Decrease In Single-channel Conductance Research Articles

Localization of the gate and selectivity filter of the full-length P2X7 receptor

The P2X7 receptor (P2X7R) belongs to the P2X family of ATP-gated cation channels. P2X7Rs are expressed in epithelial cells, leukocytes, and microglia, and they play important roles in immunological and inflammatory processes. P2X7Rs are obligate homotrimers, with each subunit having two transmembrane helices, TM1 and TM2. Structural and functional data regarding the P2X2 and P2X4 receptors indicate that the central trihelical TM2 bundle forms the intrinsic transmembrane channel of P2X receptors. Here, we studied the accessibility of single cysteines substituted along the pre-TM2 and TM2 helix (residues 327-357) of the P2X7R using as readouts (i) the covalent maleimide fluorescence accessibility of the surface-bound P2X7R and (ii) covalent modulation of macroscopic and single-channel currents using extracellularly and intracellularly applied methanethiosulfonate (MTS) reagents. We found that the channel opening extends from the pre-TM2 region through the outer half of the trihelical TM2 channel. Covalently adducted MTS ethylammonium+ (MTSEA+) strongly increased the probability that the channel was open by delaying channel closing of seven of eight responsive human P2X7R (hP2X7R) mutants. Structural modeling, as supported by experimental probing, suggested that resulting intraluminal hydrogen bonding interactions stabilize the open-channel state. The additional decrease in single-channel conductance by MTSEA+ in five of seven positions identified Y336, S339, L341C, Y343, and G345 as the narrowest part of the channel lumen. The gate and ion-selectivity filter of the P2X7R could be colocalized at and around residue S342. None of our results provided any evidence for dilation of the hP2X7R channel on sustained stimulation with ATP4.

Calcium/calmodulin-dependent kinase II activity is required for maintaining learning-induced enhancement of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated synaptic excitation.

Learning leads to changes in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic excitation. The mechanisms for maintaining such alterations needed for memory persistence remain to be clarified. Here, we report a novel molecular mechanism for maintaining learning-induced AMPAR-mediated enhancement of synaptic excitation. We show that training rats in a complex olfactory discrimination task, such that requires rule learning, leads to the enhancement of averaged amplitude of AMPAR-mediated miniature excitatory post-synaptic currents (mEPSCs) in piriform cortex pyramidal neurons for days after learning. Inhibiting calcium/calmodulin-dependent kinase II (CaMKII) using KN93 or tatCN21days after learning, reduced the averaged mEPSC amplitude in neurons in piriform cortex of trained rats to the level where they are not significantly different from mEPSC of control animals. CaMKII inhibition leads to a decrease in single channel conductance and not to changes in the number of synaptic-activated channels. We conclude that the maintenance of learning-induced enhancement of AMPAR-mediated synaptic excitation requires the activity of calcium/calmodulin-dependent kinase II. We show that training rats in a complex olfactory discrimination task leads to the enhancement of averaged amplitude of AMPA receptor-mediated miniature excitatory post-synaptic currents (mEPSCs) in piriform cortex pyramidal neurons for days after learning. Inhibiting calcium/calmodulin-dependent kinase II (CaMKII) using KN93 or tatCN21days after learning, reduced the averaged mEPSC amplitude in neurons in piriform cortex of trained rats to the level where they are not significantly different from mEPSC of control animals. CaMKII inhibition leads to a decrease in AMPAR single channel conductance. We conclude that the maintenance of learning-induced enhancement of AMPAR-mediated synaptic excitation requires the activity of CaMKII.

Topography of the TH5 Segment in the Diphtheria Toxin T-Domain Channel.

The translocation domain (T-domain) of diphtheria toxin contains 10 α helices in the aqueous crystal structure. Upon exposure to a planar lipid bilayer under acidic conditions, it inserts to form a channel and transport the attached amino-terminal catalytic domain across the membrane. The TH5, TH8, and TH9 helices form transmembrane segments in the open-channel state, with TH1-TH4 translocated across the membrane. The TH6-TH7 segment also inserts to form a constriction that occupies only a small portion of the total channel length. Here, we have examined the TH5 segment in more detail, using the substituted-cysteine accessibility method. We constructed a series of 23 mutant T-domains with single cysteine residues at positions in and near TH5, monitored their channel formation in planar lipid bilayers, and probed for an effect of thiol-specific reagents added to the solutions on either side of the membrane. For 15 of the mutants, the reagent caused a decrease in single-channel conductance, indicating that the introduced cysteine residue was exposed within the channel lumen. We also found that reaction caused large changes in ionic selectivity for some mutant channels. We determined whether reaction occurred in the open state or in the brief flicker-closed state of the channel. Finally, we compared the reaction rates from either side of the membrane. Our experiments are consistent with the hypotheses that the TH5 helix has a transmembrane orientation and remains helical in the open-channel state; they also indicate that the middle of the helix is aligned with the constriction in the channel.

The Molecular Mechanism of Cardiac Sodium Channel Current Reduction with Metabolic Stress

Background Human cardiomyopathy is associated with decreased cardiac Na + channel current (I Na ), and Na + channel changes have been implicated in the increased risk of sudden death in heart failure. We have reported that in cardiomyopathy, elevated intracellular NADH downregulates I Na acutely to a magnitude seen in Brugada syndrome through protein kinase C (PKC) activation without changing the channel membrane expression. The decrease in I Na can be ameliorated by NAD + and protein kinase A (PKA) activators. Here, we investigate the molecular mechanism by which I Na is changed during metabolic stress. Methods HEK293 cells stably expressing human cardiac channel (Na v 1.5) were used to measure single channel currents at –40, –60, and –80 mV using the cell-attached voltage-clamp configuration. Results The single channel currents and conductance of Na v 1.5 were significantly decreased by NADH (100 µM) and phorbol 12-myristate 13-acetate (PMA, 30 nM) ( P + (500 µM) or forskolin (1 µM) restored the single channel currents and conductance to the untreated level ( P >.05 vs untreated group, P Conclusions Because we have already shown no reduction in cell surface channel number with metabolic stress, the decrease of macroscopic current of cardiac Na v 1.5 induced by NADH and PKC results from a decrease of single channel conductance, whereas NAD + and PKA elevate the single channel conductance. These results are consistent with the time course of the current changes seen with NADH/NAD + .

Electrophysiology of Concatameric Pannexin 1 Channels Reveals the Stoichiometry of C-Terminal Autoinhibition

Pannexin 1 (PANX1) is a non-selective ion channel that mediates the uptake of cyanine dyes and release of nucleotides and other metabolites. PANX1 activation and ATP release/dye uptake are regulated by diverse stimuli, including physicochemical factors (e.g., stretch, K+ ions) and signaling by various G protein-coupled and ionotropic receptors. We recently described a unique regulatory mechanism for the release of ATP from apoptotic cells: PANX1 is activated by caspase cleavage of a C-terminal autoinhibitory domain. Current evidence suggests that PANX1 channels are hexameric, and cleavage-resistant subunits can interfere with caspase-dependent activation in a dominant-negative fashion. To explore the subunit stoichiometry required for C-terminal autoinhibition, we engineered concatameric PANX1 constructs with different (from 0 to 6) combinations of truncated and full-length sequences. Formation of concatemers was assessed by the use of single-molecule photobleaching and protein cross-linking. Whole-cell recordings from concatenated PANX1 constructs suggest that at least four intact C-termini are required to inhibit channel activity. In addition, as the number of intact C-termini increased, there was a progressive decrease in single channel conductance, suggesting that individual C-termini may act within the multimeric channel to inhibit channel conductance. These results provide further mechanistic insights into the regulation of PANX1 channels by the C-terminal autoinhibitory domains.

Calcium-dependent decrease in the single-channel conductance of TRPV1

TRPV1 is a Ca(2+) permeable cation channel gated by multiple stimuli including noxious heat, capsaicin, protons, and extracellular cations. In this paper, we show that Ca(2+) causes a concentration and voltage-dependent decrease in the capsaicin-gated TRPV1 single-channel conductance. This Ca(2+)-dependent effect on conductance was strongest at membrane potentials between -60 and +20mV, but was diminished at more hyperpolarised potentials. Using simultaneous recordings of membrane current and fura-2 fluorescence to measure the fractional Ca(2+) current of whole-cell currents evoked through wild-type and mutant TRPV1, we investigated a possible link between the mechanisms underlying Ca(2+) permeation and the Ca(2+)-dependent effect on conductance. Surprisingly, we found no evidence of a structural correlation, and observed that the substitution of amino acids known to regulate Ca(2+) permeability had little effect on the ability for Ca(2+) to decrease TRPV1 conductance. However, we did observe that the Ca(2+)-dependent effect on conductance was not diminished by negative hyperpolarisation for a mutant receptor with severely impaired Ca(2+) permeability, TRPV1-D646N/E648Q/E651Q. This would be consistent with the idea that Ca(2+) reduces conductance by interacting with an intra-pore binding site, and that negative hyperpolarization reduces occupancy of this site by speeding the exit of Ca(2+) into the cell. Taken together, our data show that in addition to directly and indirectly regulating channel gating, Ca(2+) also directly reduces the conductance of TRPV1. Surprisingly, the mechanism underlying this Ca(2+)-dependent effect on conductance is largely independent of mechanisms governing Ca(2+) permeability.

Regulation of TRPV5 Single-Channel Activity by Intracellular pH

The transient receptor potential channel TRPV5 contributes to the apical entry pathway for transcellular calcium reabsorption in the kidney. Acid load causes hypercalciuria in animals and humans. We have previously reported that intracellular protons directly inhibit TRPV5. Here, we examined the effects of intracellular pH on single-channel activity of TRPV5. We found that TRPV5 channels exhibit full and subconductance open states in excised inside-out patches of Chinese hamster ovary cells. The slope conductance values (Na(+) as a charge carrier, between -25 and -75 mV) for full and subconductance opening at intracellular pH 7.4 were 59 +/- 6 and 29 +/- 3 pS, respectively. Intracellular acidification caused a small decrease in single-channel conductance. Importantly, intracellular acidification decreased open probability for the full and subconductance states and increased probability for closing. To investigate how intracellular protons decrease open probability of the channel, we proposed a simple three-state model for open-subconductance-closed state transition and examined the effects of acidification on the respective forward and reverse rate constants. We found that intracellular acidification decreases opening of TRPV5 predominantly by promoting a transition from the subconductance to the closed state. Thus, intracellular acidification directly inhibits TRPV5 by causing a conformational change(s) leading to a decrease of open probability of TRPV5 as well as of the single-channel conductance.

Synaptic function and modulation of glycine receptor channels in the hypoglossal nucleus

In this review, we discuss the function and modulation of chloride-selective glycine receptor (GlyR) channels, some genetic diseases originated from dysfunction of GlyRs, and modulation of glycinergic synapses by intracellular calcium (Ca2+) with particular attention on the motoneurons of the hypoglossal nucleus. This motor nucleus is a brainstem structure implicated in the command of coordinated movements during oral behavioral phenomena, including feeding, drinking, grooming, and respiration. In this nucleus, more than 90% of its cells are motoneurons. These hypoglossal motoneurons (HMs) are involved in a variety of motor functions and exhibit two remarkable features: (i) a low endogenous Ca2+ buffering capacity, which determines the rapid dynamics of cytosolic intracellular Ca2+, and (ii) powerful glycinergic inputs, which determine the main inhibitory drive on the above cells in adult animals. Glycine receptors belong to the superfamily of Cys-loop ligand-gated ion channels. They are capable of forming functional homo-or heteromeric chloride-selective channels. Dysfunction of GlyRs results in a genetic neurological motor disorders, including hyperekplexia. These diseases originate from mutations in the GlyR gene, leading to a decrease in single channel conductance, a lower affinity to the neurotransmitter, or a low level of GlyR expression. The function of glycinergic synapses is modulated during developmental changes and strictly controlled by several feedback mechanisms at pre-and post-synaptic levels. The developmental modulation consists in switch in the GlyR subunit composition and change in the chloride homeostasis during the synaptic maturation and formation of inhibitory networks. Retrograde signalling plays an important role in the synaptic function of HMs; it provides post-synaptic neurons with efficient tools for controlling pre-synaptic afferents. Glycine receptors and glycinergic synapses are also regulated by intracellular Ca2+. The mechanisms of these modulations are discussed.

Actin cytoskeleton disassembly affects conductive properties of stretch-activated cation channels in leukaemia cells

Mechanosensitive channels in various eucaryotic cells are thought to be functionally and structurally coupled to the cortical cytoskeleton. However, the results of electrophysiological studies are rather controversial and the functional impact of cytoskeleton assembly–disassembly on stretch-activated channel properties remains unclear. Here, the possible involvement of cytoskeletal elements in the regulation of stretch-activated Ca 2+-permeable channels was studied in human leukaemia K562 cells with the use of agents that selectively modify the actin or tubulin system. F-actin disassembly resulted in a considerable reduction of the amplitude of stretch-activated currents without significant change in channel open probability. The effects of treatments with cytochalasins or latrunculin were principally similar, developed gradually and consisted a strong decrease of single channel conductance. Microtubule disruption did not affect stretch-activated channels. The data presented here are in principal agreement with the general conclusion that mechanosensitive channel functions are largely dependent on the integrity of the cortical actin cytoskeleton. Specifically, changes in conductive properties of the pore may provide an essential mechanism of channel regulation underlying functional modulation of membrane currents. Our results allow one to speculate that microfilament organization may be an important determinant in modulating biophysical characteristics of stretch-activated cation channels in cells of blood origin.

Regulation of Epithelial Na+ Channels by Actin in Planar Lipid Bilayers and in the Xenopus Oocyte Expression System

The hypothesis that actin interactions account for the signature biophysical properties of cloned epithelial Na(+) channels (ENaC) (conductance, ion selectivity, and long mean open and closed times) was tested using planar lipid bilayer reconstitution and patch clamp techniques. We found the following. 1) In bilayers, actin produced a more than 2-fold decrease in single channel conductance, a 5-fold increase in Na(+) versus K(+) permselectivity, and a substantial increase in mean open and closed times of wild-type alphabetagamma-rENaC but had no effect on a mutant form of rENaC in which the majority of the C terminus of the alpha subunit was deleted (alpha(R613X)betagamma-rENaC). 2) When alpha(R613X)betagamma-rENaC was heterologously expressed in oocytes and single channels examined by patch clamp, 12.5-pS channels of relatively low cation permeability were recorded. These characteristics were identical to those recorded in bilayers for either alpha(R613X)betagamma-rENaC or wild-type alphabetagamma-rENaC in the absence of actin. Moreover, we show that rENaC subunits tightly associate, forming either homo- or heteromeric complexes when prepared by in vitro translation or when expressed in oocytes. Finally, we show that alpha-rENaC is properly assembled but retained in the endoplasmic reticulum compartment. We conclude that actin subserves an important regulatory function for ENaC and that planar bilayers are an appropriate system in which to study the biophysical and regulatory properties of these cloned channels.

The inhibitory effect of propranolol on ATP-sensitive potassium channels in neonatal rat heart.

1. Whole cell and single channel recordings of ATP-sensitive K+ current (I(K,ATP)) were carried out in ventricular myocytes isolated from neonatal rat hearts. 2. (+/-)-Propranolol, a commonly used beta-blocker, inhibited the whole cell I(K,ATP) in a concentration-dependent manner with a half-maximal concentration (IC50) of 6.7 +/- 1.4 microM, whereas it blocked the inward rectifier K+ current (I(K,I)) only at much higher concentrations (IC50 = 102.4 +/- 20.2 microM). The inhibition was time- and voltage-independent. 3. In the outside-out patch configuration, (+/-)-propranolol inhibited I(K,ATP) (IC50 = 9.8 +/- 2.9 microM) by decreasing the open probability of the channel without inducing additional noise in the open-channel current or a decrease of single channel conductance. The single channel current of I(K,I) was also blocked by (+/-)-propranolol in the same way as I(K,ATP). 4. (+)-Propranolol, an optic isomer having no beta-blocking effect, inhibited I(K,ATP) (IC50 = 5.8 +/- 1.0 microM), whilst atenolol, a selective beta1-blocker had no effect. Neither GDPbetaS (1 mM) nor GTPgammaS (200 microM) included in the pipette solution modulated the inhibitory effect of (+/-)-propranolol. 5. We concluded that the inhibitory effect of (+/-)-propranolol was not via the beta-adrenergic signal transduction pathway, but by direct inhibition of I(K,ATP) channels.

PH sensitivity of the cardiac gap junction proteins, connexin 45 and 43.

. Intercellular communication through gap junction channels can be regulated by changes in intracellular pH (pHi). This regulation may play an important role in ischemic heart tissue. Using the dual voltage-clamp technique, we compared the pHi sensitivity of gap junction channels composed of connexin 43 (Cx43) and Cx45, two of the gap junction proteins that are expressed in heart. We made use of SKHep1 cells, endogenously expressing low levels of Cx45 and SKHep1 cells stably transfected with rat Cx43. To manipulate the pHi we applied the NH3/NH+4 pH-clamp method. At pHi 6.7 the gj of Cx45 channels was reduced to approximately 20% of control values (pHi 7.0) and at pHi 6.3 all channels closed. The gj of Cx43 channels was approximately 70% of control values at pHi 6.7 and approximately 40% at pHi 6.3. Cx43 channels closed at pHi 5.8. Single channel conductances were 17.8 pS for Cx45 and 40.8 pS for Cx43 at pHi 7.0 and did not change significantly at lower pHi. This suggests that the decrease in macroscopic conductance observed at low pHi results from the decrease in open probability of gap junctional channels rather than from a decrease in single channel conductance. Our results demonstrate that gap junction channels built of Cx45 are far more pH sensitive than channels built of Cx43.

Single amino acid substitutions within the ion permeation pathway alter single-channel conductance of the human L-type cardiac Ca2+ channel.

Voltage-dependent L-type Ca2+ channels select for Ca2+ and other divalent cations by high-affinity Ca2+ binding and ion-ion interactions in the permeation pathway. We have recently identified a series of highly conserved glutamate residues, located within the SS2 segments of each of the four repeats of the human heart Ca2+ channel alpha 1 subunit, as major determinants of ion selectivity of the channel. To further investigate the functional role of these glutamate residues in ion permeation, we have individually neutralized the glutamic acids in repeats II and IV by substitution with alanine or glutamine. Wild-type and mutant Ca2+ channels were expressed in Xenopus oocytes. Apparent affinity for external Ca2+ was assessed by measuring the block by Ca2+ of inward Li+ currents through the Ca2+ channels. Mutations reducing net negative charge at these positions resulted in a 10-fold reduction in the affinity of the channel for external Ca2+. Single-channel conductance was measured with either divalent or monovalent cations as the charge carriers. Substitution of glutamic acid at position 677 with either alanine or glutamine increased single-channel conductance, whereas the same substitutions at position 1387 resulted in a decrease in single-channel conductance with Ba2+ as the charge carrier. In contrast, the unitary current amplitude carried by Na+, in the absence of external divalent cations, was not altered by these mutations. The results suggest that these conserved glutamate residues participate in high-affinity Ca2+ binding within the pore. The different effects on Ba2+ permeation indicate an asymmetric arrangement of the glutamate residues between repeats II and IV.(ABSTRACT TRUNCATED AT 250 WORDS)

Reaction of diphtheria toxin channels with sulfhydryl-specific reagents: observation of chemical reactions at the single molecule level.

The diphtheria toxin channel is believed to be a homooligomer of its T domain in which each subunit consists of two alpha-helices, lying within the membrane, connected by a short interhelical loop of four amino acids (residues 349-352). To investigate the validity and implications of this model, we singly mutated each of these amino acids to cysteines, formed channels with the mutant T-domain proteins in planar lipid bilayers, and added to the trans compartment sulfhydryl-specific reagents [methanethiosulfonate derivatives (MTS-ER)] that introduce a positive or negative charge to reacted cysteines. The introduction of a positive charge at residue 351 or 352 (through the MTS-ER reactions) resulted in a step decrease in single-channel conductance, whereas the introduction of a negative charge resulted in a step increase. The opposite sign of these effects indicates the predominantly electrostatic nature of the phenomenon and implies that residues 351 and 352 lie close to the channel entrance. The same reactions at residue 350 resulted in very little change in channel conductance but instead changed the character of the natural rapid flickering of the channel between open and closed states to one in which the channel spent more time in the closed state; this may have resulted from the group introduced at position 350 acting as a tethered channel blocker. The MTS derivatives had no effect on channels containing a cysteine at position 349, suggesting that this residue faces away from the channel entrance. We propose that the step changes in conductance or flickering pattern result from the chemical reaction of one MTS-ER molecule with one cysteine, and thus a bimolecular chemical reaction is being witnessed at the single molecule level. From the distribution of waiting times between the appearance (i.e., the opening) of a channel and the step change in its conductance or flickering pattern, we can calculate a pseudo-first-order rate constant, which can then be converted to a second-order rate constant, for the chemical reaction.

Potentiation of nicotinic receptor response by external calcium in rat central neurons

Nicotinic acetylcholine receptor (nAChR) responses of rat medial habenular neurons are potentiated up to 3.5-fold by increasing the concentration of external Ca2+ the the millimolar range. This effect, independent of voltage, is probably due to the binding of Ca2+ to an external site. External Ca2+ decreases nAChR single-channel conductance at negative but not positive potentials, and it markedly enhances the frequency of opening of acetylcholine activated channels. The potentiating effect of Ca2+ is mimicked by Ba2+ and Sr2+, but barely by Mg2+. These data support the existence of positively acting allosteric sites for Ca2+ distinct from those involved in the decrease of single-channel conductance. A model in which external Ca2+ changes the properties of activation of the nAChR appears consistent with these data.

Mapping the site of block by tetrodotoxin and saxitoxin of sodium channel II

The SS2 and adjacent regions of the 4 internal repeats of sodium channel II were subjected to single mutations involving, mainly, charged amino acid residues. These sodium channel mutants, expressed in Xenopus oocytes by microinjection of cDNA-derived mRNAs, were tested for sensitivity to tetrodotoxin and saxitoxin and for single-channel conductance. The results obtained show that mutations involving 2 clusters of predominantly negatively charged residues, located at equivalent positions in the SS2 segment of the 4 repeats, strongly reduce toxin sensitivity, whereas mutations of adjacent residues exert much smaller or no effects. This suggests that the 2 clusters of residues, probably forming ring structures, take part in the extracellular mouth and/or the pore wall of the sodium channel. This view is further supported by our finding that all mutations reducing net negative charge in these amino acid clusters cause a marked decrease in single-channel conductance.

Human nicotinic acetylcholine receptor: the influence of second messengers on activation and desensitization

The amplitude and time course of acetylcholine(ACh)-induced membrane current were determined in cells of the human medulloblastoma cell line TE 671. ACh was applied and washed out very rapidly (about 50 ms) by a shift of a cell between two streams of solution, one of which contained the transmitter. ACh-induced current was recorded in the whole-cell mode of patch clamping. The time course of activation of the ACh-induced current could not be resolved because the method of ACh application was still too slow. Desensitization started immediately with ACh application; it could be described by two time constants. With an ACh concentration of 3 microM, the fast time constant was about 0.5 s and the slow time constant was about 3.9 s. When the ACh concentration was raised in steps to 100 mM, the peak amplitude of the current increased, reached a maximum at 1 mM and decreased again. The rate of desensitization was directly correlated with increasing ACh concentration. Current amplitude and desensitization time constants were not affected by intracellular application of cAMP or of a catalytic subunit of a cAMP-dependent protein kinase. When the intracellular calcium concentration was raised at a constant magnesium concentration, desensitization time constants remained unaffected, but the current amplitude decreased. This decrease is not caused by a decrease in single-channel conductance, therefore it may represent a decrease in the number of activatable channels.

Modification of the conductance, selectivity and concentration-dependent saturation of Pseudomonas aeruginosa protein P channels by chemical acetylation

Protein P, an anion-specific channel-forming protein from the outer membrane of Pseudomonas aeruginosa was chemically modified by acetylation and syccinylation of its accessible amino groups. The chemically modified protein retained its ability to form oligomers on sodium dodecyl sulfate polyacrylamide gels, whereas only the acetylated protein formed channels in reconstitution experiments with lipid bilayers. Acetylated protein P demonstrated a substantially reduced mean single channel conductance (25 pS at 1 M KCl) compared to the native protein P channels (250 pS at 1 M KCl) when reconstituted into black lipid bilayer membranes. The homogeneous size distribution of single-channel conductances suggested that all of the protein P molecules had been acetylated. Zero-current potential measurements demonstrated that the acetylated protein P channel was only weakly selective for anions and allowed the permeation of cations, in contrast to the native protein P channels, which were more than 100-fold selective for anions over cations. The dependence of conductance on salt concentration was changed upon acetylation, in that acetylated protein P demonstrated a linear concentration-conductance relationship, whereas native protein P channels became saturated at high salt concentrations. These data strongly suggested that the basis of anion selectivity for native protein P channels is fixed amino groups. In agreement with this, we could demonstrate a 2.5-fold decrease in single-channel conductance between pH 7 and pH 9, between which pH values the ϵ-amino groups of amino acids would start to become deprotonated. Two alternative schemes for the topography of the protein P channel and localization of the fixed amino groups are presented and discussed.