Search PubMed⌕ Search

Biomedical subjects

F Franciolini

Publications and source records attributed to F Franciolini.

At least 19 recordsLinked to original sources
PubMed2004

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.

Animals↗
PubMed2002

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.

Animals↗
PubMed2001

Verapamil block of large-conductance Ca-activated K channels in rat aortic myocytes.

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.

Animals↗
PubMed2001

Large-conductance calcium-activated potassium channels in neonatal rat intracardiac ganglion neurons.

The properties of single Ca2+-activated K+ (BK) channels in neonatal rat intracardiac neurons were investigated using the patch-clamp recording technique. In symmetrical 140 mM K+, the single-channel slope conductance was linear in the voltage range -60/+60 mV, and was 207+/-19 pS. Na+ ions were not measurably permeant through the open channel. Channel activity increased with the cytoplasmic free Ca2+ concentration ([Ca2+]i) with a Hill plot giving a half-saturating [Ca2+] (K0.5) of 1.35 microM and slope of approximately equals 3. The BK channel was inhibited reversibly by external tetraethylammonium (TEA) ions, charybdotoxin, and quinine and was resistant to block by 4-aminopyridine and apamin. Ionomycin (1-10 microM) increased BK channel activity in the cell-attached recording configuration. The resting activity was consistent with a [Ca2+]i <100 nM and the increased channel activity evoked by ionomycin was consistent with a rise in [Ca2+]i to > or =0.3 microM. TEA (0.2-1 mM) increased the action potential duration approximately equals 1.5-fold and reduced the amplitude and duration of the afterhyperpolarization (AHP) by 26%. Charybdotoxin (100 nM) did not significantly alter the action potential duration or AHP amplitude but reduced the AHP duration by approximately equals 40%. Taken together, these data indicate that BK channel activation contributes to the action potential and AHP duration in rat intracardiac neurons.

Action Potentials↗
PubMed2000

Characterization of the large-conductance Ca-activated K channel in myocytes of rat saphenous artery.

We used the patch-clamp method to characterize the BK channel in freshly isolated myocytes from the saphenous branch of the rat femoral artery. Single-channel recordings revealed that the BK channel had a conductance of 187 pS in symmetrical 150 mM KCl, was blocked by external tetraethylammonium (TEA) with a KD(TEA) of approx. 300 microM at +40 mV, and by submicromolar charybdotoxin (CTX). The sensitivity of the BK channel to Ca was especially high (KD(ca) approx. 0.1 microM at +60 mV) compared to skeletal muscle and neuronal tissues. We also investigated the macroscopic K current, which under certain conditions is essentially sustained by BK channels. This conclusion is based on the findings that the macroscopic current activated upon depolarization follows a single exponential time course and is virtually fully blocked by 100 nM CTX and 5 mM external TEA. We made use of this occurrence to assess the voltage and Ca dependence of the macroscopic BK current. In intact myocytes, the BK channel showed a strong and voltage-dependent reduction of the outward current (62% at +40 mV), most likely due to block by intracellular Ba and polyamines. The results obtained from macroscopic and unitary current indicate that approx. 2.5% of the BK channels are active under physiological conditions, sustaining approx. 20 pA of outward current. Given the high input resistance of these cells, few BK channels are required to open in order to cause a significant membrane hyperpolarization, and thus function to limit the contraction resulting from acute increases in intravascular pressure, or in response to hypertensive pathologies.

Animals↗
PubMed1999

Bimodal kinetics of a chloride channel from human fibroblasts.

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.

Cell Line↗
PubMed1999

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

1. The mechanism of verapamil block of the delayed rectifier K currents (I K(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. 2. 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. 3. 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. 4. 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.

Animals↗
PubMed1999

Mechanisms of verapamil inhibition of action potential firing in rat intracardiac ganglion neurons.

The effects of verapamil and related phenylalkylamines on neuronal excitability were investigated in isolated neurons of rat intracardiac ganglia using whole-cell perforated patch-clamp recording. Verapamil (>/=10 microM) inhibits tonic firing observed in response to depolarizing current pulses at 22 degrees C. The inhibition of discharge activity is not due to block of voltage-dependent Ca2+ channels because firing is not affected by 100 microM Cd2+. The K+ channel inhibitors charybdotoxin (100 nM), 4-aminopyridine (0.5 mM), apamin (30-100 nM), and tetraethylammonium ions (1 mM) also have no effect on firing behavior at 22 degrees C. Verapamil does not antagonize the acetylcholine-induced inhibition of the muscarine-sensitive K+ current (M-current) in rat intracardiac neurons. Verapamil inhibits the delayed outwardly rectifying K+ current with an IC50 value of 11 microM, which is approximately 7-fold more potent than its inhibition of high voltage-activated Ca2+ channel currents. These data suggest that verapamil inhibits tonic firing in rat intracardiac neurons primarily via inhibition of delayed outwardly rectifying K+ current. Verapamil inhibition of action potential firing in intracardiac neurons may contribute, in part, to verapamil-induced tachycardia.

4-Aminopyridine↗
PubMed1998

Verapamil block of the delayed rectifier K current in chick embryo dorsal root ganglion neurons.

We have used the patch-clamp method in the whole-cell configuration to investigate the mechanism of block of the delayed rectifier K current (IDRK) by verapamil in embryonic chick dorsal root ganglion (DRG) neurons. Verapamil induced a dose-dependent decay of the current, without altering its activation kinetics. This observation, together with the good description of IDRK time course at various blocker concentrations with the computer simulation of a three-state chain model (closed left and right arrow open left and right arrow open-blocked), indicates that verapamil acts as a state-dependent, open-channel blocker. To account for the double-exponential time course of recovery from block, this minimal kinetics scheme was expanded to include a closed-blocked state resulting from channel closure (at hyperpolarized voltages) with verapamil still bound to it. The apparent block and unblock rate constants assessed from verapamil-induced current decay in the presence of external Na were 0.95 +/- 0.05 ms-1mM-1 and 0.0037 +/- 0.0016 ms-1, respectively. When external Na was replaced by K, only the unblock rate constant changed, to 0.02 +/- 0.009 ms-1. Under these ionic conditions it was also observed that the recovery from block was modified from the double-exponential time course in the presence of external Na (tau1 = 160 ms; tau2 = 1600 ms), to a faster single-exponential recovery (tau = 100 ms). We tested the voltage dependence of block by applying stimulation protocols aimed at eliminating bias easily introduced by the shift of the gating equilibrium and by the coupling of channel activation and block. Under these experimental conditions the resulting block rate constant was not measurably voltage dependent.

Animals↗
PubMed1996

Characterization of a neuronal delayed rectifier K current permeant to Cs and blocked by verapamil.

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 V1/2 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 microM. 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.

Animals↗
PubMed1995

Tityus bahiensis toxin IV-5b selectively affects Na channel inactivation in chick dorsal root ganglion neurons.

A novel toxin was isolated from the venom of the Brazilian scorpion Tityus (T.) bahiensis. The N-terminal amino acid sequence of this toxin was shown to be 80% identical to the corresponding segment of T. serrulatus toxin IV-5. The new toxin was thus named toxin IV-5b. Toxin IV-5b was found to markedly slow inactivation of Na channel in dorsal root ganglion neurons from chick embryo. By contrast, Na channel activation was only negligibly delayed, and deactivation completely unaffected. Similarly unaffected by the toxin were K and Ca currents. The slowing effect of the toxin starts to appear at concentrations of c. 80 nM, and shows a KD of 143 nM. With a toxin concentration of 2.4 microM, the Na channel inactivation time constant was increased c. 3-fold with respect to the control. The slowing of inactivation was voltage dependent, and increased with depolarization.

Animals↗
PubMed1994

Anion-cation interactions in the pore of neuronal background chloride channels.

Background Cl channels in neurons and skeletal muscle are significantly permeable for alkali cations when tested with asymmetrical concentrations of the same salt. Both anion and cation permeation were proposed to require binding of an alkali cation with the pore (Franciolini, F., and W. Nonner. 1987. Journal of General Physiology. 90:453-478). We tested this hypothesis by bilaterally substituting large alkali cations for Na and found no significant changes of unitary conductance at 300 mM symmetrical concentrations. In addition, all organic cations examined were permeant in a salt gradient test (1,000 mM internal@300 mM external), including triethanolamine, benzyltrimethylamine, and bis-tris-propane (BTP, which is divalent at the tested pH of 6.2). Inward currents were detected following substitution of internal NaCl by the Na salts of the divalent anions of phosphoric, fumaric, and malic acid. Zero-current potentials in gradients of the Na and BTP salts of varied anions (propionate, F, Br, nitrate) that have different permeabilities under bi-ionic conditions, were approximately constant, as if the permeation of either cation were coupled to the permeation of the anion. These results rule out our earlier hypothesis of anion permeation dependent on a bound alkali cation, but they are consistent with the idea that the tested anions and cations form mixed complexes while traversing the Cl channel.

Animals↗
PubMed1994

A multi-ion permeation mechanism in neuronal background chloride channels.

Unitary current/voltage relationships of background Cl channels of rat hippocampal neurons were determined for varied gradients and absolute concentrations of NaCl. The channels revealed permeabilities for both Cl and Na ions. A hyperlinear increase of unitary conductance, observed for a symmetrical increase of salt concentration from 300 and 600 mM, indicated a multi-ion permeation mechanism. A variety of kinetic models of permeation were tested against the experimental current/voltage relationships. Models involving a pore occupied by mixed complexes of up to five ions were necessary to reproduce all measurements. A minimal model included four equilibrium states and four rate-limiting transitions, such that the empty pore accepts first an anion and then can acquire one or two cation/anion pairs. Three transport cycles are formed: a slow anion cycle (between the empty and single-anion states), a slow cation cycle (between the one- and three-ion states), and a fast anion cycle (between the three- and five-ion states). Thus, permeant anions are required for cation permeation, and several bound anions and cations promote a high rate of anion permeation. The optimized free-energy and electrical charge parameters yielded a self-consistent molecular interpretation, which can account for the particular order in which the pore accepts ions from the solutions. Although the model describes the mixed anion/cation permeability of the channel observed at elevated concentrations, it predicts a high selectivity for Cl anion at physiological ionic conditions.

Animals↗
PubMed1993

Effects of high salt concentration on the open probability of the background chloride channel.

We report here single channel data showing that high concentration of NaCl or CsCl at the cytoplasmic side of the membrane increases the open probability of the background Cl channel from hippocampal neurons. The open probability vs. voltage curve was shifted by 36 mV towards more negative voltages, when salt concentration was raised from 300 to 1000 mM. The steepness of the curve remained unchanged. Possible explanations for this effect are discussed.

Animals↗
PubMed1992

Newly identified steady-state potassium channels in rat hippocampal neurons.

We report two new types of potassium channels in cultured hippocampal neurons of rat. Both channels occurred in the soma membrane of these cells at very low density. They were active in steady-state conditions, within a wide voltage range that included the resting membrane potential. Their open probability was enhanced by membrane depolarization, but not influenced by Ca ions. In symmetrical 150 mM KCl the channels showed a slope conductance of ca. 40 and 80 pS, respectively. Current-voltage relations of both K channels show a negative slope at high positive voltages.

Animals↗