Search PubMed⌕ Search

Biomedical subjects

F Bezanilla

Publications and source records attributed to F Bezanilla.

At least 37 records · Page 2Linked to original sources
PubMed1998

Fast inactivation in Shaker K+ channels. Properties of ionic and gating currents.

Fast inactivating Shaker H4 potassium channels and nonconducting pore mutant Shaker H4 W434F channels have been used to correlate the installation and recovery of the fast inactivation of ionic current with changes in the kinetics of gating current known as "charge immobilization" (Armstrong, C.M., and F. Bezanilla. 1977. J. Gen. Physiol. 70:567-590.). Shaker H4 W434F gating currents are very similar to those of the conducting clone recorded in potassium-free solutions. This mutant channel allows the recording of the total gating charge return, even when returning from potentials that would largely inactivate conducting channels. As the depolarizing potential increased, the OFF gating currents decay phase at -90 mV return potential changed from a single fast component to at least two components, the slower requiring approximately 200 ms for a full charge return. The charge immobilization onset and the ionic current decay have an identical time course. The recoveries of gating current (Shaker H4 W434F) and ionic current (Shaker H4) in 2 mM external potassium have at least two components. Both recoveries are similar at -120 and -90 mV. In contrast, at higher potentials (-70 and -50 mV), the gating charge recovers significantly more slowly than the ionic current. A model with a single inactivated state cannot account for all our data, which strongly support the existence of "parallel" inactivated states. In this model, a fraction of the charge can be recovered upon repolarization while the channel pore is occupied by the NH2-terminus region.

Animals↗
PubMed1998

Voltage gating of Shaker K+ channels. The effect of temperature on ionic and gating currents.

Ionic (Ii) and gating currents (Ig) from noninactivating Shaker H4 K+ channels were recorded with the cut-open oocyte voltage clamp and macropatch techniques. Steady state and kinetic properties were studied in the temperature range 2-22 degreesC. The time course of Ii elicited by large depolarizations consists of an initial delay followed by an exponential rise with two kinetic components. The main Ii component is highly temperature dependent (Q10 > 4) and mildly voltage dependent, having a valence times the fraction of electric field (z) of 0.2-0.3 eo. The Ig On response obtained between -60 and 20 mV consists of a rising phase followed by a decay with fast and slow kinetic components. The main Ig component of decay is highly temperature dependent (Q10 > 4) and has a z between 1.6 and 2.8 eo in the voltage range from -60 to -10 mV, and approximately 0.45 eo at more depolarized potentials. After a pulse to 0 mV, a variable recovery period at -50 mV reactivates the gating charge with a high temperature dependence (Q10 > 4). In contrast, the reactivation occurring between -90 and -50 mV has a Q10 = 1.2. Fluctuation analysis of ionic currents reveals that the open probability decreases 20% between 18 and 8 degreesC and the unitary conductance has a low temperature dependence with a Q10 of 1.44. Plots of conductance and gating charge displacement are displaced to the left along the voltage axis when the temperature is decreased. The temperature data suggests that activation consists of a series of early steps with low enthalpic and negative entropic changes, followed by at least one step with high enthalpic and positive entropic changes, leading to final transition to the open state, which has a negative entropic change.

Animals↗
PubMed1998

Structural implications of fluorescence quenching in the Shaker K+ channel.

When attached to specific sites near the S4 segment of the nonconducting (W434F) Shaker potassium channel, the fluorescent probe tetramethylrhodamine maleimide undergoes voltage-dependent changes in intensity that correlate with the movement of the voltage sensor (Mannuzzu, L.M., M.M. Moronne, and E.Y. Isacoff. 1996. Science. 271:213-216; Cha, A., and F. Bezanilla. 1997. Neuron. 19:1127-1140). The characteristics of this voltage-dependent fluorescence quenching are different in a conducting version of the channel with a different pore substitution (T449Y). Blocking the pore of the T449Y construct with either tetraethylammonium or agitoxin removes a fluorescence component that correlates with the voltage dependence but not the kinetics of ionic activation. This pore-mediated modulation of the fluorescence quenching near the S4 segment suggests that the fluorophore is affected by the state of the external pore. In addition, this modulation may reflect conformational changes associated with channel opening that are prevented by tetraethylammonium or agitoxin. Studies of pH titration, collisional quenchers, and anisotropy indicate that fluorophores attached to residues near the S4 segment are constrained by a nearby region of protein. The mechanism of fluorescence quenching near the S4 segment does not involve either reorientation of the fluorophore or a voltage-dependent excitation shift and is different from the quenching mechanism observed at a site near the S2 segment. Taken together, these results suggest that the extracellular portion of the S4 segment resides in an aqueous protein vestibule and is influenced by the state of the external pore.

Animals↗
PubMed1997

RNA editing generates a diverse array of transcripts encoding squid Kv2 K+ channels with altered functional properties.

We have cloned a Kv2 potassium channel from squid optic lobe termed sqKv2. Multiple overlapping sqKv2 cDNA clones differed from one another at specific positions by purine transitions. To test whether the purine transitions were generated by RNA editing, we compared a 360 nucleotide genomic sequence with corresponding cDNA sequences (encoding S4-S6) isolated from individual animals and lying on a single gene and exon. cDNA sequences differed from genomic sequence at 17 positions, resulting in 28 unique sequences. There was invariantly an adenosine in the genomic sequence and a guanosine in the edited cDNA sequences. Two of the edits altered the rates of channel closure and slow inactivation. These results extend selective RNA editing to invertebrate taxa and represents a novel mechanism for the posttranscriptional modulation of voltage-gated ion channels.

Adenine↗
PubMed1997

Characterizing voltage-dependent conformational changes in the Shaker K+ channel with fluorescence.

We examined voltage-dependent conformational changes in three specific regions of the Shaker potassium channel with site-directed fluorescent labeling: the fourth transmembrane segment (S4), the second transmembrane segment (S2), and the putative pore region. The fluorescence changes displayed distinctive properties that correlate with gating, activation, and slow inactivation of the channel. The fluorescence signals measured near the S2 and S4 segments suggest that the S2 segment may undergo voltage-sensitive conformational changes that precede those in the S4 segment. In contrast, fluorescence changes in the pore correlated with the voltage dependence and time course of ionic activation and slow inactivation. Spectroscopy indicated that the mechanism of fluorescence change involves voltage-dependent quenching of the probe in an aqueous environment by other parts of the protein.

Animals↗
PubMed1997

Voltage-dependent proton transport by the voltage sensor of the Shaker K+ channel.

In voltage-dependent ion channels, pore opening is initiated by electrically driven movements of charged residues, and this movement generates a gating current. To examine structural rearrangements in the Shaker K+ channel, basic residues R365 and R368 in the S4 segment were replaced with histidine, and gating currents were recorded. Changes in gating charge displacement with solvent pH reveal voltage-dependent changes in exposure of the histidine to solvent protons. This technique directly monitors accessibility changes during gating, probes the environment even in confined locations, and introduces minimal interference of gating charge motion. The results indicate that charges 365 and 368 traverse the entire electric field during gating. The remarkable implication of the successive exposure of histidine to each side of the membrane is that in a pH gradient, the voltage sensor transports protons.

Amino Acid Substitution↗
PubMed1997

Total charge movement per channel. The relation between gating charge displacement and the voltage sensitivity of activation.

One measure of the voltage dependence of ion channel conductance is the amount of gating charge that moves during activation and vice versa. The limiting slope method, introduced by Almers (Almers, W. 1978. Rev. Physiol. Biochem. Pharmacol. 82:96-190), exploits the relationship of charge movement and voltage sensitivity, yielding a lower limit to the range of single channel gating charge displacement. In practice, the technique is plagued by low experimental resolution due to the requirement that the logarithmic voltage sensitivity of activation be measured at very low probabilities of opening. In addition, the linear sequential models to which the original theory was restricted needed to be expanded to accommodate the complexity of mechanisms available for the activation of channels. In this communication, we refine the theory by developing a relationship between the mean activation charge displacement (a measure of the voltage sensitivity of activation) and the gating charge displacement (the integral of gating current). We demonstrate that recording the equilibrium gating charge displacement as an adjunct to the limiting slope technique greatly improves accuracy under conditions where the plots of mean activation charge displacement and gross gating charge displacement versus voltage can be superimposed. We explore this relationship for a wide variety of channel models, which include those having a continuous density of states, nonsequential activation pathways, and subconductance states. We introduce new criteria for the appropriate use of the limiting slope procedure and provide a practical example of the theory applied to low resolution simulation data.

Electrophysiology↗
PubMed1997

Correlation between charge movement and ionic current during slow inactivation in Shaker K+ channels.

Prolonged depolarization induces a slow inactivation process in some K+ channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Delta(6-46) K+ channel and in the nonconducting mutant (Shaker H4-Delta(6-46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547-556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: (a) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and (b) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to -90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non-slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K+ channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J. 66:1011-1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation.

Animals↗
PubMed1996

A patch-clamp study of delayed rectifier currents in skeletal muscle of control and mdx mice.

1. Potassium currents were measured in the extensor digitorum longus muscle of normal and mdx mice, which lack the protein dystrophin, using the cell-attached and inside-out patch clamp techniques, in the presence of asymmetrical K+ concentrations (3 mM in the pipette, 160 mM in the bath). 2. In cell-attached patches, the delayed rectifier was the most commonly found potassium channel, with a density of roughly 8 channels microns-2. Outward macroscopic currents were activated in macropatches depolarized to potentials positive to -60 mV. The probability of opening reached half-maximal values around -40 mV for control patches and -31 mV for patches from mdx mice. 3. Tail currents were linear in the range between -60 and +20 mV, reversing close to -100 mV. The single channel current at 0 mV, estimated from non-stationary analysis of variance, was used in conjunction with the slope of the linear part of the tail current to calculate the single channel conductance, yielding a value of 19 +/- 1 pS. 4. At 0 mV, the delayed rectifier inactivated with two time constants, of 70 +/- 20 ms and 600 +/- 200 ms. Prepulses of 500 ms duration to different potentials produced incomplete inactivation with inactivation reaching 50% of its maximum at -50 mV. 5. Single channel activity was recorded using small pipettes. Both single channel conductance and kinetic behaviour were in agreement with the macroscopic current data. 6. In excised patches, the delayed rectifier current ran down, unmasking other K+ channels. A Ca(2+)-dependent K+ channel of 186 pS (BK-like channel) was found frequently in patches bathed in solutions containing appropriate concentrations of calcium, especially at stronger depolarizations. A K+ channel of 63 pS was unmasked in control excised patches bathed in solutions devoid of ATP. This channel was not observed in patches excised from mdx fibers.

Adenosine Triphosphate↗
PubMed1996

Transitions near the open state in Shaker K(+)-channel: probing with temperature.

Ionic currents from non-inactivating Shaker K(+)-channels were expressed in Xenopus oocytes and recorded with the cut-open oocyte voltage clamp technique. Kinetic properties of ionic currents were studied between 1.4 and 19 degrees C. The forward step of the last transition (...C --> O) of the activation pathway, just before the channel opening, was studied by the recovery of the ionic current after a short interval (< 500 microseconds) at a hyperpolarized potential (-90 mV) between two identical depolarizing pulses (0, 40 or 100 mV). Ionic currents under these experimental conditions are described by at least three kinetic components. The predominant kinetic component (corresponding to the ...C --> O transition) has a low temperature dependence (activation energy, Ea, around -5 kcal/mol) and it is voltage independent, suggesting that this step does not carry charge and has a mainly entropic energy barrier. The backward step (O --> C...) was studied from ionic tail currents at -120 mV after pulsing to 40 mV. The closure of the channel has a high temperature dependence (Q10 > 4), having an Ea of 24-25 kcal/mol. The closure of the channel also is voltage dependent, having a valence times fraction of the electric field, z beta of 0.5 eo. We conclude that the transition from the closed to the opened state has a net decrease in entropy and enthalpy, suggesting that the channel gets more ordered when it is open.

Animals↗
PubMed1996

Voltage-sensing residues in the S2 and S4 segments of the Shaker K+ channel.

The activation of Shaker K+ channels is steeply voltage dependent. To determine whether conserved charged amino acids in putative transmembrane segments S2, S3, and S4 contribute to the gating charge of the channel, the total gating charge movement per channel was measured in channels containing neutralization mutations. Of eight residues tested, four contributed significantly to the gating charge: E293, an acidic residue in S2, and R365, R368, and R371, three basic residues in the S4 segment. The results indicate that these residues are a major component of the voltage sensor. Furthermore, the S4 segment is not solely responsible for gating charge movement in Shaker K+ channels.

Animals↗
PubMed1995

A method for determining the unitary functional capacity of cloned channels and transporters expressed in Xenopus laevis oocytes.

The Xenopus laevis oocyte is widely used to express exogenous channels and transporters and is well suited for functional measurements including currents, electrolyte and nonelectrolyte fluxes, water permeability and even enzymatic activity. It is difficult, however, to transform functional measurements recorded in whole oocytes into the capacity of a single channel or transporter because their number often cannot be estimated accurately. We describe here a method of estimating the number of exogenously expressed channels and transporters inserted in the plasma membrane of oocytes. The method is based on the facts that the P (protoplasmic) face in water-injected control oocytes exhibit an extremely low density of endogenous particles (212 +/- 48 particles/microns2, mean, SD) and that exogenously expressed channels and transporters increased the density of particles (up to 5,000/microns2) only on the P face. The utility and generality of the method were demonstrated by estimating the "gating charge" per particle of the Na+/glucose cotransporter (SGLT1) and a nonconducting mutant of the Shaker K+ channel proteins, and the single molecule water permeability of CHIP (Channel-like In-tramembrane Protein) and MIP (Major Intrinsic Protein). We estimated a "gating charge" of approximately 3.5 electronic charges for SGLT1 and approximately 9 for the mutant Shaker K+ channel from the ratio of Qmax to density of particles measured on the same oocytes. The "gating charges" were 3-fold larger than the "effective valences" calculated by fitting a Boltzmann equation to the same charge transfer data suggesting that the charge movement in the channel and cotransporter occur in several steps. Single molecule water permeabilities (pfs) of 1.4 x 10(-14) cm3/sec for CHIP and of 1.5 x 10(-16) cm3/sec for MIP were estimated from the ratio of the whole-oocyte water permeability (Pf) to the density of particles. Therefore, MIP is a water transporter in oocytes, albeit approximately 100-fold less effective than CHIP.

Animals↗
PubMed1994

Gating current noise produced by elementary transitions in Shaker potassium channels.

Gating currents provide a direct record of the spatial rearrangement of charges occurring within the protein of voltage-sensitive ion channels. If the elementary charges move as very brief discrete pulses of current, they will produce fluctuations in the macroscopic gating current. The variance of such fluctuations in gating currents was measured in Shaker potassium channels expressed in Xenopus oocytes with a sufficiently high recording bandwidth to estimate the magnitude and time distribution of the elementary transition charge movements. Channel activation occurred in two sequential stages. The first stage consisted of numerous, fast transitions, each moving small amounts of charge that contributed little to the fluctuation in gating current, whereas the second stage, which contributed the bulk of the fluctuation, was represented by a number of discrete, correlated transitions, one or more of which carried a charge of at least 2.4 elementary charges across the membrane field.

Animals↗
PubMed1994

Chemical modification of squid axon K+ channel -SH groups with the organic mercurial compound p-hydroxymercuriphenylsulfonic acid (PHMPS).

In internally dialyzed voltage-clamped squid axons, intracellular or extracellular addition of the sulfhydryl group (-SH) specific reagent p-hydroxymercuriphenylsulfonic acid (PHMPS), causes major modifications in the magnitude and kinetic parameters of the delayed rectifier K+ current. PHMPS produces a dramatic slow-down of the macroscopic current activation kinetics with a simultaneous reduction in its amplitude. In addition, it causes a marked increase in the delay of the macroscopic current at various pre-pulse potentials (Cole-Moore shift). The main effect of PHMPS at the single channel level is a sharp decrease in the open probability (4- to 5-fold). There is, however, a small reduction in single channel conductance (20%). Gating current experiments indicate that PHMPS causes a reduction in the voltage dependence of the activation process as well as a shift of the charge/voltage relationship towards more positive potentials. This, together with an increase in the mean open time, suggests that the open state has been destabilized. The results indicate that the reaction of -SH groups with PHMPS differentially affects the gating process. All the above mentioned effects are partially reversed by either dithiotreitol or beta-mercaptoethanol, -SH group reducing agents.

Animals↗
PubMed1994

S4 mutations alter gating currents of Shaker K channels.

Activation of voltage-dependent channels involves charge-moving conformational changes of the voltage sensor that can be detected as gating currents. In Shaker K channels, the S4 sequence comprises at least part of the voltage sensor. We have measured gating currents in three S4 mutants: R368Q, R377K, and R371Q. R368Q enhances the separation of two components of charge movement and greatly reduces the valence of one component. R377K partially uncouples charge movement from channel opening. In contrast, the gating currents of R371Q resemble those of the control. Two other S4 mutations, R377Q and K374Q, make proteins that are not properly processed and transported to the cell surface and thereby eliminate the gating current. To explain the effects of R368Q, we hypothesize that R368 is part of a salt bridge that is broken early in activation. Subsequently, the S4 segment undergoes a conformational change, and, after a final, relatively voltage-independent step, the channel opens.

Amino Acid Sequence↗
PubMed1994

Gating of Shaker K+ channels: I. Ionic and gating currents.

Ionic and gating currents from noninactivating Shaker B K+ channels were studied with the cut-open oocyte voltage clamp technique and compared with the macropatch clamp technique. The performance of the cut-open oocyte voltage clamp technique was evaluated from the electrical properties of the clamped upper domus membrane, K+ tail current measurements, and the time course of K+ currents after partial blockade. It was concluded that membrane currents less than 20 microA were spatially clamped with a time resolution of at least 50 microseconds. Subtracted, unsubtracted gating currents with the cut-open oocyte voltage clamp technique and gating currents recorded in cell attached macropatches had similar properties and time course, and the charge movement properties directly obtained from capacity measurements agreed with measurements of charge movement from subtracted records. An accurate estimate of the normalized open probability Po(V) was obtained from tail current measurements as a function of the prepulse V in high external K+. The Po(V) was zero at potentials more negative than -40 mV and increased sharply at this potential, then increased continuously until -20 mV, and finally slowly increased with voltages more positive than 0 mV. Deactivation tail currents decayed with two time constants and external potassium slowed down the faster component without affecting the slower component that is probably associated with the return between two of the closed states near the open state. In correlating gating currents and channel opening, Cole-Moore type experiments showed that charge moving in the negative region of voltage (-100 to -40 mV) is involved in the delay of the conductance activation but not in channel opening. The charge moving in the more positive voltage range (-40 to -10 mV) has a similar voltage dependence to the open probability of the channel, but it does not show the gradual increase with voltage seen in the Po(V).

Animals↗
PubMed1994

Gating of Shaker K+ channels: II. The components of gating currents and a model of channel activation.

Steady-state and kinetic properties of gating currents of the Shaker K+ channels were studied in channels expressed in Xenopus oocytes and recorded with the cut-open oocyte voltage clamp. The charge versus potential (Q-V) curve reveals at least two components of charge, the first moving in the hyperpolarized region (V1/2 = -63 mV) and the second, with a larger apparent valence, moving in the more depolarized region (V1/2 = -44 mV). The kinetic analysis of gating currents revealed also two exponential decaying components that corresponded in their voltage dependence with the charge components described in the steady-state. The first component was found to correlate with the effects of prepulses that produce the Cole-Moore shift of the ionic and gating currents and seems to be occurring completely within closed conformations of the channel. The second component seems to be related to the events occurring between the closed states just preceding, but not including, the transition to the open state. The ON and OFF gating currents exhibit a pronounced rising phase at potentials at which the second component becomes important, and this region corresponds to the potential range where the channel opens. The results could not be explained with simple parallel models, but the data can be fitted to a sequential model that could be related to a first rearrangement of the putative four subunits in cooperative fashion, followed by a concerted charge movement that leads to the open channel. The first series of charge movements are produced by transitions between several closed states carrying less than two electronic charges per step, while a step carrying about 3.5 electronic charges can explain the second component. This step is followed by the transition to the open state carrying less than 0.5 electronic charges. This model is able to reproduce all the kinetic and steady-state properties of the gating currents and predicts many of the properties of the ionic currents.

Animals↗