Open Channel Noise Research Articles

Impact on AChR open channel noise by pore-peripheral salt bridge depends on voltage and divalent cations

Mechanisms behind the fluctuations in the ionic current through single acetylcholine receptor (AChR) channels have remained elusive. In a recent study of muscle AChR we showed that mutation of a conserved intramembrane salt bridge in the β- and δ-subunits markedly increased fluctuations in the open channel current that extended from low to high frequency. Here, we show that extracellular divalent cations reduce the high-frequency fluctuations and increase the low-frequency fluctuations. The low-frequency fluctuations are shown to arise from steps between two current levels, with the ratio of the time at each level changing e-fold for a 70 mV increase in membrane potential, indicating modulation by a charged element within the membrane field. Increasing the charge on the ion selectivity filter biases the ratio of current levels equivalent to a 50 mV increase in membrane potential but does not alter the voltage dependence of the ratio. The magnitudes of the voltage dependence and voltage bias allow estimates of the distance between the ion selectivity filter and the voltage-sensing element. Studies with either calcium or magnesium show that the two divalent cations synergize to increase the low-frequency fluctuations, whereas they act independently to decrease the high-frequency fluctuations, indicating multiple divalent cation binding sites. Molecular dynamics simulations applied to the structure of the Torpedo AChR reveal that mutation of the salt bridge alters the equilibrium positions and dynamics of residues local to the site of the mutation and within the adjacent ion selectivity filter in a calcium-dependent manner. Thus, disruption of a conserved intramembrane salt bridge in the muscle AChR induces fluctuations in open channel current that are sensitive to divalent cation binding at multiple sites and modulated by a charged element within the membrane field.

Bayesian inference of kinetic schemes for ion channels by Kalman filtering.

Inferring adequate kinetic schemes for ion channel gating from ensemble currents is a daunting task due to limited information in the data. We address this problem by using a parallelized Bayesian filter to specify hidden Markov models for current and fluorescence data. We demonstrate the flexibility of this algorithm by including different noise distributions. Our generalized Kalman filter outperforms both a classical Kalman filter and a rate equation approach when applied to patch-clamp data exhibiting realistic open-channel noise. The derived generalization also enables inclusion of orthogonal fluorescence data, making unidentifiable parameters identifiable and increasing the accuracy of the parameter estimates by an order of magnitude. By using Bayesian highest credibility volumes, we found that our approach, in contrast to the rate equation approach, yields a realistic uncertainty quantification. Furthermore, the Bayesian filter delivers negligibly biased estimates for a wider range of data quality. For some data sets, it identifies more parameters than the rate equation approach. These results also demonstrate the power of assessing the validity of algorithms by Bayesian credibility volumes in general. Finally, we show that our Bayesian filter is more robust against errors induced by either analog filtering before analog-to-digital conversion or by limited time resolution of fluorescence data than a rate equation approach.

Heterogeneous Idealization of Ion Channel Recordings - Open Channel Noise.

We propose a new model-free segmentation method for idealizing ion channel recordings. This method is designed to deal with heterogeneity of measurement errors. This in particular applies to open channel noise which, in general, is particularly difficult to cope with for model-free approaches. Our methodology is able to deal with lowpass filtered data which provides a further computational challenge. To this end we propose a multiresolution testing approach, combined with local deconvolution to resolve the lowpass filter. Simulations and statistical theory confirm that the proposed idealization recovers the underlying signal very accurately at presence of heterogeneous noise, even when events are shorter than the filter length. The method is compared to existing approaches in computer experiments and on real data. We find that it is the only one which allows to identify openings of the PorB porine at two different temporal scales. An implementation is available as an R package.

Phosphorylation of rat brain purified mitochondrial Voltage-Dependent Anion Channel by c-Jun N-terminal kinase-3 modifies open-channel noise

The drift kinetic energy of ionic flow through single ion channels cause vibrations of the pore walls which are observed as open-state current fluctuations (open-channel noise) during single-channel recordings. Vibration of the pore wall leads to transitions among different conformational sub-states of the channel protein in the open-state. Open-channel noise analysis can provide important information about the different conformational sub-state transitions and how biochemical modifications of ion channels would affect their transport properties. It has been shown that c-Jun N-terminal kinase-3 (JNK3) becomes activated by phosphorylation in various neurodegenerative diseases and phosphorylates outer mitochondrion associated proteins leading to neuronal apoptosis. In our earlier work, JNK3 has been reported to phosphorylate purified rat brain mitochondrial voltage–dependent anion channel (VDAC) in vitro and modify its conductance and opening probability. In this article we have compared the open-state noise profile of the native and the JNK3 phosphorylated VDAC using Power Spectral Density vs frequency plots. Power spectral density analysis of open-state noise indicated power law with average slope value α ≈1 for native VDAC at both positive and negative voltage whereas average α value < 0.5 for JNK3 phosphorylated VDAC at both positive and negative voltage. It is proposed that 1/f1 power law in native VDAC open-state noise arises due to coupling of ionic transport and conformational sub-states transitions in open-state and this coupling is perturbed as a result of channel phosphorylation.

Molecular Competition between Large and Small Polyethylene-Glycols (PEGs) Partitioning into OMPC Porin Channels

Molecular crowding by polyethylene glycols (PEGs) has been employed in a wide range of inquiries into molecular structure and dynamics. Investigating the partitioning of crowded polymers into biological nanopores reveals properties also seen in, e.g., DNA ejection and compaction into viral capsids, as well as in facilitated molecule transport. Capitalizing on our previous work on the partitioning of binary PEG mixtures into the benchmark Alpha-Hemolysin nanopore, we have now studied partitioning of mono- and polydisperse polymer solutions into comparably sized, yet differently structured, diffusion channels. We find that in the presence of large non-partitioning PEG 3400, the small, otherwise equipartitioning PEG 200 partitions disproportionately. Specifically, with 15% PEG 3400, an added 15% PEG 200 reduces channel conductance by 65% (compared with channel conductance in polymer-free solutions), while 15% PEG 200 alone reduces channel conductance by only 45%. We employed open-channel noise analysis to investigate channel diffusion properties of individual polymers, and to compare them with their properties in bulk water solution. In this way we demonstrate and quantify crowding-assisted transport through nanopores in mixed-polymer systems.

Cx46 hemichannels contribute to the sodium leak conductance in lens fiber cells

The lens is proposed to have an internal microcirculation system consisting of continuously circulating ionic fluxes that play an essential role in maintaining lens transparency. One of the key components of this system is the sodium leak conductance. Here we investigate the contribution of Cx46 hemichannels to the basal membrane permeability of peripheral fiber cells isolated from transgenic mouse lenses lacking Cx50 or both Cx50 and Cx46 (dKO) using the whole cell patch-clamp technique. Our results show that Cx46 hemichannels were largely closed at a resting voltage of -60 mV in the presence of millimolar divalent cation concentrations. However, even though the vast majority of these channels were closed at -60 mV, a small, persistent, inward current could still be detected. This current could be mostly blocked by exposure to 1 mM La(3+) and was not observed in fiber cells isolated from dKO mouse lenses suggesting that it was due to Cx46 hemichannels. In addition, Cx50(-/-) fiber cells showed increased open channel noise and a depolarized resting potential compared with dKO fiber cells. Exposure of Cx50(-/-) fiber cells to La(3+) hyperpolarized the resting potential to -58 mV, which is similar to the value of resting potential measured in dKO fiber and significantly reduced the open channel noise. In conclusion, these results suggest that Cx46 hemichannels may contribute to the sodium leak conductance in lens fiber cells.

Ca2+ block and flickering both contribute to the negative slope of the IV curve in BK channels

Single-channel current–voltage (IV) curves of human large-conductance, voltage- and Ca2+-activated K+ (BK) channels are quite linear in 150 mM KCl. In the presence of Ca2+ and/or Mg2+, they show a negative slope conductance at high positive potentials. This is generally explained by a Ca2+/Mg2+ block as by Geng et al. (2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210955) in this issue. Here, we basically support this finding but add a refinement: the analysis of the open-channel noise by means of β distributions reveals what would be found if measurements were done with an amplifier of sufficient temporal resolution (10 MHz), namely that the block by 2.5 mM Ca2+ and 2.5 mM Mg2+ per se would only cause a saturating curve up to +160 mV. Further bending down requires the involvement of a second process related to flickering in the microsecond range. This flickering is hardly affected by the presence or absence of Ca2+/Mg2+. In contrast to the experiments reported here, previous experiments in BK channels (Schroeder and Hansen. 2007. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.200709802) showed saturating IV curves already in the absence of Ca2+/Mg2+. The reason for this discrepancy could not be identified so far. However, the flickering component was very similar in the old and new experiments, regardless of the occurrence of noncanonical IV curves.

The Open Gate Structure of the Membrane-Embedded KcsA Potassium Channel Viewed From the Cytoplasmic Side

Crystallographic studies of channel proteins have provided insight into the molecular mechanisms of ion channels, even though these structures are obtained in the absence of the membrane and some structural portions have remained unsolved. Here we report the gating structure of the membrane-embedded KcsA potassium channel using atomic force microscopy (AFM). The activation gate of the KcsA channel is located on the intracellular side, and the cytoplasmic domain was truncated to clear the view of this location. Once opened, the individual subunits in the tetramer were resolved with the pore open at the center. Furthermore, AFM was able to capture the previously unsolved bulge helix at the entrance. A molecular dynamics simulation revealed that the bulge helices fluctuated dramatically at the open entryway. This dynamic behavior was observed as vigorous open-channel noise in the single-channel current recordings. The role of the bulge helices in the open gate structure is discussed.

Alternative translation initiation further increases the molecular and functional diversity of ion channels

The diversity of ion channels is attributed to the different subunit genes expressed in a given cell, to alternative splicing, to hetero-multimerization and to post-translational modifications. Another mechanism, called alternative translation initiation (ATI), was recently shown to further increase the molecular and functional complexity of K+ channels (Thomas et al. 2008; and see Simkin et al. 2008). The genes produce two or more versions of the encoded proteins from a single mRNA, and the shorter version, initiated from a downstream in-frame start codon, lacks the N-terminal fragment of the full-length isoform version. ATI is thought to occur when the ribosome skips the first translation initiation site because of a non-optimal sequence (Kozak sequence) near the initiation site and moves down to the next AUG codon to initiate translation of the protein (Fig. 1). Figure 1 Alternative translation initiation of TREK-2 K2P channel subunits (15 genes in human), including TREK-1 (K2P2.1) and TREK-2 (K2P10.1), are made of four transmembrane segments, two P domains in tandem, an extended M1P1 extracellular loop and intracellular N- and C-termini (Honore, 2007). These channels are widely expressed and present in neuronal as well as non-neuronal cells. A functional channel is a dimer of K2P subunits with possible hetero-multimerization. Cell specific alternative splicing produces different isoforms with shorter amino terminal domains (by 15 or 4 amino acids as demonstrated for TREK-1 and TREK-2), with identical functional properties (Gu et al. 2002; Xian Tao et al. 2006). TREK-1 and TREK-2 share functional properties with activation by both physical and chemical stimuli including membrane stretch, heat, polyunsaturated fatty acids, volatile and gaseous anaesthetics, and intracellular acidosis, but are inhibited by both PKA- and PKC-mediated phosphorylation (Honore, 2007). Once activated, these channels behave as typical background or leak K+ channels, thus affecting the resting conductance as well as the action potential duration. ATI produces two isoforms of TREK-1, the full length and a shorter form lacking the first 56 amino acids of the amino terminal domain (Thomas et al. 2008). The smaller and larger TREK-1 isoforms are differentially expressed throughout the brain and during development. The shorter isoform yields smaller currents besides normal suface expression and surprisingly is permeable not only to K+ but also to Na+ under physiological conditions (Thomas et al. 2008). Indeed, expression of the shorter isoforms produces depolarization in transfected rat hippocampal neurons. By contrast, the longer isoform selectively passes K+ thus producing cell hyperpolarization. TREK-1 and TREK-2 exhibit small (41 pS and 52 pS) and large (132 pS and 220 pS) conductance phenotypes when either expressed in transfected mammalian cells or recorded from native cells including cardiac myocytes (TREK-1) or cerebellar granule neurons (TREK-2) (Xian Tao et al. 2006; Kang et al. 2007). No switching between the small and the large conductance channel has ever been observed in patches containing a single channel, thus indicating that this phenomenon is not related to different gating modes resulting in subconductance states. Moreover, clustering of small conductance channels that would yield the large conductance is also unlikely as the open channel noise is higher for the small conductance. Both channel types have similar gating and regulation properties (Kang et al. 2007). In this issue of The Journal of Physiology, an article by Simkin et al. (2008) demonstrates that ATI produces TREK-2 isoforms of different size from three potential translation initiation sites within the N terminus (isoforms shorter by 54 or 66 amino acids) (Fig. 1). The longer TREK-2 isoform produces both the small and large conductance channels, while the two shorter forms yield only the large conductance channel. Importantly, in sharp contrast to TREK-1, the shorter isoforms of TREK-2 remain highly K+ selective with little or no increase in Na+ permeability. How might the N-terminus control the unitary conductance of TREK-2? Secondary structure prediction suggests that the distal end of the N terminal domain contains an α helical region. Deletion experiments indicate that the largest change in the conductance level occurs after removal of this region (55 amino acids). This region may interact with other regions of the channel, including the carboxy terminal domain that directly affects the pore properties. Alternatively, this region may interact with phospholipids of the membrane, thus stabilizing the channel in a specific conformation influencing the single channel conductance. These recent findings demonstrate that ATI plays a major role in the control of K2P channel diversity by modulating single channel conductance and/or selectivity. This mechanism is thus expected to have a major influence on cell electrogenesis.

Functional Roles of Charged Amino Acid Residues on the Wall of the Cytoplasmic Pore of Kir2.1

It is known that rectification of currents through the inward rectifier K+ channel (Kir) is mainly due to blockade of the outward current by cytoplasmic Mg2+ and polyamines. Analyses of the crystal structure of the cytoplasmic region of Kir2.1 have revealed the presence of both negatively (E224, D255, D259, and E299) and positively (R228 and R260) charged residues on the wall of the cytoplasmic pore of Kir2.1, but the detail is not known about the contribution of these charged residues, the positive charges in particular, to the inward rectification. We therefore analyzed the functional significance of these charged amino acids using single/double point mutants in order to better understand the structure-based mechanism underlying inward rectification of Kir2.1 currents. As a first step, we used two-electrode voltage clamp to examine inward rectification in systematically prepared mutants in which one or two negatively or positively charged amino acids were neutralized by substitution. We found that the intensity of the inward rectification tended to be determined by the net negative charge within the cytoplasmic pore. We then used inside-out excised patch clamp recording to analyze the effect of the mutations on blockade by intracellular blockers and on K+ permeation. We observed that a decrease in the net negative charge within the cytoplasmic pore reduced both the susceptibility of the channel to blockade by Mg2+ or spermine and the voltage dependence of the blockade. It also reduced K+ permeation; i.e., it decreased single channel conductance, increased open-channel noise, and strengthened the intrinsic inward rectification in the total absence of cytoplasmic blockers. Taken together, these data suggest that the negatively charged cytoplasmic pore of Kir electrostatically gathers cations such as Mg2+, spermine, and K+ so that the transmembrane pore is sufficiently filled with K+ ions, which enables strong voltage-dependent blockade with adequate outward K+ conductance.

Functional Expression of TRESK-2, a New Member of the Tandem-pore K+ Channel Family

A new member of the tandem-pore K+ (K(2P)) channel family has been isolated from mouse testis complementary DNA. The new K(2P) channel was named TRESK-2, as its amino acid sequence shares 65% identity with that of TRESK-1. Mouse TRESK-2 is a 394-amino acid protein and possesses four putative transmembrane segments and two pore-forming domains. TRESK-2 has a long cytoplasmic domain joining the second and third transmembrane segments and a short carboxyl terminus. In the rat, TRESK-2 mRNA transcripts were expressed abundantly in the thymus and spleen and at low levels in many other tissues, including heart, small intestine, skeletal muscle, uterus, testis, and placenta, as judged by Northern blot analysis. TRESK-2 mRNA was also expressed in mouse and human tissues. In COS-7 cells transfected with TRESK-2 DNA, a time-independent and noninactivating K+-selective current was recorded. TRESK-2 was insensitive to 1 mm tetraethylammonium, 100 nm apamin, 1 mm 4-aminopyridine, and 10 microm glybenclamide. TRESK-2 was inhibited by 10 microm quinidine, 20 microm arachidonate and acid (pH 6.3) at 49, 43, and 23%, respectively. Single channel openings of TRESK-2 showed marked open channel noise. In symmetrical 150 mm KCl, the current-voltage relationship of TRESK-2 was slightly inwardly rectifying, with the single channel conductance 13 picosiemens (pS) at +60 mV and 16 pS at -60 mV. In inside-out patches, TRESK-2 was unaffected by the intracellular application of 10 microm guanosine 5'-O-(thiotriphosphate). These results show that TRESK-2 is a functional member of the K(2P) channel family and contributes to the background K+ conductance in many types of cells.

Residue Ionization and Ion Transport through OmpF Channels

Single trimeric channels of the general bacterial porin, OmpF, were reconstituted into planar lipid membranes and their conductance, selectivity, and open-channel noise were studied over a wide range of proton concentrations. From pH 1 to pH 12, channel transport properties displayed three characteristic regimes. First, in acidic solutions, channel conductance is a strong function of pH; it increases by approximately threefold as the proton concentration decreases from pH 1 to pH 5. This rise in conductance is accompanied by a sharp increase in cation transport number and by pronounced open-channel low-frequency current noise with a peak at ∼pH 2.5. Random stepwise transients with amplitudes at ∼1/5 of the monomer conductance are major contributors to this noise. Second, over the middle range (pH 5 ÷ pH 9), channel conductance and selectivity stay virtually constant; open channel noise is at its minimum. Third, over the basic range (pH 9 ÷ pH 12), channel conductance and cation selectivity start to grow again with an onset of a higher frequency open-channel noise. We attribute these effects to the reversible protonation of channel residues whose pH-dependent charge influences transport by direct interactions with ions passing through the channel.

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.

Evidence for non-independent gating of P2X2 receptors expressed in Xenopus oocytes

BackgroundP2X2 receptor is an ATP-activated ion channel which is widely expressed in the nervous system, and mediates synaptic transmission.ResultsWe recorded currents of P2X2 receptors expressed in Xenopus oocytes from outside-out patches and have found that currents recorded from patches containing a single or multiple P2X2 channels differ in a manner suggesting positive cooperativity. First, the currents from multichannel patches exhibit simultaneous transitions more frequently than predicted from the activity of independent channels. Second, the mean open lifetime at the current level of a single channel in a multichannel burst is about six times longer than the open time of currents from single channel patches, a trend opposite to what is expected of independent channels. These results indicate that the channels have positive cooperativity and that the longer opening is due to a slower closing rate. Third, from kinetic analysis the likelihood of the cooperative model is significantly larger than that of the independent model. Fourth, the open channel noise of currents from patches containing multiple channels is less than half that from a single channel, which is consistent with the channel properties being different when they are active in groups.ConclusionTaken together, our results suggest that P2X2 receptors are non-independent, but interact with positive cooperativity.

Hidden Markov Modeling for Single Channel Kinetics with Filtering and Correlated Noise

Hidden Markov modeling (HMM) can be applied to extract single channel kinetics at signal-to-noise ratios that are too low for conventional analysis. There are two general HMM approaches: traditional Baum's reestimation and direct optimization. The optimization approach has the advantage that it optimizes the rate constants directly. This allows setting constraints on the rate constants, fitting multiple data sets across different experimental conditions, and handling nonstationary channels where the starting probability of the channel depends on the unknown kinetics. We present here an extension of this approach that addresses the additional issues of low-pass filtering and correlated noise. The filtering is modeled using a finite impulse response (FIR) filter applied to the underlying signal, and the noise correlation is accounted for using an autoregressive (AR) process. In addition to correlated background noise, the algorithm allows for excess open channel noise that can be white or correlated. To maximize the efficiency of the algorithm, we derive the analytical derivatives of the likelihood function with respect to all unknown model parameters. The search of the likelihood space is performed using a variable metric method. Extension of the algorithm to data containing multiple channels is described. Examples are presented that demonstrate the applicability and effectiveness of the algorithm. Practical issues such as the selection of appropriate noise AR orders are also discussed through examples.

Asymmetric and independent contribution of the second transmembrane segment 12' residues to diliganded gating of acetylcholine receptor channels: a single-channel study with choline as the agonist.

Mutagenesis studies have suggested that the second transmembrane segment (M2) plays a critical role during acetylcholine receptor liganded gating. An adequate description of the relationship between gating and structure of the M2 domain, however, has been hampered by the fact that many M2 mutations increase the opening rate constant to levels that, in the presence of acetylcholine, are unresolvably fast. Here, we show that the use of saturating concentrations of choline, a low-efficacy agonist, is a convenient tool to circumvent this problem. In the presence of 20 mM choline: (a) single-channel currents occur in clusters; (b) fast blockade by choline itself reduces the single-channel conductance by approximately 50%, yet the excess open-channel noise is only moderate; (c) the kinetics of gating are fitted best by a single-step, C <--> O model; and (d) opening and closing rate constants are within a well resolvable range. Application of this method to a series of recombinant adult mouse muscle M2 12' mutants revealed that: (a) the five homologous M2 12' positions make independent and asymmetric contributions to diliganded gating, the delta subunit being the most sensitive to mutation; (b) mutations at delta12' increase the diliganded gating equilibrium constant in a manner that is consistent with the sensitivity of the transition state to mutation being approximately 30% like that of the open state and approximately 70% like that of the closed state; (c) the relationship between delta12' amino acid residue volume, hydrophobicity or alpha-helical tendency, and the gating equilibrium constant of the corresponding mutants is not straightforward; however, (d) rate and equilibrium constants for the mutant series are linearly correlated (on log-log plots), which suggests that the conformational rearrangements upon mutation are mostly local and that the position of the transition state along the gating reaction coordinate is unaffected by these mutations.

K+ Binding Sites and Interactions between Permeating K+ Ions at the External Pore Mouth of an Inward Rectifier K+ Channel (Kir2.1)

The arginine at position 148 is highly conserved in the inward rectifier K+ channel family. Increases of external pH decrease the single-channel conductance in mutant R148H of the Kir2.1 channel (arginine is mutated into histidine) but not in the wild type channel. Moreover, in 100 mM external K+, varying external pH induced biphasic changes of open channel noise, which peaks at around pH 7.4 in the R148H mutant but not in the wild type channel. The maximum single-channel conductances are higher in the wild type channel and R148H mutant at pH 6.0 than those in the R148H mutant at pH 7.4. However, the maximal conductance is achieved with much lower external [K+] for the latter. Interestingly, the single-channel conductances and open channel noise of the wild type channel at pH 6. 0 and the R148H mutant at pH 6.0 and 7.4 become the same in [K+] = 10 mM. These results indicate that the residue at position 148 is accessible to the external H+ and probably is involved in the formation of two K+ binding sites in the external pore mouth. Effective repulsion between permeating K+ ions in this area requires a positive charge at position 148, and such K+-K+ interaction is the essential mechanism underlying high K+ conduction rate through the Kir2.1 channel pore.

Getting the most out of noise in the central nervous system

Rather than merely a nuisance, noise in biological systems is a useful property. Before patch-clamp methods were invented, analysis of membrane current noise provided the first solid, if indirect, evidence for the existence of ion-conducting pores with discrete conductance levels. Although supplanted by single-channel recording techniques for most tasks, analysis of current membrane noise remains useful for certain problems, such as determining the properties of channels with rapid kinetics that open with a high probability and desensitize, channels localized at synapses, channels with an unusually low unitary conductance and open-channel noise. In addition, the role of noise in information processing in the CNS is increasingly being recognized. In this article, we summarize the analysis of current membrane noise with an emphasis on what the technique is still useful for, and discuss the role for noise in information processing.

A conserved cytoplasmic region of ROMK modulates pH sensitivity, conductance, and gating.

ROMK channels play a key role in overall K balance by controlling K secretion across the apical membrane of mammalian cortical collecting tubule. In contrast to the family of strong inward rectifiers (IRKs), ROMK channels are markedly sensitive to intracellular pH. Using Xenopus oocytes, we have confirmed this pH sensitivity at both the single-channel and whole cell level. Reduction of oocyte pH from 6.8 to 6.4 (using a permeant acetate buffer) reduced channel open probability from 0.76 +/- 0.02 to near zero (n = 8), without altering single-channel conductance. This was due to the appearance of a long-lived closed state at low internal pH. We have confirmed that a lysine residue (K61 on ROMK2; K80 on ROMK1), NH2 terminal to the first putative transmembrane segment (M1), is primarily responsible for conferring a steep pH sensitivity to ROMK (B. Fakler, J. Schultz, J. Yang, U. Schulte, U. Bråandle, H. P. Zenner, L. Y. Jan, and J. P. Ruppersberg. EMBO J. 15: 4093-4099, 1996). However, the apparent pKa of ROMK also depends on another residue in a highly conserved, mildly hydrophobic area: T51 on ROMK2 (T70 on ROMK1). Replacing this neutral threonine (T51) with a negatively charged glutamate shifted the apparent pKa for inward conductance from 6.5 +/- 0.01 (n = 8, wild type) to 7.0 +/- 0.02 (n = 5, T51E). On the other hand, replacing T51 with a positively charged lysine shifted the apparent pKa in the opposite direction, from 6.5 +/- 0.01 (n = 8, wild type) to 6.0 +/- 0.02 (n = 9, T51K). The opposite effects of the glutamate and lysine substitutions at position 51 (ROMK2) are consistent with a model in which T51 is physically close to K61 and alters either the local pH or the apparent pKa via an electrostatic mechanism. In addition to its effects on pH sensitivity, the mutation T51E also decreased single-channel conductance from 34.0 +/- 1.0 pS (n = 8, wild type) to 17.4 +/- 1 pS (n = 9, T51E), reversed the voltage gating of the channel, and significantly increased open-channel noise. These effects on single-channel currents suggest that the T51 residue, located in a mildly hydrophobic area of ROMK2, also interacts with the hydrophobic region of the permeation pathway.

1/f-Noise of open bacterial porin channels.

General diffusion pores and specific porin channels from outer membranes of gram-negative bacteria were reconstituted into lipid bilayer membranes. The current noise of the channels was investigated for the different porins in the open state and in the ligand-induced closed state using fast Fourier transformation. The open channel noise exhibited 1/f-noise for frequencies up to 200 Hz. The 1/f-noise was investigated using the Hooge formula (Hooge, Phys. Lett. 29A: 139-140 (1969)), and the Hooge parameter alpha was calculated for all bacterial porins used in this study. The 1/f-noise was in part caused by slow inactivation and activation of porin channels. However, when care was taken that during the noise measurement no opening or closing of porin channels occurred, the Hooge Parameter alpha was a meaningful number for a given channel. A linear relationship was observed between alpha and the single-channel conductance, g, of the different porins. This linear relation between single-channel conductance and the Hooge parameter alpha could be qualitatively explained by assuming that the passing of an ion through a bacterial porin channel is-to a certain extent-influenced by nonlinear effects between channel wall and passing ion.