Single-channel Behavior Research Articles

Functional Characterization of Mammalian Inositol 1,4,5-Trisphosphate Receptor Isoforms

Inositol 1,4,5-trisphosphate receptors (InsP3R) play a key role in intracellular calcium (Ca2+) signaling. Three mammalian InsP3R isoforms—InsP3R type 1 (InsP3R1), InsP3R type 2 (InsP3R2), and InsP3R type 3 (InsP3R3) are expressed in mammals, but the functional differences between the three mammalian InsP3R isoforms are poorly understood. Here we compared single-channel behavior of the recombinant rat InsP3R1, InsP3R2, and InsP3R3 expressed in Sf9 cells, reconstituted into planar lipid bilayers and recorded with 50mM Ba2+ as a current carrier. We found that: 1), for all three mammalian InsP3R isoforms the size of the unitary current is 1.9 pA and single-channel conductance is 74–80 pS; 2), in optimal recording conditions the maximal single-channel open probability for all three mammalian InsP3R isoforms is in the range 30–40%; 3), in optimal recording conditions the mean open dwell time for all three mammalian InsP3R isoforms is 7–8ms, the mean closed dwell time is ∼10ms; 4), InsP3R2 has the highest apparent affinity for InsP3 (0.10μM), followed by InsP3R1 (0.27μM), and then by InsP3R3 (0.40μM); 5), InsP3R1 has a high-affinity (0.13mM) ATP modulatory site, InsP3R2 gating is ATP independent, and InsP3R3 has a low-affinity (2mM) ATP modulatory site; 6), ATP modulates InsP3R1 gating in a noncooperative manner (nHill=1.3); 7), ATP modulates InsP3R3 gating in a highly cooperative manner (nHill=4.1). Obtained results provide novel information about functional properties of mammalian InsP3R isoforms.

BER Estimation of Optical WDM RZ-DPSK Systems Through the Differential Phase<tex>$Q$</tex>

The optical return-to-zero differential phase-shift keying system is analyzed to determine the accuracy of the differential phase Q method in estimating the bit-error rate (BER). It is found that it consistently underestimates the BER though it successfully predicts the qualitative behavior of single-channel and wavelength-division-multiplexed systems for back-to-back and point-to-point configurations. A simple modification reduced the underestimation and produced accurate results.

Single-Channel Behavior of Heteromeric α1β Glycine Receptors: An Attempt to Detect a Conformational Change before the Channel Opens

The alpha1beta heteromeric receptors are likely to be the predominant synaptic form of glycine receptors in the adult. Their activation mechanism was investigated by fitting putative mechanisms to single-channel recordings obtained at four glycine concentrations (10-1000 microm) from rat alpha1beta receptors, expressed in human embryonic kidney 293 cells. The adequacy of each mechanism, with its fitted rate constants, was assessed by comparing experimental dwell time distributions, open-shut correlations, and the concentration-open probability (P(open)) curve with the predictions of the model. A good description was obtained only if the mechanism had three glycine binding sites, allowed both partially and fully liganded openings, and predicted the presence of open-shut correlations. A strong feature of the data was the appearance of an increase in binding affinity as more glycine molecules bind, before the channel opens. One interpretation of this positive binding cooperativity is that binding sites interact, each site sensing the state of ligation of the others. An alternative, and novel, explanation is that agonist binding stabilizes a higher affinity form of the receptor that is produced by a conformational change ("flip") that is separate from, and precedes, channel opening. Both the "interaction" scheme and the flip scheme describe our data well, but the latter has fewer free parameters and above all it offers a mechanism for the affinity increase. Distinguishing between the two mechanisms will be important for our understanding of the structural dynamics of activation in the nicotinic superfamily and is important for our understanding of mutations in these receptors.

Dopamine transporters depolarize neurons by a channel mechanism

Neurotransmitter transporters generate larger currents than expected if one assumes fixed stoichiometry models. It remains controversial, however, whether these depolarizing currents arise from high density and rapid turnover rates of a classical transporter, or whether transporters exhibit bona fide channel behavior. Although heterologously expressed transporters show single-channel behavior and noise analysis in native cells strongly suggests channel behavior, no directly observed single-channel events associated with transporters have been reported thus far in native cells. We describe single-channel events arising directly from the Caenorhabditis elegans dopamine transporter (DAT-1) as evidenced by DA-induced channel activity blocked by a high-affinity DAT-1 inhibitor, increased channel activity in neurons that overexpress DAT-1, and loss of channels in dat-1 knockout neurons. Our data indicate that authentic transporter channels underlie depolarizing whole-cell currents. Thus, DA transporters not only transport DA but also exhibit a channel mode of conduction that directly modulates membrane potential and neuronal function.

Single sodium channels from human skeletal muscle in planar lipid bilayers: characterization and response to pentobarbital.

To investigate the response to general anesthetics of different sodium-channel subtypes, we examined the effects of pentobarbital, a close thiopental analogue, on single sodium channels from human skeletal muscle and compared them to existing data from human brain and human ventricular muscle channels. Sodium channels from a preparation of human skeletal muscle were incorporated into planar lipid bilayers, and the steady-state behavior of single sodium channels and their response to pentobarbital was examined in the presence of batrachotoxin, a sodium-channel activator. Single-channel currents were recorded before and after the addition of pentobarbital (0.34-1.34 mM). In symmetrical 500 mM NaCl, human skeletal muscle sodium channels had an averaged single-channel conductance of 21.0 +/- 0.6 pS, and the channel fractional open time was 0.96 +/- 0.04. The activation midpoint potential was -96.2 +/- 1.6 mV. Extracellular tetrodotoxin blocked the channel with a half-maximal concentration (k1/2) of 60 nM at 0 mV. Pentobarbital reduced the time-averaged conductance of single skeletal muscle sodium channels in a concentration-dependent manner (inhibitory concentration 50% [IC50] = 0.66 mM). The steady-state activation was shifted to more hyperpolarized potentials (-16.7 mV at 0.67 mM pentobarbital). In the planar lipid bilayer system, skeletal muscle sodium channels have some electrophysiological properties that are significantly different compared with those of sodium channels from cardiac or from central nervous tissue. In contrast to the control data, these different human sodium channel subtypes showed the same qualitative and quantitative response to the general anesthetic pentobarbital. The implication of these effects for overall anesthesia will depend on the role the individual channels play within their neuronal networks, but suppression of both central nervous system and peripheral sodium channels may add to general anesthetic effects.

Mitochondrial Uniporter Identified

Kirichok et al . have identified a novel calcium (Ca 2+ )-selective ion channel as the elusive mitochondrial Ca 2+ uniporter. Mitochondrial accumulation of Ca 2+ shapes intracellular Ca 2+ signals, regulates energy production, and has been implicated in cell death. The nature of the mitochondrial Ca 2+ uniporter that enables this uptake, however, has been unclear. Kirichok et al . patch-clamped "mitoplasts" (vesicles derived from the inner mitochondrial membranes) from COS-7 cells and measured an inwardly rectifying Ca 2+ current in whole mitoplasts that, like the mitochondrial Ca 2+ uniporter, was inhibited by Ruthenium 360 and Ruthenium Red. This current, which the authors termed I MiCa , also showed the same cation selectivity and order of divalent cation permeability as the mitochondrial Ca 2+ uniporter. The behavior of single MiCa channels analyzed by recording from inside-out mitoplast patches could account for the properties of the macroscopic I MiCa . MiCa was voltage-dependent, was highly selective for Ca 2+ , and carried sufficient Ca 2+ current to account for mitochondrial Ca 2+ uptake. Thus, the authors concluded that the MiCa channel functioned as the mitochondrial Ca 2+ uniporter. Y. Kirichok, G. Krapivinsky, D. E. Clapham, The mitochondrial calcium uniporter is a highly selective ion channel. Nature 427 , 360-364 (2004). [Online Journal]

Ion channels: their structure, function and control – an overview

In the past two decades, investigations on ion channels have made remarkable progress. Since the development of patch clamp recording techniques in 1980s, investigators have been able to detect the real‐time behaviour of single channels on many kinds of cell membrane. Rapid advances in molecular biology techniques led to the cloning and identification of a variety of ion channel genes, many of which have been the targets of knock‐outs or disruptions. Recent significant progress in structural biology has enabled us to grasp the molecular architecture of some ion channels, and their structure–function relationship. In this special issue, knowledge from these various approaches are summarized and presented by seven specialists in the respective fields. This discussion took place on 24 March, 2003, during the joint annual scientific sessions of the 80th Japanese Physiological and the 76th Pharmacological Society meetings in Fukuoka, Japan. The symposium was held just after the outbreak of hostilities in Iraq, and some of the speakers originally invited could not participate. Fortunately, two excellent substitute speakers took part (Drs Zamponi and Findlay on Ca2+ channels).

Imaging single-channel calcium microdomains by total internal reflection microscopy

The microdomains of Ca2+ in the cytosol around the mouth of open Ca2+ channels are the basic 'building blocks' from which cellular Ca2+ signals are constructed. Moreover, the kinetics of local [Ca2+] closely reflect channel gating, so their measurement holds promise as an alternative to electrophysiological patch-clamp recording as a means to study single channel behavior. We have thus explored the development of optical techniques capable of imaging single-channel Ca2+ signals with good spatial and temporal resolution, and describe results obtained using total internal reflection fluorescence microscopy to monitor Ca2+ influx through single N-type channels expressed in Xenopus oocytes.

Ion channels in the inner mitochondrial membrane as tools for studying apoptosis and neuroprotection

Since several years ion channels of the inner mitochondrial membrane are known to be of significance for apoptosis or neuroprotection. It was shown that the blockade of the largest of them, the permeability transition pore (PTP) can suppress apoptotic cell death (Andrabi et al., FASEB J, in press) and that keeping potassium channels open, like the K(Ca)- or the K(ATP)-channel, protects cells from ischemic preconditioning. We prepared mitoplasts i.e. vesicles of inner mitochondrial membrane by a hypotonic treatment of the mitochondria and studied four different ion channels by means of the patch-clamp method. We show here that the K(Ca)-channel is the dominant channel in astrocyte and in glioma mitochondria. Voltage dependence, Ca-sensitivity and pharmacological behaviour resemble that of their counterpart in the plasma membrane. Similar to this channel, the mtK(ATP)-channel was shown to protect cells from ischemic injury. This channel was described as a single channel only once in 13 years until we have rediscovered it now (Dahlem et al., BBA-Bioenergetics, in press). We show the biophysical and some pharmacological properties, in particular block by NO. Furthermore, we studied the single-channel behaviour of the PTP and found that the channel could be directly blocked by two substances that have clearly neuroprotective properties, the dopamin-D2-agonist Pramipexole (Sifrol) with an IC50 of 500 nM and melatonin (IC50=800nM). We could show at least for melatonin that this effect is due to a reduced rate of neuronal cell death. Finally, a 108 pS Cl-channel that was shown to be identical with inner membrane anion channel (IMAC) can be blocked by fatty acids.

Control of mitochondrial protein import by pH

Protein import into mitochondria is inhibited by protons (IC 50 pH 6.5). The channels of the import machinery were examined to further investigate this pH dependence. TOM and TIM23 are the protein translocation channels of the mitochondrial outer and inner membranes, respectively, and their single channel behaviors at various pHs were determined using patch-clamp techniques. While not identical, increasing H + concentration decreases the open probability of both TIM23 and TOM channels. The pattern of the pH dependences of protein import and channel properties suggests TIM23 open probability can limit import of nuclear-encoded proteins into the matrix of yeast mitochondria.

Properties and modulation of the G protein-coupled K+ channel in rat cerebellar granule neurons: ATP versus phosphatidylinositol 4,5-bisphosphate.

Cerebellar granule (CG) neurons express a G protein-gated K+ current (GIRK) that is involved in the neurotransmitter regulation of the excitatory input to the Purkinje fibres of the cerebellum. Here, we characterized the single-channel behaviour of GIRK in CG neurons, and examined the effects of several known modulators of GIRK and their putative physiological roles. Whole-cell GIRKs were activated by baclofen, a GABAB receptor agonist. In cell-attached patches, baclofen activated GIRK with a single-channel conductance of 34 pS and a mean open time of 0.5 ms. In inside-out patches, application of GTPgammaS to the cytoplasmic side activated GIRK with similar kinetic properties. Addition of 2 mM ATP resulted in a marked increase in GIRK activity and induced longer-lived openings with a mean open time of 2.3 ms (ATP-dependent gating). Brain cytosolic fraction or free fatty acids inhibited this effect of ATP, and this was reversed by addition of purified recombinant brain fatty acid binding protein. Applying phosphatidylinositol 4,5-bisphosphate (PIP2) to inside-out patches in place of ATP also increased GIRK activity; however, only an increase in the frequency of opening was observed. The stimulatory effect of PIP2 on GIRK activity was not inhibited by the cytosolic fraction. Following maximal activation by PIP2, ATP caused an additional 2.2-fold increase in GIRK activity. These results show that GIRKs in CG neurons are regulated by positive and negative modulators that affect frequency as well as open time duration. The net effect is that the ligand-activated GIRK is in the 'low activity' state associated with short-lived openings, mainly due to strong action of the cytosolic inhibitor of ATP-dependent gating. Our results also show that intracellular ATP modulates GIRK via pathways different from that of PIP2 in CG neurons.

Cardiac L-type Calcium Channel β-Subunits Expressed in Human Heart Have Differential Effects on Single Channel Characteristics

l-Type calcium channels are multiprotein complexes composed of pore-forming (CaV1.2) and modulatory auxiliary alpha2delta- and beta-subunits. We demonstrate expression of two different isoforms for the beta2-subunit (beta2a, beta2b) and the beta3-subunit (beta3a, beta3trunc) in human non-failing and failing ischemic myocardium. Quantitatively, in the left ventricle expression of beta2b transcripts prevails in the order of > beta3 >> beta2a. The expressed cardiac full-length beta3-subunit is identical to the beta3a-isoform, and beta3trunc results from deletion of exon 6 (20 nn) entailing a reading frameshift and translation stop at nucleotide position 495. In failing ischemic myocardium beta3trunc expression increases whereas overall beta3 expression remains unchanged. Heterologous coexpression studies demonstrated that beta2 induced larger currents through rabbit and human cardiac CaV1.2 pore subunits than beta3 isoforms. All beta-subunits increased channel availability at single channel level, but beta2 exerted an additional, marked stimulation of rapid gating (open and closed times, first latency), leading to higher peak current values. We conclude that cardiac beta-subunit isoforms differentially modulate calcium inward currents because of regulatory effects within the channel protein complex. Moreover, differences in the various beta-subunit gene products present in human heart might account for altered single channel behavior found in human heart failure.

Carbachol-Activated Monovalent Cation-Selective Channels in the Murine Small Intestine

1 Bogomolets Institute of Physiology, National Academy of Sciences of Ukraine, Kyiv, Ukraine. 2 St. George’s Hospital Medical School, London, Great Britain. Correspondence should be addressed to A. V. Zholos (e-mail: zholos@serv.biph.kiev.ua). A cation current (I cat ), which is selective with respect to monovalent cations, has been observed in several types of smooth muscles [1, 2]. In visceral smooth muscles, carbachol (CCh) produces depolarization by activation of the monovalent cation-selective channels and subsequent opening of the voltagegated Ca channels. So far, there is no information on the action of CCh on single smooth muscle cells of the murine ileum. Using symmetrical Cs solutions, both the external application of 50 μM CCh and the internal action of 200 μM GTPγS evoked an inward current with a peak amplitude exceeding 0.5 nA (n = 50). The current-voltage (I-V) relationship for this current was Ushaped at negative potentials, and the reversal potential, E rev , was close to 0 mV. At least two types of monovalent cation-selective channels with unitary conductances of 17 and 70 pS (n = 12) were identified in outside-out patches. The open probability (P o ) for the 70 pS channel increased with depolarization, and the U shape of the I-V relationship of the whole-cell current at negative potentials was mostly determined by the voltage-dependent P o typical of this channel. External application of 20 μM quinine completely blocked single channel currents evoked by CCh or GTPγS in the outside-out patch configuration. Single channel openings in outside-out patches were rather stable and could be observed for more than 10 min after patch excision. The number of active channels in a patch was usually from one to four, and outside-out patches generally had either no channels or more than one active channel. The effects of external Ca and Mg ions on the behavior of single channels were also tested. Addition of Mg and Ca at 2.0, 2.5, or 10 mM concentrations to the bath solution reduced the unitary cationic channel conductance from 70 pS down to 42 pS and 32 pS, respectively, and somewhat increased the open channel noise. Our data suggest that the CCh-evoked I cat in smooth muscle cells of the murine ileum is mediated by at least two types of cationic channels with conductances of 17 or 70 pS. The 70 pS channel plays a major role in the generation of the whole-cell CCh-evoked I cat , and the U-shaped I-V relationship at negative potentials is mostly determined by the voltage dependence of P o typical of this channel.

Connexin43 phosphorylation at S368 is acute during S and G2/M and in response to protein kinase C activation.

Phorbol esters such as 12-O-tetradeconylphorbol-13-acetate (TPA) activate protein kinase C, increase Connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. Previous work has implicated protein kinase C (PKC) in the direct phosphorylation of Cx43 at S368, which results in a change in single channel behavior that contributes to a decrease in intercellular communication. We have examined Cx43 phosphorylation in several cell lines with an antibody specific for phosphorylated S368. We show that this antibody detects Cx43 only when it is phosphorylated at S368 and, consistent with previous results, TPA treatment causes a dramatic increase in phosphorylation at S368. However, in some cell types, the increased phosphorylation at S368 did not cause a detectable shift in migration as compared with the nonphosphorylated Cx43. Immunofluorescence showed increased S368 immunolabeling in cytoplasmic and plasma membrane structures in response to TPA. Immunoblot analysis of synchronized cells showed increased phosphorylation at S368 during S and G2/M phases of the cell cycle. S-phase cells contained more total Cx43 but assembled fewer functional gap junctional channels than G0-phase cells. Since M-phase cells also communicate poorly and contain few assembled gap junctions, phosphorylation at S368 appears to be negatively correlated with gap junction assembly. Thus, both gap junctional communication and S368 phosphorylation change during S phase and G2/M, implying that phosphorylation at S368 might play a role in key cell-cycle events.

Single-channel properties of native and cloned rat vanilloid receptors.

The responses of single-channel currents to capsaicin were recorded using the giga-seal patch-clamp technique in cell-attached and excised (inside-out/outside-out) patches from embryonic rat dorsal root ganglion (DRG) neurones in culture and in Xenopus oocytes heterologously expressing the rat vanilloid receptor (rVR1). Native and cloned vanilloid receptor (VR)-mediated currents exhibited outward rectification. In both the DRG neurones and oocytes expressing VR1, the chord conductances at -60 and +60 mV were approximately 50 and approximately 100 pS, respectively. At positive potentials, the channel exhibited a single conductance state. In contrast, at negative potentials, brief sojourns to subconductance states were apparent. The probability of the channel being open (P(o)) was dependent on the transmembrane voltage and the patch configuration (i.e. cell-attached vs. excised). In both DRG neurones and oocytes, the P(o) was greater at positive (+60 mV) than at negative (-60 mV) potentials. In cell-attached patches, the P(o) was approximately twofold higher, regardless of the applied potential. Most likely, the outward rectification observed in whole-cell currents is due to the voltage dependence of single-channel conductance and P(o). The open-time distributions of single-channel currents recorded from native and cloned VRs in the presence of low agonist concentrations (0.01-0.03 microM) were best fitted with three exponential components. The closed-time distributions were best fitted by five exponential components. At higher concentrations (0.5-1 microM), an additional component was required to fit the open-time distribution, and the number of exponential components needed to fit the closed-time distributions was reduced to two. The overall mean open time at +60 mV was approximately 4 ms, compared to approximately 1.2 ms at -60 mV. However, the overall mean closed time was not voltage dependent. There were no significant differences between the native and cloned receptors. A comparison of single-channel properties of native and heterologously expressed VR channels indicates that expression of the rVR1 subunit alone can account for the single-channel behaviour of the majority of the native VRs. These results suggest that either native VRs are made up of VR1 subunits, or the incorporation of subunits other than VR1 does not influence the functional properties. The responses of single-channel currents to capsaicin were recorded using the giga-seal patch-clamp technique in cell-attached and excised (inside-out/outside-out) patches from embryonic rat dorsal root ganglion (DRG) neurones in culture and in Xenopus oocytes heterologously expressing the rat vanilloid receptor (rVR1). Native and cloned vanilloid receptor (VR)-mediated currents exhibited outward rectification. In both the DRG neurones and oocytes expressing VR1, the chord conductances at -60 and +60 mV were approximately 50 and approximately 100 pS, respectively. At positive potentials, the channel exhibited a single conductance state. In contrast, at negative potentials, brief sojourns to subconductance states were apparent. The probability of the channel being open (P(o)) was dependent on the transmembrane voltage and the patch configuration (i.e. cell-attached vs. excised). In both DRG neurones and oocytes, the P(o) was greater at positive (+60 mV) than at negative (-60 mV) potentials. In cell-attached patches, the P(o) was approximately twofold higher, regardless of the applied potential. Most likely, the outward rectification observed in whole-cell currents is due to the voltage dependence of single-channel conductance and P(o). The open-time distributions of single-channel currents recorded from native and cloned VRs in the presence of low agonist concentrations (0.01-0.03 microM) were best fitted with three exponential components. The closed-time distributions were best fitted by five exponential components. At higher concentrations (0.5-1 microM), an additional component was required to fit the open-time distribution, and the number of exponential components needed to fit the closed-time distributions was reduced to two. The overall mean open time at +60 mV was approximately 4 ms, compared to approximately 1.2 ms at -60 mV. However, the overall mean closed time was not voltage dependent. There were no significant differences between the native and cloned receptors. A comparison of single-channel properties of native and heterologously expressed VR channels indicates that expression of the rVR1 subunit alone can account for the single-channel behaviour of the majority of the native VRs. These results suggest that either native VRs are made up of VR1 subunits, or the incorporation of subunits other than VR1 does not influence the functional properties.

Functional Characterization of the Type 1 Inositol 1,4,5-Trisphosphate Receptor Coupling Domain SII(±) Splice Variants and the Opisthotonos Mutant Form

The type 1 inositol (1,4,5)-trisphosphate receptor (InsP 3R1) plays a critical role in Ca 2+ signaling in cells. Neuronal and nonneuronal isoforms of the InsP 3R1 differ by alternative splicing in the coupling domain of the InsP 3R1 (SII site) (Danoff et al., 1991). Deletion of 107 amino acids from the coupling domain of the InsP 3R1 results in epileptic-like behaviors in opisthotonos ( opt) spontaneous mouse mutant (Street et al., 1997). Using Spodoptera frugiperda cells expression system, we compared single-channel behavior of recombinant InsP 3R1-SII(+), InsP 3R1-SII(−), and InsP 3R1- opt channels in planar lipid bilayers. The main results of our study are: 1) the InsP 3R1-SII(−) has a higher conductance (94 pS) and the InsP 3R1- opt has a lower conductance (64 pS) than the InsP 3R1-SII(+) (81 pS); 2) the bell-shaped Ca 2+-dependence peaks at 200–300 nM Ca 2+ for all three InsP 3R1 isoforms; 3) the bell-shaped Ca 2+-dependence is wider for the InsP 3R1-SII(+) and narrower for the InsP 3R1-SII(−) and InsP 3R1- opt; 4) the apparent affinity for ATP is sixfold lower for the InsP 3R1-SII(−) (1.4 mM) and 20-fold lower for the InsP 3R1- opt (5.3 mM) than for the InsP 3R1-SII(+) (0.24 mM); 5) the InsP 3R1-SII(−) is approximately twofold more active than the InsP 3R1-SII(+) in the absence of ATP. Obtained results provide novel information about the molecular determinants of the InsP 3R1 function.

Role of the carboxyl terminal of connexin43 in transjunctional fast voltage gating.

Previous studies show that chemical regulation of connexin43 (Cx43) gap junction channels depends on the integrity of the carboxyl terminal (CT) domain. Experiments using Xenopus oocytes show that truncation of the CT domain alters the time course for current inactivation; however, correlation with the behavior of single Cx43 channels has been lacking. Furthermore, whereas chemical gating is associated with a "ball-and-chain" mechanism, there is no evidence whether transjunctional voltage regulation for Cx43 follows a similar model. We provide data on the properties of transjunctional currents from voltage-clamped pairs of mammalian tumor cells expressing either wild-type Cx43 or a mutant of Cx43 lacking the carboxyl terminal domain (Cx43M257). Cx43 transjunctional currents showed bi-exponential decay and a residual steady-state conductance of approximately 35% maximum. Transjunctional currents recorded from Cx43M257 channels displayed a single, slower exponential decay. Long transjunctional voltage pulses caused virtual disappearance of the residual current at steady state. Single channel data revealed disappearance of the residual state, increase in the mean open time, and slowing of the transition times between open and closed states. Coexpression of CxM257 with Cx43CT in a separate fragment restored the lower conductance state. We propose that Cx43CT is an effector of fast voltage gating. Truncation of Cx43CT limits channel transitions to those occurring across the higher energy barrier that separates open and closed states. We further propose that a ball-and-chain interaction provides the fast component of voltage-dependent gating between CT domain and a receptor affiliated with the pore.

Dissociation of the store‐operated calcium current ICRAC and the Mg‐nucleotide‐regulated metal ion current MagNuM

Rat basophilic leukaemia cells (RBL-2H3-M1) were used to study the characteristics of the store-operated Ca(2+) release-activated Ca(2+) current (I(CRAC)) and the magnesium-nucleotide-regulated metal cation current (MagNuM) (which is conducted by the LTRPC7 channel). Pipette solutions containing 10 mM BAPTA and no added ATP induced both currents in the same cell, but the time to half-maximal activation for MagNuM was about two to three times slower than that of I(CRAC). Differential suppression of I(CRAC) was achieved by buffering free [Ca(2+)](i) to 90 nM and selective inhibition of MagNuM was accomplished by intracellular solutions containing 6 mM Mg.ATP, 1.2 mM free [Mg(2+)](i) or 100 microM GTP-gamma-S, allowing investigations on these currents in relative isolation. Removal of extracellular Ca(2+) and Mg(2+) caused both currents to be carried significantly by monovalent ions. In the absence or presence of free [Mg(2+)](i), I(CRAC) carried by monovalent ions inactivated more rapidly and more completely than MagNuM carried by monovalent ions. Since several studies have used divalent-free solutions on either side of the membrane to study selectivity and single-channel behaviour of I(CRAC), these experimental conditions would have favoured the contribution of MagNuM to monovalent conductance and call for caution in interpreting results where both I(CRAC) and MagNuM are activated.

Single sodium channels from human ventricular muscle in planar lipid bilayers.

Sodium channels from human ventricular muscle membrane vesicles were incorporated into planar lipid bilayers and the steady-state behavior of single sodium channels were examined in the presence of batrachotoxin. In symmetrical 500 mM NaCl the averaged single channel conductance was 24.7 +/- 1.3 pS and the channel fractional open time was 0.85 +/- 0.04. The activation midpoint potential was -99.5 +/- 3.1 mV. Extracellular tetrodotoxin blocked the channel with a k(1/2) of 414 nM at 0 mV. In 7 out of 13 experiments subconductance states were observed (9.2 +/- 1.2 pS). When sodium chloride concentration was lowered to 100 mM, single channel conductance decreased to 19.0 +/- 0.9 pS, steady-state activation shifted by -17.3 +/- 5.1 mV, tetrodotoxin sensitivity increased to 324 nM, and sub-conductance states were invariably observed in single channel records (7.9 +/- 0.7 pS). In the planar lipid bilayer system the properties of cardiac sodium channels from different species are not very different, but there are significant differences between sodium channels from human heart and from human CNS.

Gramicidin derivatives as membrane-based pH sensors

Ion channels provide a means for sensitive pH measurement at membrane interfaces. Detailed knowledge of the structure and function of gramicidin channels permits the engineering of pH-sensitive derivatives. Two derivatives, gramicidin-ethylenediamine and gramicidin-histamine, are shown to exhibit pH-dependent single-channel behaviour over the pH ranges 9–11 and 6.5–8.5, respectively. Thermal isomerization of a carbamate group at the entrance of the channels leads to a pattern of steps in single-channel recordings. The size of the steps depends on the time-averaged degree of protonation of the appended group (ethylenediamine or histamine). Measurement of the size of the steps thus permits single-molecule pH sensing under symmetrical pH conditions or in the presence of a pH gradient.