggest that the channel plays a role in adjusting K⁺ secretion to Na⁺ absorption in the fine regulation of cation excretion in renal collecting ducts.     

Ketamine blocks voltage-gated K+ channels and causes membrane depolarization in rat mesenteric artery myocytes

Clinical doses of ketamine typically increase blood pressure, heart rate, and cardiac output. However, the precise mechanism by which ketamine produces these cardiovascular effects remains unclear. The voltage-gated K⁺ (K_V) channel is the major regulator of resting membrane potential (E _m) and vascular tone in many arteries. Therefore, we sought to evaluate the effects of ketamine on K_V currents using the standard whole-cell patch clamp recordings in single myocytes, enzymatically dispersed from rat mesenteric arteries. Ketamine [(±)-racemic mixture] inhibited K_V currents reversibly and concentration dependently with a K _d of 566.7 ± 32.3 μM and Hill coefficient of 0.75 ± 0.03. The inhibition of K_V currents by ketamine was voltage independent, and the time courses of channel activation and inactivation were little affected. The effects of ketamine on steady-state activation and inactivation curves were also minimal. Use-dependent inhibition was not observed either. S(+)-ketamine inhibited K_V currents with similar potency and efficacy as the racemic mixture. The average resting E _m in rat mesenteric artery myocytes was −44.1 ± 4.2 mV, and both racemic and S(+)-ketamine induced depolarization of E _m (15.8 ± 3.6 and 24.3 ± 5.0 mV at 100 μM, respectively). We conclude that ketamine induces E _m depolarization in vascular myocytes by blocking K_V channels in a state-independent manner, which may contribute to the increased vascular tone and blood pressure produced by this drug under a clinical setting.     

A newly identified Ca2+ dependent K+ channel in the smooth muscle membrane of single cells dispersed from the rabbit portal vein

We found a new type of Ca²⁺-dependent K⁺ channel in smooth muscle cell membranes of single cells of the rabbit portal vein. A slope conductance of the current was 180 pS when 142 mM K⁺ solution was exposed to both sides of the membrane (this channel was named the K_M channel, in comparison to the known K_L and K_S channels from the same membrane patch; Inoue et al. 1985). This K_M channel was less sensitive to the cytoplasmic Ca²⁺ concentration, [Ca²⁺]_i, but was sensitive to the extracellular Ca²⁺, [Ca²⁺]_o, e.g. in the outside-out membrane patch, lowering the [Ca²⁺]_o in the bath markedly reduced the open probability of this channel, and also in cell-attached configuration, lowering of the [Ca²⁺]_o using the internally perfused patch clamp electrode device reduced the opening of K_M channel. TEA⁺ (1–10 mM) reduced the amplitude of the elementary current through the K_M channel applied from each side of the membrane, but this agent inhibited the K_M channel to a greater extent when applied to the inner than to the outer surface of the membrane. Furthermore, this K_M channel had a weak voltage dependency, and the open probability of the channel remained much the same within a wide range of potential (from –60 mV to +60 mV). Whereas most Ca²⁺-dependent K⁺ channels are regulated mainly by [Ca²⁺]_i and possess a voltage dependency, these properties of the K_M channel differed from other Ca²⁺-dependent K⁺ channels. The elucidation of this K_M channel should facilitate explanations of the actions of external Ca²⁺ or TEA⁺ on the membrane potential, in the smooth muscles of the rabbit portal vein.     

Characterization of Ca2+-activated K+ channels in excised patches of human T lymphocytes

Ca²⁺-activated K⁺ [K(Ca)] channels were studied in excised patches of resting and activated human peripheral blood T lymphocytes. The K(Ca) channel had a single-channel conductance of 50±6 pS in symmetrical high-K⁺ solutions in the potential range of –100 to –10 mV and was inwardly rectifying at more depolarized potentials. The channel was sensitive to block by charybdotoxin (10 nM) and insensitive to apamin (3 nM). Half-maximum activation occurred at an internal free Ca²⁺ concentration of 360±110 nM. The concentration-effect curve had a slope factor of 0.83±0.12, suggesting a 11 interaction of Ca²⁺ ions with the channel. Ca²⁺ affects the open time probability of the K(Ca) channels, mainly by modulating the frequency of channel opening. The open probability did not show voltage dependence. The kinetics of the channel could be described assuming one open state and two closed states. The time constant of the exponential describing the open time distribution amounted to 2.8±1.2 ms, whereas the closed time distribution could be described with two exponentials with time constants of 0.2±0.05 ms and 8.0±2.1 ms, respectively. Resting T lymphocytes expressed a low number of channels but the density of channels increased dramatically during chronic phytohaemagglutinin stimulation.     

Inactivation determined by a single site in K+ pores

An N-terminus peptide or a C-terminus mechanism involving a single residue in transmembrane segment 6 produces inactivation in voltage-dependent K⁺ channels. Here we show that a single position in the pore of K⁺ channels can produce inactivation having characteristics distinct from either N- or C-type inactivation. In a chimeric K⁺ channel (CHM), the point reversion CHM V 369I produced fast inactivation and CHM V 369S had the additional effect of halving K⁺ conductance consistent with a position in the pore. The result was not restricted to CHM; mutating position 369 in the naturally occurring channel Kv2.1 also produced fast inactivation. Like N- and C-types of inactivation, pore or P-type inactivation was characterized by short bursts terminated by rapid entry into the inactivated state. Unlike C-type inactivation, in which external tetraethylammonium (TEA) produced a simple blockade that slowed inactivation and reduced currents, in P-type inactivation external TEA increased currents. Unlike N-type inactivation, internal TEA produced a simple reduction in current and K⁺ occupancy of the pore had no effect. External TEA was not the only cation to increase current; external K⁺ enhanced channel availability and recovery from inactivation. Additional features of P-type inactivation were residue-specific effects on the extent of inactivation and removal of inactivation by a point reversion at position 374, which also regulates conductance. The demonstration of P-type inactivation indicates that pore residues in K⁺ channels may be part of the inactivation gating machinery.