F. Franciolini

Blocking of the squid axon K+ channel by noxiustoxin: a toxin from the venom of the scorpionCentruroides noxius

We have studied the selective effects of noxiustoxin (NTX), a fraction of the venom of the scorpionCentruroides noxius, on the K currents of perfused squid giant axons using the voltage-clamp technique. At concentrations below 1.5 μM, NTX blocked K currents in a voltage-independent manner, with little effect on their turning-on and turning-off kinetics. Above 1.5 μM, the block by NTX became voltagedependent and could be partially removed by repetitive pulsing and strong depolarizations. Long repolarizations and more negative holding potentials favoured the slow restoration of channel block. Reduction of K currents by internally perfusing the fibers with solutions of low K+ concentration (200 mM), affected very little the removal of NTX-block during repetitive pulsing, suggesting that block removal depended on membrane potential and not on outward movements of K+ ions through open channels. In high extracellular K+ (300 mM) the blocking action of NTX was reduced and the instantaneous I–V characteristics showed a marked outward rectification. At 20 μM NTX, inward tail currents measured on step repolarizations to −70 mV were fully blocked, suggesting a direct interaction of the toxin with the open channel. The effects of the total venomCentruroides noxius Hoffmann was also studied. External application of 0.25 mg/ml of the venom caused a marked reduction of both Na and K currents, an effect similar to that of other scorpion venoms.

Verapamil Block of Large-Conductance Ca-Activated K Channels in Rat Aortic Myocytes

Abstract. The effects of verapamil on the large conductance Ca-activated K (BK) channel from rat aortic smooth muscle cells were examined at the single channel level. Micromolar concentrations of verapamil produced a reversible flickering block of the BK channel activity. Kinetic analysis showed that verapamil decreased markedly the time constants of the open states, without any significant change in the time constants of the closed states. The appearance of an additional closed state — specifically, a nonconducting, open-blocked state — was also observed, whose time constant would reflect the mean residence time of verapamil on the channel. These observations are indicative of a state-dependent, open-channel block mechanism. Dedicated kinetic (group) analysis confirmed the state-dependent block exerted by verapamil. D600 (gallopamil), the methoxy derivative of verapamil, was also tested and found to exert a similar type of block, but with a higher affinity than verapamil. The permanently charged and membrane impermeant verapamil analogue D890 was used to address other important features of verapamil block, such as the sidedness of action and the location of the binding site on the channel protein. D890 induced a flickering block of BK channels similar to that observed with verapamil only when applied to the internal side of the membrane, indicating that D890 binds to a site accessible from the cytoplasmic side. Finally, the voltage dependence of D890 block was assessed. The experimental data fitted with a Langmuir equation incorporating the Woodhull model for charged blockers confirms that the D890-binding site is accessed from the internal mouth of the BK channel, and locates it approximately 40% of the membrane voltage drop along the permeation pathway.

Characterization of a Neuronal Delayed Rectifier K Current Permeant to Cs and Blocked by Verapamil

Abstract. We have used the patch-clamp method in the whole-cell configuration to characterize the delayed rectifier K current (IDRK) in embryonic chick dorsal root ganglion (DRG) neurons. The IDRK is activated by depolarizing pulses positive to −40 mV, and its V½ is near −20 mV. The slope factor of 10.4 mV for an e-fold change in conductance indicates an equivalent gating charge of 2.4e. Inactivation during sustained depolarizing pulses displays two distinct time constants of 200–300 msec and 6–9 sec, respectively. Outward current through the delayed rectifier K (DRK) channels could also be carried by internal Cs, which however exerts mild block when in mixtures with K, as evidenced by the anomalous mole fraction effect. The relative permeability of Cs vs. K, PCs/PK, as calculated from reversal potential measurements, is 0.25. Rb likewise permeates the DRK channel (PRb/PK= 0.67). The IDRK was effectively suppressed by external application of the Ca channel blocker Verapamil, with apparent dissociation constant of ca. 4 μm. The time course of Verapamil block, its good description by equations derived from open-channel block kinetic scheme, and the frequency-dependent effect of the blocker indicate that Verapamil can bind to the channel only when it is in the open state.

Histamine activates a background, arachidonic acid-sensitive K channel in embryonic chick dorsal root ganglion neurons

Histamine has been proposed to be an important modulator of developing neurons, but its mechanism of action remains unclear. In embryonic chick dorsal root ganglion neurons we found that histamine activates, through the pyrilamine-sensitive H1 receptor, a K-selective, background channel. The K channel activated by histamine was also activated by arachidonic acid in a dose-dependent way, with a KD of 4 microM and a slope of 2.5, had a unitary conductance of about 150 pS (symmetrical 140 KCl) and a moderate voltage dependence. The channel was insensitive to the classical K channel blockers tetraethylammonium, charybdotoxin, 4-aminopyridine, but inhibited by millimolar Ba2+. Channel activity could also be increased by lowering the intracellular pH from 7.2 to 5.5, or by applying negative pressure pulses through the patch pipette. Experiments aimed at delineating the metabotropic pathway leading to K channel activation by histamine indicated the involvement of a pertussis toxin-insensitive G protein, and a quinacrine-sensitive cytosolic phospholipase A2. The histamine-induced K channel activation was observed only with elevated internal Ca2+ (achieved using 0.5 microM ionomycin or elevated external KCl). An increase in the histamine-induced phosphoinositide hydrolysis was also observed upon internal Ca2+ elevation, showing the presence of a Ca2+ dependent step upstream to inositol 1,4,5-triphosphate production. In view of the functional importance of K conductances during cell differentiation, we propose that histamine activation of this K channel may have a significant role during normal development of embryonic chick neurons.

Mechanism of verapamil block of a neuronal delayed rectifier K channel: active form of the blocker and location of its binding domain

The mechanism of verapamil block of the delayed rectifier K currents (IK(DR)) in chick dorsal root ganglion (DRG) neurons was investigated using the whole‐cell patch clamp configuration. In particular we focused on the location of the blocking site, and the active form (neutral or charged) of verapamil using the permanently charged verapamil analogue D890. Block by D890 displayed similar characteristics to that of verapamil, indicating the same state‐dependent nature of block. In contrast with verapamil, D890 was effective only when applied internally, and its block was voltage dependent (136 mV/e‐fold change of the on rate). Given that verapamil block is insensitive to voltage ( Trequattrini et al., 1998 ), these observations indicate that verapamil reaches its binding site in the uncharged form, and accesses the binding domain from the cytoplasm. In external K and saturating verapamil we recorded tail currents that did not decay monotonically but showed an initial increase (hook). As these currents can only be observed if verapamil unblock is significantly voltage dependent, it has been suggested ( DeCoursey, 1995 ) that neutral drug is protonated upon binding. We tested this hypothesis by assessing the voltage dependence of the unblock rate from the hooked tail currents for verapamil and D890. The voltage dependence of the off rate of D890, but not of verapamil, was well described by adopting the classical Woodhull (1973) model for ionic blockage of Na channels. The voltage dependence of verapamil off rate was consistent with a kinetic scheme where the bound drug can be protonated with rapid equilibrium, and both charged and neutral verapamil can unbind from the site, but with distinct kinetics and voltage dependencies.

Bimodal kinetics of a chloride channel from human fibroblasts.

Abstract. Excised patches were used to study the kinetics of a Cl channel newly identified in cultured human fibroblasts (L132). The conductance of ca. 70 pS in 150 mm symmetrical Cl, and the marked outward rectification ascribe this channel to the ICOR family. Long single-channel recordings (>30 min) revealed that the channel spontaneously switches from a kinetic mode characterized by high voltage dependence (with activity increasing with depolarization; mode 1), into a second mode (mode 2) insensitive to voltage, and characterized by a high activity in the voltage range ±120 mV. On patch excision the channel always appeared in mode 1, which was maintained for a variable time (5–20 min). In most instances the channels then switched into mode 2, and never were seen to switch back, in spite of the eight patches that cumulatively dwelled in this mode 2.33-fold as compared to mode 1. Stability plots of long recordings showed that the channel was kinetically stable in both modes, allowing standard analysis of steady-state kinetics to be performed. Open and closed time distributions of mode 1 and mode 2 revealed that the apparent number of kinetic states of the channel was the same in the two modes. The transition from mode 1 into mode 2 was not instantaneous, but required a variable time in the range 5–60 sec. During the transition the channel mean open time was intermediate between mode 1 and mode 2. The intermediate duration in the stability plot however is not to be interpreted as if the channel, during the transition, rapidly switches between mode 1 and mode 2, but represents a distinct kinetic feature of the transitional channel.

A fast transient outward monovalent current in rat saphenous myocytes passing through Ca2+ channels.

Transient outward currents in rat saphenous arterial myocytes were studied using the perforated configuration of the patch-clamp method. When myocytes were bathed in a Na-gluconate solution containing TEA to block large-conductance Ca2+-activated K+ (BK) currents, depolarizing pulses positive to +20 mV from a holding potential of ?100 mV induced fast transient outward currents. The activation and inactivation time constants of the current were voltage dependent, and at +40 mV were 3.6 ± 0.8 ms and 23.9 ± 6.4 ms (n = 4), respectively. The steady-state inactivation of the transient outward current was steeply voltage dependent (z = 1.7), with 50% of the current inactivated at ?55 mV. The current was insensitive to the A-type K+ channel blocker 4-AP (1–5 mM), and was modulated by external Ca, decreasing to approximately 0.85 of control values upon raising Ca2+ from 1 to 10 mM, and increasing approximately 3-fold upon lowering it to 0.1 mM. Transient outward currents were also recorded following replacement of internal K+ with either Na+ or Cs+, raising the possibility that the current was carried by monovalent ions passing through voltage-gated Ca2+ channels. This hypothesis was supported by the finding that the transient outward current had the same inactivation rate as the inward Ba2+ current, and that both currents were effectively blocked by the L-type Ca2+ channel blocker, nifedipine and enhanced by the agonist BAYK8644.