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Biomedical subjects

Francisco Bezanilla

Publications and source records attributed to Francisco Bezanilla.

9 recordsLinked to original sources
PubMed2003

Fast gating in the Shaker K+ channel and the energy landscape of activation.

An early component of the gating current in Shaker K+ channels with a time constant of approximately 12 microsec has been recorded with a high-speed patch-clamp setup. This fast component was found to be part of the gating current associated with the opening and closing of the channel. With regard to an energy-landscape interpretation of protein kinetics, the voltage and temperature dependence of the fast component may be explained by a combination of drift diffusion and barrier jumping in the initial stages of channel activation. The data were modeled by a gating particle undergoing Brownian motion in a one-dimensional diffusion landscape that featured diminishing electrical resistance and entropy in the direction of channel activation. The final open state of the channel was reasoned to be narrow and deep to account for successful subtraction of linear-charge displacements at positive potentials. The overall picture of gating that emerges from these studies is that the channel experiences incremental organization from a relaxed state in the early steps of activation to a rigidly structured open state.

Animals↗
PubMed2003

A fluorometric approach to local electric field measurements in a voltage-gated ion channel.

Site-specific electrostatic measurements have been limited to soluble proteins purified for in vitro spectroscopic characterization or proteins of known structure; however, comparable measurements have not been made for functional membrane bound proteins. Here, using an electrochromic fluorophore, we describe a method to monitor localized electric field changes in a voltage-gated potassium channel. By coupling the novel probe Di-1-ANEPIA to cysteines in Shaker and tracking field-induced optical changes, in vivo electrostatic measurements were recorded with submillisecond resolution. This technique reports dynamic changes in the electric field during the gating process and elucidates the electric field profile within Shaker. The extension of this method to other membrane bound proteins, including transporters, will yield insight into the role of electrical forces on protein function.

Animals↗
PubMed2003

A physical model of potassium channel activation: from energy landscape to gating kinetics.

We have developed a method for rapidly computing gating currents from a multiparticle ion channel model. Our approach is appropriate for energy landscapes that can be characterized by a network of well-defined activation pathways with barriers. To illustrate, we represented the gating apparatus of a channel subunit by an interacting pair of charged gating particles. Each particle underwent spatial diffusion along a bistable potential of mean force, with electrostatic forces coupling the two trajectories. After a step in membrane potential, relaxation of the smaller barrier charge led to a time-dependent reduction in the activation barrier of the principal gate charge. The resulting gating current exhibited a rising phase similar to that measured in voltage-dependent ion channels. Reduction of the two-dimensional diffusion landscape to a circular Markov model with four states accurately preserved the time course of gating currents on the slow timescale. A composite system containing four subunits leading to a concerted opening transition was used to fit a series of gating currents from the Shaker potassium channel. We end with a critique of the model with regard to current views on potassium channel structure.

Adaptation, Physiological↗
PubMed2002

Extensive editing of mRNAs for the squid delayed rectifier K+ channel regulates subunit tetramerization.

We report the extensive editing of mRNAs that encode the classical delayed rectifier K+ channel (SqK(v)1.1A) in the squid giant axon. Using a quantitative RNA editing assay, 14 adenosine to guanine transitions were identified, and editing efficiency varied tremendously between positions. Interestingly, half of the sites are targeted to the T1 domain, important for subunit assembly. Other sites occur in the channel's transmembrane spans. The effects of editing on K+ channel function are elaborate. Edited codons affect channel gating, and several T1 sites regulate functional expression as well. In particular, the edit R87G, a phylogenetically conserved position, reduces expression close to 50-fold by regulating the channel's ability to form tetramers. These data suggest that RNA editing plays a dynamic role in regulating action potential repolarization in the giant axon.

Action Potentials↗
PubMed2002

Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation.

The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluorescence signals from the sites proximal to each of the four S4 segments of a rat skeletal muscle sodium channel were monitored simultaneously with either gating or ionic currents. At least one of the kinetic components of fluorescence from every S4 segment correlates with movement of gating charge. The fast kinetic component of fluorescence from sites S216C (S4 domain I), S660C (S4 domain II), and L1115C (S4 domain III) is comparable to the fast component of gating currents. In contrast, the fast component of fluorescence from the site S1436C (S4 domain IV) correlates with the slow component of gating. In all the cases, the slow component of fluorescence does not have any apparent correlation with charge movement. The fluorescence signals from sites reflecting the movement of S4s in the first three domains initiate simultaneously, whereas the fluorescence signals from the site S1436C exhibit a lag phase. These results suggest that the voltage-dependent movement of S4 domain IV is a later step in the activation sequence. Analysis of equilibrium and kinetic properties of fluorescence over activation voltage range indicate that S4 domain III is likely to move at most hyperpolarized potentials, whereas the S4s in domain I and domain II move at more depolarized potentials. The kinetics of fluorescence changes from sites near S4-DIV are slower than the activation time constants, suggesting that the voltage-dependent movement of S4-DIV may not be a prerequisite for channel opening. These experiments allow us to map structural features onto the kinetic landscape of a sodium channel during activation.

Algorithms↗
PubMed2002

A comparison of propagated action potentials from tropical and temperate squid axons: different durations and conduction velocities correlate with ionic conductance levels.

To determine which physiological properties contribute to temperature adaptation in the squid giant axon, action potentials were recorded from four species of squid whose habitats span a temperature range of 20 degrees C. The environments of these species can be ranked from coldest to warmest as follows: Loligo opalescens>Loligo pealei>Loligo plei>Sepioteuthis sepioidea. Action potential conduction velocities and rise times, recorded at many temperatures, were equivalent for all Loligo species, but significantly slower in S. sepioidea. By contrast, the action potential's fall time differed among species and correlated well with the thermal environment of the species ('warmer' species had slower decay times). The biophysical underpinnings of these differences were examined in voltage-clamped axons. Surprisingly, no differences were found between the activation kinetics or voltage-dependence of Na(+) and K(+) currents. Conductance levels, however, did vary. Maximum Na(+) conductance (g(Na)) in S. sepiodea was significantly less than in the Loligo species. K(+) conductance (gK) was highest in L. pealei, intermediate in L. plei and smallest in S. sepiodea. The time course and magnitude of g(K) and g(Na) were measured directly during membrane action potentials. These data reveal clear species-dependent differences in the amount of g(K) and g(Na) recruited during an action potential.

Acclimatization↗