RESUMEN
The molecular basis of a severe developmental and neurological disorder associated with a de novo G375R variant of the tetrameric BK channel is unknown. Here, we address this question by recording from single BK channels expressed to mimic a G375R mutation heterozygous with a WT allele. Five different types of functional BK channels were expressed: 3% were consistent with WT, 12% with homotetrameric mutant, and 85% with three different types of hybrid (heterotetrameric) channels assembled from both mutant and WT subunits. All channel types except WT showed a marked gain-of-function in voltage activation and a smaller decrease-of-function in single-channel conductance, with both changes in function becoming more pronounced as the number of mutant subunits per tetrameric channel increased. The net cellular response from the five different types of channels comprising the molecular phenotype was a shift of -120 mV in the voltage required to activate half of the maximal current through BK channels, giving a net gain-of-function. The WT and homotetrameric mutant channels in the molecular phenotype were consistent with genetic codominance as each displayed properties of a channel arising from only one of the two alleles. The three types of hybrid channels in the molecular phenotype were consistent with partial dominance as their properties were intermediate between those of mutant and WT channels. A model in which BK channels randomly assemble from mutant and WT subunits, with each subunit contributing increments of activation and conductance, approximated the molecular phenotype of the heterozygous G375R mutation.
Asunto(s)
Canalopatías , Canales de Potasio de Gran Conductancia Activados por el Calcio , Humanos , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Mutación , FenotipoRESUMEN
BK type Ca2+-activated K+ channels activate in response to both voltage and Ca2+. The membrane-spanning voltage sensor domain (VSD) activation and Ca2+ binding to the cytosolic tail domain (CTD) open the pore across the membrane, but the mechanisms that couple VSD activation and Ca2+ binding to pore opening are not clear. Here we show that a compound, BC5, identified from in silico screening, interacts with the CTD-VSD interface and specifically modulates the Ca2+ dependent activation mechanism. BC5 activates the channel in the absence of Ca2+ binding but Ca2+ binding inhibits BC5 effects. Thus, BC5 perturbs a pathway that couples Ca2+ binding to pore opening to allosterically affect both, which is further supported by atomistic simulations and mutagenesis. The results suggest that the CTD-VSD interaction makes a major contribution to the mechanism of Ca2+ dependent activation and is an important site for allosteric agonists to modulate BK channel activation.
Asunto(s)
Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Membrana Celular/metabolismo , Calcio/metabolismoRESUMEN
Large-conductance Ca2+ and voltage-activated K+ (BK) channels control membrane excitability in many cell types. BK channels are tetrameric. Each subunit is composed of a voltage sensor domain (VSD), a central pore-gate domain, and a large cytoplasmic domain (CTD) that contains the Ca2+ sensors. While it is known that BK channels are activated by voltage and Ca2+, and that voltage and Ca2+ activations interact, less is known about the mechanisms involved. We explore here these mechanisms by examining the gating contribution of an interface formed between the VSDs and the αB helices located at the top of the CTDs. Proline mutations in the αB helix greatly decreased voltage activation while having negligible effects on gating currents. Analysis with the Horrigan, Cui, and Aldrich model indicated a decreased coupling between voltage sensors and pore gate. Proline mutations decreased Ca2+ activation for both Ca2+ bowl and RCK1 Ca2+ sites, suggesting that both high-affinity Ca2+ sites transduce their effect, at least in part, through the αB helix. Mg2+ activation also decreased. The crystal structure of the CTD with proline mutation L390P showed a flattening of the first helical turn in the αB helix compared to wild type, without other notable differences in the CTD, indicating that structural changes from the mutation were confined to the αB helix. These findings indicate that an intact αB helix/VSD interface is required for effective coupling of Ca2+ binding and voltage depolarization to pore opening and that shared Ca2+ and voltage transduction pathways involving the αB helix may be involved.
Asunto(s)
Calcio/metabolismo , Activación del Canal Iónico/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Dominios Proteicos/genética , Regulación Alostérica , Animales , Cationes Bivalentes/metabolismo , Membrana Celular/metabolismo , Cristalografía por Rayos X , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/ultraestructura , Potenciales de la Membrana , Mutagénesis Sitio-Dirigida , Oocitos , Técnicas de Placa-Clamp , Prolina/genética , Conformación Proteica en Hélice alfa/genética , Relación Estructura-Actividad , Xenopus laevisRESUMEN
To fertilize an oocyte, sperm must first undergo capacitation in which the sperm plasma membrane becomes hyperpolarized via activation of potassium (K+) channels and resultant K+ efflux. Sperm-specific SLO3 K+ channels are responsible for these membrane potential changes critical for fertilization in mouse sperm, and they are only sensitive to pH i However, in human sperm, the major K+ conductance is both Ca2+- and pH i -sensitive. It has been debated whether Ca2+-sensitive SLO1 channels substitute for human SLO3 (hSLO3) in human sperm or whether human SLO3 channels have acquired Ca2+ sensitivity. Here we show that hSLO3 is rapidly evolving and reveal a natural structural variant with enhanced apparent Ca2+ and pH sensitivities. This variant allele (C382R) alters an amino acid side chain at a principal interface between the intramembrane-gated pore and the cytoplasmic gating ring of the channel. Because the gating ring contains sensors to intracellular factors such as pH and Ca2+, the effectiveness of transduction between the gating ring and the pore domain appears to be enhanced. Our results suggest that sperm-specific genes can evolve rapidly and that natural genetic variation may have led to a SLO3 variant that differs from wild type in both pH and intracellular Ca2+ sensitivities. Whether this physiological variation confers differences in fertility among males remains to be established.
Asunto(s)
Alelos , Calcio/metabolismo , Evolución Molecular , Activación del Canal Iónico/genética , Mutación Missense , Canales de Potasio con Entrada de Voltaje , Espermatozoides/metabolismo , Sustitución de Aminoácidos , Animales , Fertilidad/genética , Humanos , Concentración de Iones de Hidrógeno , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Masculino , Ratones , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/metabolismoRESUMEN
Large conductance Ca2+-activated K+ channels (BK channels) gate open in response to both membrane voltage and intracellular Ca2+ The channel is formed by a central pore-gate domain (PGD), which spans the membrane, plus transmembrane voltage sensors and a cytoplasmic gating ring that acts as a Ca2+ sensor. How these voltage and Ca2+ sensors influence the common activation gate, and interact with each other, is unclear. A previous study showed that a BK channel core lacking the entire cytoplasmic gating ring (Core-MT) was devoid of Ca2+ activation but retained voltage sensitivity (Budelli et al. 2013. Proc. Natl. Acad. Sci. USA http://dx.doi.org/10.1073/pnas.1313433110). In this study, we measure voltage sensor activation and pore opening in this Core-MT channel over a wide range of voltages. We record gating currents and find that voltage sensor activation in this truncated channel is similar to WT but that the coupling between voltage sensor activation and gating of the pore is reduced. These results suggest that the gating ring, in addition to being the Ca2+ sensor, enhances the effective coupling between voltage sensors and the PGD. We also find that removal of the gating ring alters modulation of the channels by the BK channel's ß1 and ß2 subunits.
Asunto(s)
Activación del Canal Iónico/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Potenciales de la Membrana/fisiología , Modelos Teóricos , Animales , Calcio/metabolismo , Oocitos , Técnicas de Placa-Clamp , Xenopus laevisRESUMEN
Single-channel kinetics has proven a powerful tool to reveal information about the gating mechanisms that control the opening and closing of ion channels. This introductory review focuses on the gating of large conductance Ca(2+)- and voltage-activated K(+) (BK or Slo1) channels at the single-channel level. It starts with single-channel current records and progresses to presentation and analysis of single-channel data and the development of gating mechanisms in terms of discrete state Markov (DSM) models. The DSM models are formulated in terms of the tetrameric modular structure of BK channels, consisting of a central transmembrane pore-gate domain (PGD) attached to four surrounding transmembrane voltage sensing domains (VSD) and a large intracellular cytosolic domain (CTD), also referred to as the gating ring. The modular structure and data analysis shows that the Ca(2+) and voltage dependent gating considered separately can each be approximated by 10-state two-tiered models with five closed states on the upper tier and five open states on the lower tier. The modular structure and joint Ca(2+) and voltage dependent gating are consistent with a 50 state two-tiered model with 25 closed states on the upper tier and 25 open states on the lower tier. Adding an additional tier of brief closed (flicker states) to the 10-state or 50-state models improved the description of the gating. For fixed experimental conditions a channel would gate in only a subset of the potential number of states. The detected number of states and the correlations between adjacent interval durations are consistent with the tiered models. The examined models can account for the single-channel kinetics and the bursting behavior of gating. Ca(2+) and voltage activate BK channels by predominantly increasing the effective opening rate of the channel with a smaller decrease in the effective closing rate. Ca(2+) and depolarization thus activate by mainly destabilizing the closed states.
RESUMEN
High-conductance Ca(2+)- and voltage-activated K(+) (Slo1 or BK) channels (KCNMA1) play key roles in many physiological processes. The structure of the Slo1 channel has two functional domains, a core consisting of four voltage sensors controlling an ion-conducting pore, and a larger tail that forms an intracellular gating ring thought to confer Ca(2+) and Mg(2+) sensitivity as well as sensitivity to a host of other intracellular factors. Although the modular structure of the Slo1 channel is known, the functional properties of the core and the allosteric interactions between core and tail are poorly understood because it has not been possible to study the core in the absence of the gating ring. To address these questions, we developed constructs that allow functional cores of Slo1 channels to be expressed by replacing the 827-amino acid gating ring with short tails of either 74 or 11 amino acids. Recorded currents from these constructs reveals that the gating ring is not required for either expression or gating of the core. Voltage activation is retained after the gating ring is replaced, but all Ca(2+)- and Mg(2+)-dependent gating is lost. Replacing the gating ring also right-shifts the conductance-voltage relation, decreases mean open-channel and burst duration by about sixfold, and reduces apparent mean single-channel conductance by about 30%. These results show that the gating ring is not required for voltage activation but is required for Ca(2+) and Mg(2+) activation. They also suggest possible actions of the unliganded (passive) gating ring or added short tails on the core.
Asunto(s)
Activación del Canal Iónico/fisiología , Canal de Potasio Kv1.4/química , Canal de Potasio Kv1.4/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/química , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Animales , Calcio/metabolismo , Humanos , Activación del Canal Iónico/efectos de los fármacos , Cinética , Canal de Potasio Kv1.4/antagonistas & inhibidores , Canal de Potasio Kv1.4/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Magnesio/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Oligonucleótidos/genética , Oocitos/metabolismo , Técnicas de Placa-Clamp , Péptidos/farmacología , Tetraetilamonio/farmacología , XenopusRESUMEN
Fitting dwell-time distributions with sums of exponentials is widely used to characterize histograms of open- and closed-interval durations recorded from single ion channels, as well as for other physical phenomena. However, it can be difficult to identify the contributing exponential components. Here we extend previous methods of exponential sum-fitting to present a maximum-likelihood approach that consistently detects all significant exponentials without the need for user-specified starting parameters. Instead of searching for exponentials, the fitting starts with a very large number of initial exponentials with logarithmically spaced time constants, so that none are missed. Maximum-likelihood fitting then determines the areas of all the initial exponentials keeping the time constants fixed. In an iterative manner, with refitting after each step, the analysis then removes exponentials with negligible area and combines closely spaced adjacent exponentials, until only those exponentials that make significant contributions to the dwell-time distribution remain. There is no limit on the number of significant exponentials and no starting parameters need be specified. We demonstrate fully automated detection for both experimental and simulated data, as well as for classical exponential-sum-fitting problems.
Asunto(s)
Estadística como Asunto/métodos , Activación del Canal Iónico , Canales Iónicos/metabolismo , Funciones de Verosimilitud , Factores de TiempoRESUMEN
Large-conductance, voltage- and Ca(2+)-activated K(+) (BK) channels display near linear current-voltage (I-V) plots for voltages between -100 and +100 mV, with an increasing sublinearity for more positive potentials. As is the case for many types of channels, BK channels are blocked at positive potentials by intracellular Ca(2+) and Mg(2+). This fast block progressively reduces single-channel conductance with increasing voltage, giving rise to a negative slope in the I-V plots beyond about +120 mV, depending on the concentration of the blockers. In contrast to these observations of pronounced differences in the magnitudes and shapes of I-V plots in the absence and presence of intracellular blockers, Schroeder and Hansen (2007. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.200709802) have reported identical I-V plots in the absence and presence of blockers for BK channels, with both plots having reduced conductance and negative slopes, as expected for blockers. Schroeder and Hansen included both Ca(2+) and Mg(2+) in the intracellular solution rather than a single blocker, and they also studied BK channels expressed from α plus ß1 subunits, whereas most previous studies used only α subunits. Although it seems unlikely that these experimental differences would account for the differences in findings between previous studies and those of Schroeder and Hansen, we repeated the experiments using BK channels comprised of α plus ß1 subunits with joint application of 2.5 mM Ca(2+) plus 2.5 mM Mg(2+), as Schroeder and Hansen did. In contrast to the findings of Schroeder and Hansen of identical I-V plots, we found marked differences in the single-channel I-V plots in the absence and presence of blockers. Consistent with previous studies, we found near linear I-V plots in the absence of blockers and greatly reduced currents and negative slopes in the presence of blockers. Hence, studies of conductance mechanisms for BK channels should exclude intracellular Ca(2+)/Mg(2+), as they can reduce conductance and induce negative slopes.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/antagonistas & inhibidores , Bloqueadores de los Canales de Potasio/farmacología , Animales , Calcio/farmacología , Espacio Intracelular/química , Activación del Canal Iónico/efectos de los fármacos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Magnesio/farmacología , Potenciales de la Membrana , Ratones , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/metabolismo , XenopusRESUMEN
Transient receptor potential (TRP) channels couple various environmental factors to changes in membrane potential, calcium influx, and cell signaling. They also integrate multiple stimuli through their typically polymodal activation. Thus, although the TRPM8 channel has been extensively investigated as the major neuronal cold sensor, it is also regulated by various chemicals, as well as by several short channel isoforms. Mechanistic understanding of such complex regulation is facilitated by quantitative single-channel analysis. We have recently proposed a single-channel mechanism of TRPM8 regulation by voltage and temperature. Using this gating mechanism, we now investigate TRPM8 inhibition in cell-attached patches using HEK293 cells expressing TRPM8 alone or coexpressed with its short sM8-6 isoform. This is compared with inhibition by the chemicals N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide (BCTC) and clotrimazole or by elevated temperature. We found that within the seven-state single-channel gating mechanism, inhibition of TRPM8 by short sM8-6 isoforms closely resembles inhibition by increased temperature. In contrast, inhibition by BCTC and that by clotrimazole share a different set of common features.
Asunto(s)
Antifúngicos/farmacología , Clotrimazol/farmacología , Calor , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Pirazinas/farmacología , Piridinas/farmacología , Canales Catiónicos TRPM/antagonistas & inhibidores , Canales Catiónicos TRPM/metabolismo , Células HEK293 , Humanos , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Canales Catiónicos TRPM/genética , Termorreceptores/metabolismoRESUMEN
BK channels are activated by intracellular Ca(2+) and Mg(2+) as well as by depolarization. Such activation is possible because each of the four subunits has two high-affinity Ca(2+) sites, one low-affinity Mg(2+) site, and a voltage sensor. This study further investigates the mechanism of Mg(2+) activation by using single-channel recording to determine separately the action of Mg(2+) on the open and closed states of the channel. To limit Mg(2+) action to the Mg(2+) sites, the two high-affinity Ca(2+) sites are disabled by mutation. When the voltage is stepped from negative holding potentials to +100 mV, we find that 10 mM Mg(2+) decreases the mean closed latency to the first channel opening 2.1-fold, decreases the mean closed interval duration 8.7-fold, increases mean burst duration 10.1-fold, increases the number of openings per burst 4.4-fold, and increases mean open interval duration 2.3-fold. Hence, Mg(2+) can bind to closed BK channels, increasing their opening rates, and to open BK channels, decreasing their closing rates. To explore the relationship between Mg(2+) action and voltage sensor activation, we record single-channel activity in macropatches containing hundreds of channels. Open probability (P(o)) is dramatically increased by 10 mM Mg(2+) when voltage sensors are activated with either depolarization or the mutation R210C. The increased P(o) arises from large decreases in mean closed interval durations and moderate increases in mean open interval durations. In contrast, 10 mM Mg(2+) has no detectable effects on P(o) or interval durations when voltage sensors are deactivated with very negative potentials or the mutation R167E. These observations are consistent with a model in which Mg(2+) can bind to and alter the gating of both closed and open states to increase P(o), provided that one or more voltage sensors are activated.
Asunto(s)
Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Magnesio/metabolismo , Animales , Sitios de Unión , Femenino , Activación del Canal Iónico/fisiología , Cinética , Potenciales de la Membrana/fisiología , Mutación , Oocitos/metabolismo , Técnicas de Placa-Clamp , Xenopus laevisRESUMEN
Presynaptic short-term plasticity (STP) dynamically modulates synaptic strength in a reversible manner on a timescale of milliseconds to minutes. For low basal vesicular release probability (prob0), four components of enhancement, F1 and F2 facilitation, augmentation (A), and potentiation (P), increase synaptic strength during repetitive nerve activity. For release rates that exceed the rate of replenishment of the readily releasable pool (RRP) of synaptic vesicles, depression of synaptic strength, observed as a rundown of postsynaptic potential amplitudes, can also develop. To understand the relationship between enhancement and depression at the frog (Rana pipiens) neuromuscular synapse, data obtained over a wide range of prob0 using patterned stimulation are analyzed with a hybrid model to reveal the components of STP. We find that F1, F2, A, P, and depletion of the RRP all contribute to STP during repetitive nerve activity at low prob0. As prob0 is increased by raising Ca(o)(2+) (extracellular Ca2+), specific components of enhancement no longer contribute, with first P, then A, and then F2 becoming undetectable, even though F1 continues to enhance release. For levels of prob0 that lead to appreciable depression, only F1 and depletion of the RRP contribute to STP during rundown, and for low stimulation rates, F2 can also contribute. These observations place prob0-dependent limitations on which components of enhancement contribute to STP and suggest some fundamental mechanistic differences among the components. The presented model can serve as a tool to readily characterize the components of STP over wide ranges of prob0.
Asunto(s)
Calcio/fisiología , Unión Neuromuscular/fisiología , Plasticidad Neuronal/fisiología , Transmisión Sináptica/fisiología , Animales , Estimulación Eléctrica , Electrofisiología , Potenciales Postsinápticos Excitadores/fisiología , Modelos Neurológicos , Rana pipiens , Vesículas Sinápticas/fisiologíaRESUMEN
Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels have the largest conductance (250-300 pS) of all K(+)-selective channels. Yet, the contributions of the various parts of the ion conduction pathway to the conductance are not known. Here, we examine the contribution of the entrance to the inner cavity to the large conductance. Residues at E321/E324 on each of the four α subunits encircle the entrance to the inner cavity. To determine if 321/324 is accessible from the inner conduction pathway, we measured single-channel current amplitudes before and after exposure and wash of thiol reagents to the intracellular side of E321C and E324C channels. MPA(-) increased currents and MTSET(+) decreased currents, with no difference between positions 321 and 324, indicating that side chains at 321/324 are accessible from the inner conduction pathway and have equivalent effects on conductance. For neutral amino acids, decreasing the size of the entrance to the inner cavity by substituting large side-chain amino acids at 321/324 decreased outward single-channel conductance, whereas increasing the size of the entrance with smaller side-chain substitutions had little effect. Reductions in outward conductance were negated by high [K(+)](i). Substitutions had little effect on inward conductance. Fitting plots of conductance versus side-chain volume with a model consisting of one variable and one fixed resistor in series indicated an effective diameter and length of the entrance to the inner cavity for wild-type channels of 17.7 and 5.6 Å, respectively, with the resistance of the entrance â¼7% of the total resistance of the conduction pathway. The estimated dimensions are consistent with the structure of MthK, an archaeal homologue to BK channels. Our observations suggest that BK channels have a low resistance, large entrance to the inner cavity, with the entrance being as large as necessary to not limit current, but not much larger.
Asunto(s)
Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Proteínas de Unión al ADN , Electroquímica , Transporte Iónico , Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Modelos Moleculares , Oocitos/fisiología , Potasio/metabolismo , Conformación Proteica , Proteínas ViralesRESUMEN
Transient receptor potential (TRP) channels play critical roles in cell signaling by coupling various environmental factors to changes in membrane potential that modulate calcium influx. TRP channels are typically activated in a polymodal manner, thus integrating multiple stimuli. Although much progress has been made, the underlying mechanisms of TRP channel activation are largely unknown. The TRPM8 cation channel has been extensively investigated as a major neuronal cold sensor but is also activated by voltage, calcium store depletion, and some lipids as well as by compounds that produce cooling sensations, such as menthol or icilin. Several models of TRPM8 activation have been proposed to explain the interaction between these diverse stimuli. However, a kinetic scheme is not yet available that can describe the detailed single-channel kinetics to gain further insight into the underlying gating mechanism. To work toward this goal, we investigated voltage-dependent single-channel gating in cell-attached patches at two different temperatures (20 and 30 °C) using HEK293 cells stably expressing TRPM8. Both membrane depolarization and cooling increased channel open probability (P(o)) mainly by decreasing the duration of closed intervals, with a smaller increase in the duration of open intervals. Maximum likelihood analysis of dwell times at both temperatures indicated gating in a minimum of five closed and two open states, and global fitting over a wide range of voltages identified a seven-state model that described the voltage dependence of P(o), the single-channel kinetics, and the response of whole-cell currents to voltage ramps and steps. The major action of depolarization and cooling was to accelerate forward transitions between the same two sets of adjacent closed states. The seven-state model provides a general mechanism to account for TRPM8 activation by membrane depolarization at two temperatures and can serve as a starting point for further investigations of multimodal TRP activation.
Asunto(s)
Activación del Canal Iónico/fisiología , Canales Catiónicos TRPM/fisiología , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Potenciales de la Membrana/fisiología , TemperaturaRESUMEN
Voltage-dependent gating mechanisms of large conductance Ca(2+) and voltage-activated (BK) channels were investigated using two-dimensional maximum likelihood analysis of single-channel open and closed intervals. To obtain sufficient data at negative as well as positive voltages, single-channel currents were recorded at saturating Ca(2+) from BK channels mutated to remove the RCK1 Ca(2+) and Mg(2+) sensors. The saturating Ca(2+) acting on the Ca(2+) bowl sensors of the resulting BK(B) channels increased channel activity while driving the gating into a reduced number of states, simplifying the model. Five highly constrained idealized gating mechanisms based on extensions of the Monod-Wyman-Changeux model for allosteric proteins were examined. A 10-state model without coupling between the voltage sensors and the opening/closing transitions partially described the voltage dependence of Po but not the single-channel kinetics. With allowed coupling, the model gave improved descriptions of Po and approximated the single-channel kinetics; each activated voltage sensor increased the opening rate approximately an additional 23-fold while having little effect on the closing rate. Allowing cooperativity among voltage sensors further improved the description of the data: each activated voltage sensor increased the activation rate of the remaining voltage sensors approximately fourfold, with little effect on the deactivation rate. The coupling factor was decreased in models with cooperativity from approximately 23 to approximately 18. Whether the apparent cooperativity among voltage sensors arises from imposing highly idealized models or from actual cooperativity will require additional studies to resolve. For both cooperative and noncooperative models, allowing transitions to five additional brief (flicker) closed states further improved the description of the data. These observations show that the voltage-dependent single-channel kinetics of BK(B) channels can be approximated by highly idealized allosteric models in which voltage sensor movement increases Po mainly through an increase in channel opening rates, with limited effects on closing rates.
Asunto(s)
Calcio/fisiología , Activación del Canal Iónico/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Modelos Moleculares , Animales , Células Cultivadas , Humanos , Riñón/citología , Riñón/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Ratones , Técnicas de Placa-Clamp , TransfecciónRESUMEN
Discrete state Markov models have proven useful for describing the gating of single ion channels. Such models predict that the dwell-time distributions of open and closed interval durations are described by mixtures of exponential components, with the number of exponential components equal to the number of states in the kinetic gating mechanism. Although the exponential components are readily calculated (Colquhoun and Hawkes, 1982, Phil. Trans. R. Soc. Lond. B. 300:1-59), there is little practical understanding of the relationship between components and states, as every rate constant in the gating mechanism contributes to each exponential component. We now resolve this problem for simple models. As a tutorial we first illustrate how the dwell-time distribution of all closed intervals arises from the sum of constituent distributions, each arising from a specific gating sequence. The contribution of constituent distributions to the exponential components is then determined, giving the relationship between components and states. Finally, the relationship between components and states is quantified by defining and calculating the linkage of components to states. The relationship between components and states is found to be both intuitive and paradoxical, depending on the ratios of the state lifetimes. Nevertheless, both the intuitive and paradoxical observations can be described within a consistent framework. The approach used here allows the exponential components to be interpreted in terms of underlying states for all possible values of the rate constants, something not previously possible.
Asunto(s)
Simulación por Computador , Activación del Canal Iónico/fisiología , Modelos Biológicos , Cinética , Cadenas de Markov , MatemáticaRESUMEN
Micromolar zinc potentiates neuronal nicotinic acetylcholine receptors (nAChRs) in a subtype-dependent manner. Zinc potentiates receptor function even at saturating agonist concentrations, without altering the receptor desensitization rate. Potentiation could occur through an increase in the number of available receptors, an increase in single-channel current amplitude, or an increase in single-channel open probability. To distinguish among these possibilities, we examined rat neuronal nAChRs expressed in Xenopus oocytes. Blockade of a large fraction of ACh activated alpha4beta4 or alpha4beta2 receptors by the open channel blocker hexamethonium failed to change the extent of potentiation by zinc, suggesting that zinc does not change the number of available receptors. The single-channel amplitudes of ACh (1 microM) activated alpha4beta4 receptors in outside-out patches were similar in the absence and the presence of 100 microM zinc (3.0 +/- 0.1 and 2.9 +/- 0.1 pA, respectively). To determine the effect of zinc on single-channel open probability, we examined alpha4beta4 receptors in cell-attached patches. The open probability at 100 nM ACh (0.011 +/- 0.002) was increased 4.5-fold by 100 microM zinc (0.050 +/- 0.008), accounting for most of the potentiation observed at the whole cell level. The increase in open probability was due to an increase in burst duration, which increased from 207 +/- 38 ms in the absence of zinc to 830 +/- 189 ms in the presence of zinc. Our results suggest that potentiation of neuronal nAChRs by zinc is due to a stabilization of the bursting states of the receptor.
Asunto(s)
Potenciales de la Membrana/efectos de los fármacos , Receptores Nicotínicos/fisiología , Oligoelementos/farmacología , Zinc/farmacología , Acetilcolina/farmacología , Animales , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Estimulación Eléctrica/métodos , Hexametonio/farmacología , Activación del Canal Iónico/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Antagonistas Nicotínicos/farmacología , Oocitos , Técnicas de Placa-Clamp , Probabilidad , Receptores Nicotínicos/genética , Factores de Tiempo , Transfección , Xenopus laevisRESUMEN
Patch-clamp recording has revolutionized the study of ion channels, transporters, and the electrical activity of small cells. Vital to this method is formation of a tight seal between glass recording pipette and cell membrane. To better understand seal formation and improve practical application of this technique, we examine the effects of divalent ions, protons, ionic strength, and membrane proteins on adhesion of membrane to glass and on seal resistance using both patch-clamp recording and atomic force microscopy. We find that H(+), Ca(2+), and Mg(2+) increase adhesion force between glass and membrane (lipid and cellular), decrease the time required to form a tight seal, and increase seal resistance. In the absence of H(+) (10(-10) M) and divalent cations (<10(-8) M), adhesion forces are greatly reduced and tight seals are not formed. H(+) (10(-7) M) promotes seal formation in the absence of divalent cations. A positive correlation between adhesion force and seal formation indicates that high resistance seals are associated with increased adhesion between membrane and glass. A similar ionic dependence of the adhesion of lipid membranes and cell membranes to glass indicates that lipid membranes without proteins are sufficient for the action of ions on adhesion.