Pipette Potential Research Articles

Unusual properties of α7 nicotinic acetylcholine receptor ion channels in B lymphocyte-derived SP-2/0 cells

This study demonstrates the presence of α7 nicotinic acetylcholine receptors (nAChR) in B lymphocyte-derived SP-2/0 cells by means of flow cytometry and immunocytochemistry. According to lectin and sandwich ELISA, the α7 subunits expressed in SP-2/0 cells are more glycosylated compared to those expressed in the brain or normal B lymphocytes and are combined with β2 subunits. At zero and negative pipette potentials, either acetylcholine or α7-specific agonist PNU282987 stimulated the ion channel activity in SP-2/0 cells revealed by single channel patch-clamp recordings. The conductivity was within the range of 19 to 39 pS and reversal potential was between −17 mV and +28 mV, the currents were potentiated by α7-specific positive allosteric modulator PNU120596 and were partially blocked by α7-specific antagonist methyllicaconitine (MLA). However, they were oriented downwards suggesting that the channels mediated the cation outflux rather than influx. As shown by Ca2+ imaging studies, PNU282987 did not stimulate immediate Ca2+ influx into SP-2/0 cells. Instead, Ca2+ influx through Ca-release-activated channels (CRACs) was observed within minutes after either PNU282987 or MLA application. It is concluded that SP-2/0 express α7β2 nAChRs, which mediate the cation outflux under negative pipette potentials applied, possibly, due to depolarized membrane or negative surface charge formed by carbohydrate residues. In addition, α7β2 nAChRs may influence CRACs in ion-independent way.

Severe Hypoxia Activates BK channel in Rat Carotid Body Glomus Cells

Large-conductance Ca2+-activated K+ channel (BK) is highly expressed in rat carotid body glomus cells. Previous studies have reported that hypoxia inhibits BK, but the effect of hypoxia in intact glomus cells is not well defined. As hypoxia depolarizes glomus cells and elevates intracellular [Ca2+], we tested the hypothesis that hypoxia activates BK in intact cells. In cell-attached patches with 140 mM KCl in the pipette and 5 mM KCl in the bath solution, BK was always in the closed state, and hypoxia (0% O2) activated BK in ~10% of patches tested. In cell-attached patches, bath application of 1 mM NaCN also activated BK in some patches. When BK was first activated by depolarizing the patch membrane by changing the pipette potential or by perfusing the cells with high KCl (10-15 mM), hypoxia increased the BK activity reversibly. The stimulatory effect of hypoxia on BK was also observed in cell-attached patches with 5 mM KCl in the pipette, if the patch membrane was depolarized first. Study of the relationship among [Ca2+]i, cell Em and BK activity indicated that BK begins to activate when cell depolarizes ~30 mV and [Ca2+]i increases above ~500 nM (from a basal level of ~100 nM). Thus, severe hypoxia activates BK in glomus cells, and this presumably helps to limit over-excitation.

Cell-attached recordings of responses evoked by photorelease of GABA in the immature cortical neurons

We present a novel non-invasive technique to measure the polarity of GABAergic responses based on cell-attached recordings of currents activated by laser-uncaging of GABA. For these recordings, a patch pipette was filled with a solution containing RuBi-GABA, and GABA was released from this complex by a laser beam conducted to the tip of the patch pipette via an optic fiber. In cell-attached recordings from neocortical and hippocampal neurons in postnatal days P2-5 rat brain slices in vitro, we found that laser-uncaging of GABA activates integral cell-attached currents mediated by tens of GABA(A) channels. The initial response was inwardly directed, indicating a depolarizing response to GABA. The direction of the initial response was dependent on the pipette potential and analysis of its slope-voltage relationships revealed a depolarizing driving force of +11 mV for the currents through GABA channels. Initial depolarizing responses to GABA uncaging were inverted to hyperpolarizing in the presence of the NKCC1 blocker bumetanide. Current-voltage relationships of the currents evoked by RuBi-GABA uncaging using voltage-ramps at the peak of responses not only revealed a bumetanide-sensitive depolarizing reversal potential of the GABA(A) receptor mediated responses, but also showed a strong voltage-dependent hysteresis. Upon desensitization of the uncaged-GABA response, current-voltage relationships of the currents through single GABA(A) channels revealed depolarizing responses with the driving force values similar to those obtained for the initial response. Thus, cell-attached recordings of the responses evoked by local intrapipette GABA uncaging are suitable to assess the polarity of the GABA(A)-Rs mediated signals in small cell compartments.

Riluzole blocks human muscle acetylcholine receptors

Riluzole, the only drug available against amyotrophic lateral sclerosis (ALS), has recently been shown to block muscle ACh receptors (AChRs), raising concerns about possible negative side-effects on neuromuscular transmission in treated patients. In this work we studied riluzole's impact on the function of muscle AChRs in vitro and on neuromuscular transmission in ALS patients, using electrophysiological techniques. Human recombinant AChRs composed of α(1)β(1)δ subunits plus the γ or ε subunit (γ- or ε-AChR) were expressed in HEK cells or Xenopus oocytes. In both preparations, riluzole at 0.5 μm, a clinically relevant concentration, reversibly reduced the amplitude and accelerated the decay of ACh-evoked current if applied before coapplication with ACh. The action on γ-AChRs was more potent and faster than on ε-AChRs. In HEK outside-out patches, riluzole-induced block of macroscopic ACh-evoked current gradually developed during the initial milliseconds of ACh presence. Single channel recordings in HEK cells and in human myotubes from ALS patients showed that riluzole prolongs channel closed time, but has no effect on channel conductance and open duration. Finally, compound muscle action potentials (CMAPs) evoked by nerve stimulation in ALS patients remained unaltered after a 1 week suspension of riluzole treatment. These data indicate that riluzole, while apparently safe with regard to synaptic transmission, may affect the function of AChRs expressed in denervated muscle fibres of ALS patients, with biological consequences that remain to be investigated.

Measuring the Firing Rate of High-Resistance Neurons with Cell-Attached Recording

Cell-attached recording is extensively used to study the firing rate of mammalian neurons, but potential limitations of the method have not been investigated in detail. Here we perform cell-attached recording of molecular layer interneurons in cerebellar slices from rats and mice, and we study how experimental conditions influence the measured firing rate. We find that this rate depends on time in cell-attached mode, on pipette potential, and on pipette ionic composition. In the first minute after sealing, action currents are variable in shape and size, presumably reflecting membrane instability. The firing rate remains approximately constant during the first 4 min after sealing and gradually increases afterward. Making the pipette potential more positive leads to an increase in the firing rate, with a steeper dependence on voltage if the pipette solution contains K(+) as the main cation than if it contains Na(+). Ca(2+) imaging experiments show that establishing a cell-attached recording can result in an increased somatic Ca(2+) concentration, reflecting an increased firing rate linked to an increase in the pipette-cell conductance. Pipette effects on cell firing are traced to a combination of passive electrical coupling, opening of voltage- and Ca(2+)-sensitive K(+) channels (BK channels) after action potentials, and random activation of voltage-insensitive, presumably mechanosensitive, cationic channels. We conclude that, unless experimental conditions are optimized, cell-attached recordings in small neurons may report erroneous firing rates.

A Mechanosensitive Channel (MscS) with Multiple Conducting States Shows Stronger Selectivity for Anions in the Presence of Divalent Cations

The mechanosensitive channel of small conductance (MscS) in E. coli plays an integral role in osmoprotection[1]. The channel gates in response to an increase in membrane tension as a result of hypo-osmotic shock and acts as an ‘emergency release valve’ preventing cell lysis. Very little is known about the permeation of divalent cations through MscS. This was addressed by reconstituting MscS into azolectin liposomes (protein:lipid - 1:10,000) using the dehydration-rehydration method as previously described[2]. Single-channel recordings were performed in symmetrical 100 mM BaCl2, CaCl2, and MgCl2. At least 5 distinct conducting states were present at +50 mV pipette potential. A further increase in pipette potential to +70 mV resulted in 8 distinct conducting states which were conserved up to the highest voltage tested (+150 mV). It is not known whether these sub-conducting states are physiologically relevant. In addition to these sub-conducting states the substitution of K+ by Ba2+, Ca2+ and Mg2+ increased MscS rectification at negative pipette potentials. The rectification increased as the hydrated ionic radius of the major permeating cation increased (Mg2+ = Ca2+ = Ba2+ > Li+>Na+>K+>Rb+>Cs+). It was hypothesised that divalent cations interact and screen electronegative charges surrounding the portals of the cytoplasmic domain of MscS. If this were the case then divalent cations would increase the anion selectivity of MscS. In line with this prediction in asymmetric BaCl2 the calculated permeability ratio was PCl/PBa = ∼11.5 as opposed to PCl/PK = ∼3.5 in asymmetric KCl. The significance of the effects of divalent ions on MscS gating and ion selectivity is unknown at present, however they may give us a new insight into the global gating mechanism of MscS and importance of calcium in osmoregulation of bacterial cells.

Outward Currents Through “low” Conductance Non-Mechanosensitive Channels in Escherichia Coli Membranes

Mechanosensitive high conductance channels in bacteria have received considerable attention (e.g.1,2) and have a presumed role against osmotic stress. In contrast non-mechanosensitive channels are less well described. In patch-clamp on-spheroplast recordings from E. coli (BW25113) with high K+ (200 mM) in both the pipette and bath solution MscL, MscK, MscS and MscM channels are reproducibly seen, and appear to be the only channels present. In on-spheroplast recordings with high Ca2+ or Mg2+ (100 mM) in the pipette and bath solution both MscL and MscM channels are observed. In addition, lower conductance channels of < 30 pS (n = 12) and 80 - 100 pS (n = 12) respectively are observed at depolarised potentials (pipette potential - 55 mV and above) in the absence of applied pressure in these solutions. These openings are long duration, non-flickery (in the case of the < 30 pS channel) and shorter duration, flickery (in the case of the 80-100 pS channel). The I-V relationships appear linear over a limited voltage range (pipette potentials −55 to −90 mV). The “low” conductance channels are seen in the absence of Cl- ions (sulphate as replacement) in the pipette and bath solutions. These channels are not seen in excised inside-out patches (n = 6). The identity, ionic selectivity and role of these channels remain to be characterised. An anion preferring 100 pS conductance channel in the absence of applied pressure has been reported previously in excised patches at both depolarised and hyperpolarised potentials in high K+ (150) solutions (3).1. Martinac, B. et al. PNAS, 84, 2297-2301 (1987)2. Booth, I.R. et al. Nature Rev. Microbiol., 5, 431-440 (2007).3. Kubalski, A. Biochim. et Biophys. Acta, 1238, 177-182 (1995)1CDC is supported by a Cardiff University Endowment Scholarship.

Ankyrin-B Regulates Kir6.2 Membrane Expression and Function in Heart

Ankyrin polypeptides are critical for normal membrane protein expression in diverse cell types, including neurons, myocytes, epithelia, and erythrocytes. Ankyrin dysfunction results in defects in membrane expression of ankyrin-binding partners (including ion channels, transporters, and cell adhesion molecules), resulting in aberrant cellular function and disease. Here, we identify a new role for ankyrin-B in cardiac cell biology. We demonstrate that cardiac sarcolemmal K(ATP) channels directly associate with ankyrin-B in heart via the K(ATP) channel alpha-subunit Kir6.2. We demonstrate that primary myocytes lacking ankyrin-B display defects in Kir6.2 protein expression, membrane expression, and function. Moreover, we demonstrate a secondary role for ankyrin-B in regulating K(ATP) channel gating. Finally, we demonstrate that ankyrin-B forms a membrane complex with K(ATP) channels and the cardiac Na/K-ATPase, a second key membrane transporter involved in the cardiac ischemia response. Collectively, our new findings define a new role for cardiac ankyrin polypeptides in regulation of ion channel membrane expression in heart.

Extracellular Allosteric Regulatory Subdomain within the γ Subunit of the Epithelial Na+ Channel

The activity of the epithelial Na(+) channel (ENaC) is modulated by Na(+) self-inhibition, a down-regulation of the open probability of ENaC by extracellular Na(+). A His residue within the extracellular domain of gammaENaC (gammaHis(239)) was found to have a critical role in Na(+) self-inhibition. We investigated the functional roles of residues in the vicinity of this His by mutagenesis and analyses of Na(+) self-inhibition responses in Xenopus oocytes. Significant changes in the speed and magnitude of Na(+) self-inhibition were observed in 16 of the 47 mutants analyzed. These 16 mutants were distributed within a 22-residue tract. We further characterized this scanned region by examining the accessibility of introduced Cys residues to the sulfhydryl reagent MTSET. External MTSET irreversibly increased or decreased currents in 13 of 47 mutants. The distribution patterns of the residues where substitutions significantly altered Na(+) self-inhibition or/and conferred sensitivity to MTSET were consistent with the existence of two helices within this region. In addition, single channel recordings of the gammaH239F mutant showed that, in the absence of Na(+) self-inhibition and with an increased open probability, ENaCs still undergo transitions between open and closed states. We conclude that gammaHis(239) functions within an extracellular allosteric regulatory subdomain of the gamma subunit that has an important role in conferring the response of the channel to external Na(+).

Voltage‐gated chloride channels in storage cells of the anhydrobiotic eutardigrade Richtersius coronifer

Tardigrades comprise polyextremophilic species that can survive in an anhydrobiotic state for years. Anhydrobiosis may be dependent on properties of the free floating storage cells found in the body cavity of all tardigrades. We studied ion channels in the storage cell membrane with patch clamp technique, using native cells from the eutardigrade Richtersius coronifer.In cell‐attached configuration the most frequently encountered ion channel had a linear i/V‐relationship with a reversal potential (Vrev) of ~0.4 mV (n ≥6). The single‐channel conductance was 82.4±0.0 pS (mean±SE, n≥6). Binomial analysis of patches with 2–4 active channels showed stochastic behavior. The open‐state probability increased with the pipette potential, indicating voltage‐gating. In whole‐cell configuration the currents showed outward rectification with Vrev = −18.2±1.1 mV (mean±SE, n=24), closest to the equilibrium potential for Cl−. Perfusion with Na‐gluconate Ringer right‐shifted Vrev from −19.5±1.9 to 4.6±1.3 mV, and the current decreased significantly (mean±SE, n=5), confirming that a significant component of the whole‐cell current is attributed to Cl− channels. This was verified by perfusion with KCl Ringer (n=5) and addition of BaCl2 (n=7), neither of which significantly changed Vrev. Future studies are needed to firmly establish the identity and function of the demonstrated ion channel in storage cells.

The N Terminal M2 Cap of Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors (AChRs) isomerize (‘gate’) between a low affinity/non-conducting (R) and a high affinity/ion-conducting (R∗) conformation. Many different residues in this large, heteropentameric membrane protein have been shown to contribute to the free energy difference between the R and R∗ structures. Previously we showed that at the N-terminal ‘cap’ of the M2, pore-lining helix (positions 18′-27′) there are large and early energy changes in the α subunit whereas in the e subunit the residues are mostly iso-energetic. We measured the energy sensitivity (computed from the apparent range in diliganded equilibrium constant E2) and relative timing (Φ values) of the isomerization movements of residues in the N-terminal ‘cap’ domain of the δ and β subunits. We used cell attached, single-channel analysis to quantify the energetic consequences of point mutations (mouse α2βδe, HEK 293 cells, +70 mV pipette potential, cell-attached, 22° C , activated by 20 mM choline or 0.5 mM ACh). The probed positions were δ: I18′, S19′, K20′, R21′, L22′, P23′, A24′, T25′ and M27′ (a total of 43 mutants) and β:A19′, K21′, V22′, P23′, E24′, S26′, and L27′ (a total of 34 mutants). Of these, only one position, δS19′, showed a >2 kcal/mol range-energy (Φ = 0.20 ± 0.07). The only residues having a >10-fold range in E2 (1.35 kcal/mol) were: δ (18′, 19′, 20′, 21′ and 23′) and β (21′, 24′ and 27′). These all showed approximately the same Φ value: 0.30. Comparing this value to those in the ‘cap’ region of other subunits, the Φ-order is α > e > δ = β. The high Φ and large energy changes are apparently only in the α subunit M2-cap, which indicates that domain plays a special role in AChR gating. NIH (NS 23513, 064969)

Biophysics and Structure of the Patch and the Gigaseal

Interpreting channel behavior in patches requires an understanding of patch structure and dynamics, especially in studies of mechanosensitive channels. High resolution optical studies show that patch formation occurs via blebbing that disrupts normal membrane structure and redistributes in situ components including ion channels. There is a 1–2 μm region of the seal below the patch where proteins are excluded and this may consist of extracted lipids that form the gigaseal. Patch domes often have complex geometries with inhomogeneous stresses due to the membrane-glass adhesion energy (Ea), cytoskeletal forces, and possible lipid subdomains. The resting tension in the patch dome ranges from 1–4 mN/m, a significant fraction of the lytic tension of a bilayer (∼10 mN/m). Thus, all patch experiments are conducted under substantial, and uneven, resting tension that may alter the kinetics of many channels. Ea seems dominated by van der Waals attraction overlaid with a normally repulsive Coulombic force. High ionic strength pipette saline increased Ea and, surprisingly, increased cytoskeletal rigidity in cell-attached patches. Low pH pipette saline also increased Ea and reduced the seal selectivity for cations, presumably by neutralizing the membrane surface charge. The seal is a negatively charged, cation selective, space with a resistance of ∼7 gigohm/μm in 100 mM KCl, and the high resistivity of the space may result from the presence of high viscosity glycoproteins. Patches creep up the pipette over time with voltage independent and voltage dependent components. Voltage-independent creep is expected from the capillary attraction of Ea and the flow of fresh lipids from the cell. Voltage-dependent creep seems to arise from electroosmosis in the seal. Neutralization of negative charges on the seal membrane with low pH decreased the creep rate and reversed the direction of creep at positive pipette potentials.

Aromatic Residues ∈Trp-55 and δTrp-57 and the Activation of Acetylcholine Receptor Channels

The two transmitter binding sites of the neuromuscular acetylcholine (ACh) receptor channel contain several aromatic residues, including a tryptophan located on the complementary, negative face of each binding pocket. These two residues, Trp-55 in the epsilon subunit and Trp-57 in the delta subunit, were mutated (AEFHILRVY), and for most constructs the rate constants for acetylcholine binding and channel gating were estimated by using single channel kinetic analyses. The rate constants for unliganded channel opening and closing were also estimated for some mutants. From these measurements we calculated all of the equilibrium constants of the "allosteric" cycle as follows: diliganded gating, unliganded gating, dissociation from the C(losed) conformation, and dissociation from the O(pen) conformation. The results indicate the following. (i) These aromatic side chains play a relatively minor role in ACh receptor channel activation. (ii) The main consequence of mutations is to reduce the affinity of the O conformation of the binding site for ACh, with the effect being greater at the epsilon subunit. (iii) In epsilon (but not delta) the aromatic nature of the side chain is important in determining affinity, to a slightly greater degree in the O conformation. Phi value analyses (of both tryptophan residues) show Phi approximately 1 for both the ACh binding and diliganded gating reactions. (iv) This suggests that the structural boundaries of the dynamic elements of the gating conformational change may not be subunit-delimited, and (v) the mutated tryptophan residues experience energy changes that occur relatively early in both the ligand-binding and channel-gating reactions.

Effects of extracellular chloride ion on epithelial sodium channel (ENaC) in arginine vasotocin (AVT)-stimulated renal epithelial cells

The epithelial Na(+) channel (ENaC) contributes to control of blood pressure by reabsorbing Na(+) in the cortical collecting duct of the kidney. The luminal Cl(-) concentration in the duct varies under physiological conditions. As the body Na(+) content is lower, the luminal Cl(-) concentration in the duct becomes lower. Thus, we hypothesized that the extracellular Cl(-) elevates ENaC activity in AVT-stimulated renal epithelial A6 cells (a model cell line of the cortical collecting duct) leading to recovery from a low body Na(+) content. To clarify this point, we studied effects of extracellular Cl(-) concentration on ENaC activity using cell-attached patch clamp technique. We found that ENaC had a single-channel conductance of 4.6 +/- 0.1 pS (mean +/- SE) and channel activity (open probability, Po) of 0.30 +/- 0.02 at a pipette potential of 60 mV. Lowering pipette Cl(-) concentration diminished Po to 0.23 +/- 0.02 associated with a significant decrease in open time from 0.78 +/- 0.03 to 0.61 +/- 0.02 s with no significant change in closed time, and shifted the current-voltage relationship leftward. These results suggest that the extracellular Cl(-) regulates the ENaC-mediated Na(+) reabsorption by affecting ENaC properties in AVT-stimulated renal epithelial cells.

A Pore-blocking Hydrophobic Motif at the Cytoplasmic Aperture of the Closed-state Nav1.7 Channel Is Disrupted by the Erythromelalgia-associated F1449V Mutation

Sodium channel Na(v)1.7 has recently elicited considerable interest as a key contributor to human pain. Gain-of-function mutations of Na(v)1.7 produce painful disorders, whereas loss-of-function Na(v)1.7 mutations produce insensitivity to pain. The inherited erythromelalgia Na(v)1.7/F1449V mutation, within the C terminus of domain III/transmembrane helix S6, shifts channel activation by -7.2 mV and accelerates time to peak, leading to nociceptor hyperexcitability. We constructed a homology model of Na(v)1.7, based on the KcsA potassium channel crystal structure, which identifies four phylogenetically conserved aromatic residues that correspond to DIII/F1449 at the C-terminal end of each of the four S6 helices. The model predicted that changes in side-chain size of residue 1449 alter the pore's cytoplasmic aperture diameter and reshape inter-domain contact surfaces that contribute to closed state stabilization. To test this hypothesis, we compared activation of wild-type and mutant Na(v)1.7 channels F1449V/L/Y/W by whole cell patch clamp analysis. All but the F1449V mutation conserve the voltage dependence of activation. Compared with wild type, time to peak was shorter in F1449V, similar in F1449L, but longer for F1449Y and F1449W, suggesting that a bulky, hydrophobic residue is necessary for normal activation. We also substituted the corresponding aromatic residue of S6 in each domain individually with valine, to mimic the naturally occurring Na(v)1.7 mutation. We show that DII/F960V and DIII/F1449V, but not DI/Y405V or DIV/F1752V, regulate Na(v)1.7 activation, consistent with well established conformational changes in DII and DIII. We propose that the four aromatic residues contribute to the gate at the cytoplasmic pore aperture, and that their ring side chains form a hydrophobic plug which stabilizes the closed state of Na(v)1.7.

Small Molecule Activator of the Human Epithelial Sodium Channel

The epithelial sodium channel (ENaC), a heterotrimeric complex composed of alpha, beta, and gamma subunits, belongs to the ENaC/degenerin family of ion channels and forms the principal route for apical Na(+) entry in many reabsorbing epithelia. Although high affinity ENaC blockers, including amiloride and derivatives, have been described, potent and specific small molecule ENaC activators have not been reported. Here we describe compound S3969 that fully and reversibly activates human ENaC (hENaC) in an amiloride-sensitive and dose-dependent manner in heterologous cells. Mechanistically, S3969 increases hENaC open probability through interactions requiring the extracellular domain of the beta subunit. hENaC activation by S3969 did not require cleavage by the furin protease, indicating that nonproteolyzed channels can be opened. Function of alphabetaG37Sgamma hENaC, a channel defective in gating that leads to the salt-wasting disease pseudohypoaldosteronism type I, was rescued by S3969. Small molecule activation of hENaC may find application in alleviating human disease, including pseudohypoaldosteronism type I, hypotension, and neonatal respiratory distress syndrome, when improved Na(+) flux across epithelial membranes is clinically desirable.

Detrended fluctuation analysis as a statistical method to study ion single channel signal

The scaling behaviors of ion single channel current signal time series could be analyzed by means of detrended fluctuation analysis. Nevertheless, the statistical analysis of an ionic current signal recorded from voltage dependence K+ single channel is presented. The detrended fluctuation analysis (DFA) exponent alpha is significantly larger than 0.5, as (DFA) exponents were calculated for 4 different pipette potentials in rat dorsal root ganglion neurons. alpha=0.9475+/-0.006 for V=-30 mV; alpha=0.958+/-0.004 for V=-40 mV; alpha=0.966+/-0.005 for V=-50 mV; alpha=0.971+/-0.03 for V=-60 mV. The scaling exponents for different pipette potentials reveal the existence of memory in ion channels, and at the same time, the memory in ion channel depends on the pipette potential. The result of Markovian model data showed that it had different DFA exponent alpha which indicates that long-range correlation effect is present amongst the continued conducting states of the ion channel. Thus a scaling exponent description is found to characterize the fluctuation properties of the non-linear behaviors of ion channel kinetics regardless of whether the channel is in open or closed state.

Conformational Changes in a Pore-lining Helix Coupled to Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating

Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ATP-binding cassette transporters in that it functions as an ion channel. In CFTR, ATP binding opens the channel, and its subsequent hydrolysis causes channel closure. We studied the conformational changes in the pore-lining sixth transmembrane segment upon ATP binding by measuring state-dependent changes in accessibility of substituted cysteines to methanethiosulfonate reagents. Modification rates of three residues (resides 331, 333, and 335) near the extracellular side were 10-1000-fold slower in the open state than in the closed state. Introduction of a charged residue by chemical modification at two of these positions (resides 331 and 333) affected CFTR single-channel gating. In contrast, modifications of pore-lining residues 334 and 338 were not state-dependent. Our results suggest that ATP binding induces a modest conformational change in the sixth transmembrane segment, and this conformational change is coupled to the gating mechanism that regulates ion conduction. These results may establish a structural basis of gating involving the dynamic rearrangement of transmembrane domains necessary for vectorial transport of substrates in ATP-binding cassette transporters.

Genetic Demonstration That the Plasma Membrane Maxianion Channel and Voltage-dependent Anion Channels Are Unrelated Proteins

The maxianion channel is widely expressed in many cell types, where it fulfills a general physiological function as an ATP-conductive gate for cell-to-cell purinergic signaling. Establishing the molecular identity of this channel is crucial to understanding the mechanisms of regulated ATP release. A mitochondrial porin (voltage-dependent anion channel (VDAC)) located in the plasma membrane has long been considered as the molecule underlying the maxianion channel activity, based upon similarities in the biophysical properties of these two channels and the purported presence of VDAC protein in the plasma membrane. We have deleted each of the three genes encoding the VDAC isoforms individually and collectively and demonstrate that maxianion channel (approximately 400 picosiemens) activity in VDAC-deficient mouse fibroblasts is unaltered. The channel activity is similar in VDAC1/VDAC3-double-deficient cells and in double-deficient cells with the VDAC2 protein depleted by RNA interference. VDAC deletion slightly down-regulated, but never abolished, the swelling-induced ATP release. The lack of correlation between VDAC protein expression and maxianion channel activity strongly argues against the long held hypothesis of plasmalemmal VDAC being the maxianion channel.

Correlation character of ionic current fluctuations: Analysis of ion current through a voltage-dependent potassium single channel

The gating of ion channels has widely been modeled by assuming the transition between open and closed states is a memoryless process. Nevertheless, the statistical analysis of an ionic current signal recorded from voltage dependence K + single channel is presented. Calculating the sample auto-correlation function of the ionic current based on the digitized signals, rather than the sequence of open and closed states duration time. The results provide evidence for the existence of memory. For different voltages, the ion channel current fluctuation has different correlation attributions. The correlations in data generated by simulation of two Markov models, on one hand, auto-correlation function of the ionic current shows a weaker memory, after a delayed period of time, the attribute of memory does not exist; on the other hand, the correlation depends on the number of states in the Markov model. For V p = − 60 mV pipette potential, spectral analysis of ion channel current was conducted, the result indicates that the spectrum is not a flat spectrum, the data set from ionic current fluctuations shows considerable variability with a broad 1/f -like spectrum, α = 1.261 ± 0.24. Thus the ion current fluctuations give information about the kinetics of the channel protein, the results suggest the correlation character of ion channel protein nonlinear kinetics regardless of whether the channel is in open or closed state.