Phosphatidylinositol 4,5-bisphosphate and intracellular pH regulate the ROMK1 potassium channel via separate but interrelated mechanisms

ROMK channels are responsible for K(+) secretion in kidney. The activity of ROMK is regulated by intracellular pH (pH(i)) with acidification causing channel closure (effective pK(a) approximately 6.9). Recently, we and others reported that a direct interaction of the channels with phosphatidyl-4,5-bisphosphate (PIP(2)) is critical for opening of the inwardly rectifying K(+) channels. Here, we investigate the relationship between the mechanisms for regulation of ROMK by PIP(2) and by pH(i). We find that disruption of PIP(2)-ROMK1 interaction not only decreases single-channel open probability (P(o)) but gives rise to a ROMK1 subconductance state. This state has an increased sensitivity to intracellular protons (effective pK(a) shifted to pH approximately 7.8), such that the subconductance channels are relatively quiescent at physiological pH(i). Open probability for the subconductance channels can then be increased by intracellular alkalinization to supra-physiological pH. This increase in P(o) for the subconductance channels by alkalinization is not associated with an increase in PIP(2)-channel interaction. Thus, direct interaction with PIP(2) is critical for ROMK1 to open at full conductance. Disruption of this interaction increases pH(i) sensitivity for the channels via emergence of the subconductance state. The control of open probability of ROMK1 by pH(i) occurs via a mechanism distinct from the regulation by PIP(2).     

Mechanism and molecular determinant for regulation of rabbit transient receptor potential type 5 (TRPV5) channel by extracellular pH

The transient receptor potential type 5 (TRPV5) channel is present in kidney and intestine and important for transepithelial (re)absorption of calcium in these tissues. We report that in whole-cell patch clamp recording extracellular acidification inhibited rabbit TRPV5 with apparent pKa approximately 6.55. The two extracellular loops between the fifth and sixth transmembrane segments of TRPV5 presumably form part of the outer opening of the pore and likely are important in binding and regulation by external protons. We found that mutation of glutamate 522 to glutamine (E522Q) decreased the sensitivity of the channel to extracellular acidification. Mutations of other titratable amino acids within the two extracellular loops to non-titratable amino acids had no effect on pH sensitivity. Substitutions of aspartate or other titratable amino acids for glutamate 522 conferred an increase in pH sensitivity. The pH sensitivity mediated by glutamate 522 was independent of extracellular or intracellular Mg2+. Single channel analysis revealed that extracellular acidification reduced single channel conductance as well as open probability of the wild type channel. In contrast to wild type channel, extracellular acidification did not reduce open probability for E522Q mutant. Methanethiosulfonate reagents inhibited the activity of glutamine 522 to cysteine mutant channel with a reaction rate constant approaching that with free thiols in solution, suggesting that glutamate 522 is located on the surface of the channel. These data suggest that glutamate 522 of the rabbit TRPV5 is a "pH sensor," and extracellular protons inhibit TRPV5 likely by altering conformation of the channel protein.     

Ca2+-activated K+ channels from cultured renal medullary thick ascending limb cells: Effects of pH

Summary Ca²⁺-activated K⁺ channels were studied in cultured medullary thick ascending limb cells (MTAL) using the patch-clamp technique. The purpose was to determine the effect of acidic pH on channel properties in excised patches of apical cell membrane. At pH 7.4, increasing Ca²⁺ on the intracellular side or applying positive voltages increases channel open probability. Reducing pH to 5.8 on the intracellular face of the channel decreases channel open probability at each voltage and Ca²⁺ concentration. Channel mean open times display two distributions and mean closed times display three distributions. Increasing Ca²⁺ or applying depolarizing voltages lengthens each of the mean open times and shortens each of the closed times. Lowering pH to 5.8 decreases the mean open times and increases mean closed times at each Ca²⁺ and voltage with the greatest effect on the mean closed times. In contrast, both single-channel conductance and channel kinetics are unaffected when pH is reduced to 5.8 on the extracellular face of the membrane. We conclude that protons interfere with Ca²⁺ binding to the gate of Ca²⁺-activated K⁺ channels reducing the probability of channel opening.     

Cytoplasmic acidosis induces multiple conductance states in ATP-sensitive potassium channels of cardiac myocytes

We studied the effect of cytoplasmic acidosis on the ionic conducting states of ATP-sensitive potassium channels in heart ventricular cells of guinea pigs and rabbits by using a patch-clamp technique with inside-out patch configuration. Under normal conditions (pH 7.4), the channel alternated between a closed state and a main open state in the absence of nucleotides on the cytoplasmic side. As internal pH was reduced below 6.5, the single channel current manifested distinct subconductance levels. The probability of the appearance of these subconductance levels was pH dependent with a greater probability of subconductance states at lower pH. A variance-mean amplitude analysis technique revealed two subconductance levels approximately equally spaced between the main open level and the closed level (63 and 33%). A current-voltage plot of the two subconductance levels and the main level showed that they had similar reversal potentials and rectification properties. An intrinsic flickering gating property characteristic of these ATP-sensitive channels was found unchanged in the 63% subconductance state, suggesting that this subconductance state and the main conductance state share similar ion pore properties (including ion selection and block) and similar gating mechanisms. The appearance of the subconductance states decreased as ionic strength was increased, and the subconductance states were also slightly voltage dependent, suggesting an electrostatic interaction between the protons and the negative surface charge in the vicinity of the binding sites, which may be close to the inner entrance of the ion pore. Proteolytic modification of the channel on the cytoplasmic side with trypsin did not abolish the subconductance levels. External acidosis did not induce subconductance levels. These results suggest that protons bound to the negatively charged group at the inner entrance of the channel ion pore may induce conformational changes, leading to partially reduced conductance states.     

Characterization of the channel properties of tetanus toxin in planar lipid bilayers.

A detailed characterization of the properties of the channel formed by tetanus toxin in planar lipid bilayers is presented. Channel formation proceeds at neutral pH. However, an acidic pH is required to detect the presence of channels in the membrane rapidly and effectively. Acid pH markedly lowers the single-channel conductance, for phosphatidylserine at 0.5 M KCl gamma = 89 pS at pH 7.0 while at pH 4.8, gamma = 30 pS. The toxin channel is cation selective without significant selectivity between potassium and sodium (gamma [K+]/gamma [Na+] greater than or equal to 1.35). In all the lipids studied gamma is larger at positive than at negative voltages. The toxin channel is voltage dependent both at neutral and acidic pH: for phosphatidylserine membranes, the probability of the channel being open is much greater at positive than at negative voltage. In different phospholipids the channel exhibits different voltage dependence. In phosphatidylserine membranes the channel is inactivated at negative voltages, whereas in diphytanoylphosphatidylcholine membranes channels are more active at negative voltages than at positive. The presence of acidic phospholipids in the bilayers increases both the single-channel conductance as well as the probability of the channel being open at positive voltage. A subconductance state is readily identifiable in the single-channel recordings. Accordingly, single-channel conductance histograms are best fitted with a sum of 3 Gaussian distributions corresponding to the closed state, the open subconductance state and the full open state. Channel activity occurs in bursts of openings separated by long closings. Probability density analysis of the open dwell times of the toxin channel indicate the existence of a single open state with a lifetime greater than or equal to 1 ms in all lipids studied. Analysis of intra-bursts closing lifetimes reveals the existence of two components; the slow component is of the order of 1 ms, the fast one is less than or equal to 0.5 ms. The channel activity induced by tetanus toxin in lipid bilayers suggests a mechanism for its neurotoxicity: a voltage dependent, cation selective channel inserted in the postsynaptic membrane would lead to continuous depolarization and, therefore, persistent activation of the postsynaptic cell.     

Complex voltage-dependent behavior of single unliganded calcium-sensitive potassium channels

study and characterization of unliganded openings is of central significance for the elucidation of gating mechanisms for allosteric ligand-gated ion channels. Unliganded openings have been reported for many channel types, but their low open probability can make it difficult to study their kinetics in detail. Because the large conductance calcium-activated potassium channel mSlo is sensitive to both intracellular calcium and to membrane potential, we have been able to obtain stable unliganded single-channel recordings of mSlo with relatively high opening probability. We have found that the single-channel gating behavior of mSlo is complex, with multiple open and closed states, even when no ligand is present. Our results rule out a Monod-Wyman-Changeux allosteric mechanism with a central voltage-dependent concerted step, and they support the existence of quaternary states with less than the full number of voltage sensors activated, as has been suggested by previous work involving measurements of gating currents.     

On the Role of Pore Helix in Regulation of TRPV5 by Extracellular Protons

The transient receptor potential channel TRPV5 is localized to the apical membrane of the distal renal tubule and plays an important role in the regulation of transepithelial Ca²⁺ reabsorption in kidney. We have previously reported that extracellular protons inhibit TRPV5 by binding to glutamate-522 (E522) in the extracellular domain of the channel. We suggested that E522 is an extracellular “pH sensor” and its titration by extracellular protons inhibits TRPV5 via conformational change(s) of the pore helix. We now report that mutation of a pore helix residue glutamate-535 to glutamine (E535Q) enhances the sensitivity of the channel to inhibition by extracellular protons (i.e., shifting the apparent pKa for inhibition by extracellular protons to the more alkaline extracellular pH). The enhancement of extracellular proton-mediated inhibition of E535Q mutant is also dependent on E522. We have also reported that intracellular acidification enhances the sensitivity of TRPV5 to inhibition by extracellular protons. We now find that modulation of the extracellular proton-mediated inhibition by intracellular acidification is preserved in the E535Q mutant. These results provide further support for the idea that pore helix is involved in the regulation of TRPV5 by extracellular protons. Inhibition of TRPV5 by extracellular protons may contribute to hypercalciuria in diseases associated with high acid load.     

Hidden Markov model analysis of intermediate gating steps associated with the pore gate of shaker potassium channels.

Cooperativity among the four subunits helps give rise to the remarkable voltage sensitivity of Shaker potassium channels, whose open probability changes tenfold for a 5-mV change in membrane potential. The cooperativity in these channels is thought to arise from a concerted structural transition as the final step in opening the channel. Recordings of single-channel ionic currents from certain other channel types, as well as our previous recordings from T442S mutant Shaker channels, however, display intermediate conductance levels in addition to the fully open and closed states. These sublevels might represent stepwise, rather than concerted, transitions in the final steps of channel activation. Here, we report a similar fine structure in the closing transitions of Shaker channels lacking the mutation. Describing the deactivation time course with hidden Markov models, we find that two subconductance levels are rapidly traversed during most closing transitions of chimeric, high conductance Shaker channels. The lifetimes of these levels are voltage-dependent, with maximal values of 52 and 22 micros at -100 mV, and the voltage dependences of transitions among these states suggest that they arise from equivalent conformational changes occurring in individual subunits. At least one subconductance level is found to be traversed in normal conductance Shaker channels. We speculate that voltage-dependent conformational changes in the subunits give rise to changes in a "pore gate" associated with the selectivity filter region of the channel, producing the subconductance states. As a control for the hidden Markov analysis, we applied the same procedures to recordings of the recovery from N-type inactivation in Shaker channels. These transitions are found to be instantaneous in comparison.     

Protons activate homomeric Kir6.2 channels by selective suppression of the long and intermediate closures

The ATP-sensitive K+ channels (KATP) play an important role in regulating membrane excitability. These channels are regulated by H+ in addition to ATP, ADP, and phospholipids. To understand how protons affect the single-channel properties, Kir6.2DeltaC36 currents were studied in excised inside-out patches. We chose to study the homomeric Kir6.2 channel with 36 amino acids deleted at the C-terminal end, as there are ADP/ATP-binding sites in the SUR subunit, which may obscure the understanding of the channel-gating process. In the absence of ATP, moderate intracellular acidosis (pH 6.8) augmented P(open) with small suppression (by approximately 10%) of the single-channel conductance. The long and intermediate closures were selectively inhibited, leading to a shortening of the mean closed time without significant changes in the mean open time. Stronger acidification (相似文献   

Sodium channel subconductance levels measured with a new variance-mean analysis

The currents through single Na+ channels were recorded from dissociated cells of the flexor digitorum brevis muscle of the mouse. At 15 degrees C the prolonged bursts of Na+ channel openings produced by application of the drug DPI 201-106 had brief sojourns to subconductance levels. The subconductance events were relatively rare and brief, but could be identified using a new technique that sorts amplitude estimates based on their variance. The resulting "levels histogram" had a resolution of the conductance levels during channel activity that was superior to that of standard amplitude histograms. Cooling the preparation to 0 degrees C prolonged the subconductance events, and permitted further quantitative analysis of their amplitudes, as well as clear observations of single-channel subconductance events from untreated Na+ channels. In all cases the results were similar: a subconductance level, with an amplitude of roughly 35% of the fully open conductance and similar reversal potential, was present in both drug-treated and normal Na+ channels. Drug-treated channels spent approximately 3-6% of their total open time in the subconductance state over a range of potentials that caused the open probability to vary between 0.1 and 0.9. The summed levels histograms from many channels had a distinctive form, with broader, asymmetrical open and substate distributions compared with those of the closed state. Individual subconductance events to levels other than the most common 35% were also observed. I conclude that subconductance events are a normal subset of the open state of Na+ channels, whether or not they are drug treated. The subconductance events may represent a conformational alteration of the channel that occurs when it conducts ions.     

Conformational changes of pore helix coupled to gating of TRPV5 by protons

The transient receptor potential channel TRPV5 constitutes the apical entry pathway for transepithelial Ca2+ transport. We showed that TRPV5 was inhibited by both physiological intra- and extracellular acid pH. Inhibition of TRPV5 by internal protons was enhanced by extracellular acidification. Similarly, inhibition by external protons was enhanced by intracellular acidification. Mutation of either an extra- or an intracellular pH sensor blunted the cross-inhibition by internal and external protons. Both internal and external protons regulated the selectivity filter gate. Using the substituted cysteine accessibility method, we found that intracellular acidification of TRPV5 caused a conformational change of the pore helix consistent with clockwise rotation along its long axis. Thus, rotation of pore helix caused by internal protons facilitates closing of TRPV5 by external protons. This regulation by protons likely contributes to pathogenesis of disturbances of Ca2+ transport in many diseased states. Rotation of pore helix may be a common mechanism for cross-regulation of ion channels by extra- and intracellular signals.     

Subconductance states in calcium-activated potassium channels from canine airway smooth muscle

The single-channel patch clamp technique was used to analyze subconductance states in the 260 pS calcium-activated potassium channel from canine airway smooth muscle. More than sixty minutes of single channel data (greater than 87,000 events) from five excised patches were analyzed. Six subconductance amplitudes were clearly established to be 17, 33, 41, 52, 63 and 72% of the full conductance. Subconductance openings were usually brief (milliseconds) and represented less than 5% of the total channel open time, but they also persisted for several seconds on rare occasions. They appeared to be unaffected by voltage or time after seal formation, but may have increased in occurrence with decreasing calcium concentration. Irregular amplitude intervals, and the presence of ramp-like, analog transitions between conductance states, suggest a model for maxi-K subconductance states in which the channel protein undergoes random conformational changes causing a variable pore size.     

Intracellular pH of crab and crayfish muscle fibre types during GABA application and inhibitory nerve stimulation

The inhibitory motoneurons of crustaceans form synapses both with the sarcolemma of muscle fibres and with the very distal branchings of the excitatory motoneurons. The transmitter of these synapses is GABA (γ-aminobutyric acid) which is known to open Cl⁻ channels. Studies on the dactyl opener muscle of crayfish suggest that application of GABA not only leads to an increase in the Cl⁻ permeability but also to a considerable HCO⁻ ₃ conductance that causes an intracellular acidification. To investigate possible physiological implications, we measured the intracellular pH of various muscle fibre types of crayfish and crab using pH-sensitive microelectrodes. Independent of the presence or absence of inhibitory innervation, bath application of 10⁻⁵ mol l⁻¹ GABA led to acidification in all fibre types (pH change: 0.14 ± 0.08, n=11). In no preparation was a change in intracellular pH observed upon stimulation of specific or common inhibitory motoneurons with 10–40 pulses s⁻¹ for 2–5 min. The results suggest that HCO⁻ ₃ conductance cannot be activated through synaptic GABA receptors. However, all crustacean muscle fibre types seem to possess extrasynaptic GABA-sensitive channels that exhibit a considerable HCO⁻ ₃ conductance. The physiological importance of these channels remains to be elucidated. Accepted: 13 July 2000     

Multiple Conductance Substates in Pharmacologically Untreated Na+ Channels Generating Persistent Openings in Rat Entorhinal Cortex Neurons

Na⁺-channel activity recorded in cell-attached patches from entorhinal cortex neurons in the absence of gating-modifying drugs was examined to determine the possible occurrence of substate openings. Brief sojourns to subconductance levels were occasionally observed within prolonged (“persistent”) burst openings. Subconductance occurrence and amplitude were determined following two distinct, complementary approaches: (1) direct visual inspection and (2) automated detection by application of a method that exploits the current variance of fixed-width tracing segments to sort amplitude estimations. The two approaches led to comparable results. At least six subconductance levels in addition to the full open state were revealed, with amplitudes that were approximately 20%, 30%, 40%, 50%, 60% and 75% that of full openings. The global probability of subconductance opening occurrence within a burst as well as the probability of observing one particular subconductance level within a burst showed no clear dependence upon membrane potential in the −40 to +10 mV range. Open- and closed-time distributions of substate openings could either be similar to those observed in burst full openings or show distinct patterns. Low-amplitude late openings were also observed in isolation, separately from full-size openings. These openings corresponded to conductance levels very similar to those of the substates observed within full-size burst openings; therefore, they were interpreted as isolated subconductance openings. Early, transient openings responsible for the fast-inactivating whole-cell Na⁺-current component also manifested distinct conductance levels, the two most prominent of which were in an approximate 75:100 amplitude ratio. Interestingly, the 75% conductance level observed among early openings occurred much more frequently than in “persistent” burst openings. We conclude that pharmacologically untreated Na⁺ channels from native neurons generate substate openings that may influence differently the multiple gating modes displayed by these channels. Angel Alonso is deceased.     

Biophysical and molecular mechanisms underlying the modulation of heteromeric Kir4.1-Kir5.1 channels by CO2 and pH

CO2 chemoreception may be related to modulation of inward rectifier K+ channels (Kir channels) in brainstem neurons. Kir4.1 is expressed predominantly in the brainstem and inhibited during hypercapnia. Although the homomeric Kir4.1 only responds to severe intracellular acidification, coexpression of Kir4.1 with Kir5.1 greatly enhances channel sensitivities to CO2 and pH. To understand the biophysical and molecular mechanisms underlying the modulation of these currents by CO2 and pH, heteromeric Kir4. 1-Kir5.1 were studied in inside-out patches. These Kir4.1-Kir5.1 currents showed a single channel conductance of 59 pS with open-state probability (P(open)) approximately 0.4 at pH 7.4. Channel activity reached the maximum at pH 8.5 and was completely suppressed at pH 6.5 with pKa 7.45. The effect of low pH on these currents was due to selective suppression of P(open) without evident effects on single channel conductance, leading to a decrease in the channel mean open time and an increase in the mean closed time. At pH 8.5, single-channel currents showed two sublevels of conductance at approximately 1/4 and 3/4 of the maximal openings. None of them was affected by lowering pH. The Kir4.1-Kir5.1 currents were modulated by phosphatidylinositol-4,5-bisphosphate (PIP2) that enhanced baseline P(open) and reduced channel sensitivity to intracellular protons. In the presence of 10 microM PIP2, the Kir4.1-Kir5.1 showed a pKa value of 7.22. The effect of PIP2, however, was not seen in homomeric Kir4.1 currents. The CO2/pH sensitivities were related to a lysine residue in the NH2 terminus of Kir4.1. Mutation of this residue (K67M, K67Q) completely eliminated the CO2 sensitivity of both homomeric Kir4.1 and heteromeric Kir4.1-Kir5.1. In excised patches, interestingly, the Kir4.1-Kir5.1 carrying K67M mutation remained sensitive to low pHi. Such pH sensitivity, however, disappeared in the presence of PIP2. The effect of PIP2 on shifting the titration curve of wild-type and mutant channels was totally abolished when Arg178 in Kir5.1 was mutated. Thus, these studies demonstrate a heteromeric Kir channel that can be modulated by both acidic and alkaline pH, show the modulation of pH sensitivity of Kir channels by PIP2, and provide information of the biophysical and molecular mechanisms underlying the Kir modulation by intracellular protons.     

Mutant TRPV4-mediated toxicity is linked to increased constitutive function in axonal neuropathies

Mutations in TRPV4 have been linked to three distinct axonal neuropathies. However, the pathogenic mechanism underlying these disorders remains unclear. Both gain and loss of calcium channel activity of the mutant TRPV4 have been suggested. Here, we show that the three previously reported TRPV4 mutant channels have a physiological localization and display an increased calcium channel activity, leading to increased cytotoxicity in three different cell types. Patch clamp experiments showed that cells expressing mutant TRPV4 have much larger whole-cell currents than those expressing the wild-type TRPV4 channel. Single channel recordings showed that the mutant channels have higher open probability, due to a modification of gating, and no change in single-channel conductance. These data support the hypothesis that a "gain of function" mechanism, possibly leading to increased intracellular calcium influx, underlies the pathogenesis of the TRPV4-linked axonal neuropathies, and may have immediate implications for designing rational therapies.     

Tetanus toxin channel in phosphatidylserine planar bilayers: conductance states and pH dependence

Tetanus toxin (TeTx) forms ionic channel in phosphatidylserine bilayers. TeTx channels exhibit different modes of channel bursting activity, from a closed state to well defined open states of different amplitudes. At positive applied voltages, TeTx channels flicker continuously between a closed state and the various distinct open states. Furthermore, fast transitions into subconductance states are discernible within the bursts of channel activity. Elementary conductance steps submultiple of the open states were not identified in single channel records owing to rapid transitions between different states. However, statistical analysis shows that conductances cluster with amplitudes multiple of an elementary value: e.g. 25–30 pS at neutral pH. Single channel current amplitudes decrease with the pH of the bulk electrolyte solution. Conductance decrements can be accounted for by the relative decrease of permeant cation concentration at the membrane-water interface, by a relative enrichment of protons that block the channel or by the stabilization of a conformational state of the channel protein. Offprint requests to: F. Gambale     

Actin modifies Ca2+ block of epithelial Na+ channels in planar lipid bilayers

The mechanism by which the cytoskeletal protein actin affects the conductance of amiloride-sensitive epithelial sodium channels (ENaC) was studied in planar lipid bilayers. In the presence of monomeric actin, we found a decrease in the single-channel conductance of alpha-ENaC that did not occur when the internal [Ca2+]free was buffered to <10 nM. An analysis of single-channel kinetics demonstrated that Ca2+ induced the appearance of long-lived closed intervals separating bursts of channel activity, both in the presence and in the absence of actin. In the absence of actin, the duration of these bursts and the time spent by the channel in its open, but not in its short-lived closed state, were inversely proportional to [Ca2+]. This, together with a lengthening of the interburst intervals, translated into a dose-dependent decrease in the single-channel open probability. In contrast, a [Ca2+]-dependent decrease in alpha-ENaC conductance in the presence of actin was accompanied by lengthening of the burst intervals with no significant changes in the open or closed (both short- and long-lived) times. We conclude that Ca2+ acts as a "fast-to-intermediate" blocker when monomeric actin is present, producing a subsequent attenuation of the apparent unitary conductance of the channel.     

Characterization of a Chloride-Selective Channel from Rough Endoplasmic Reticulum Membranes of Rat Hepatocytes: Evidence for a Block by Phosphate

We have characterized the conduction and blocking properties of a chloride channel from rough endoplasmic reticulum membranes of rat hepatocytes after incorporation into a planar lipid bilayer. Our experiments revealed the existence of a channel with a mean conductance of 164 ± 5 pS in symmetrical 200 mm KCl solutions. We determined that the channel was ten times more permeable for Cl⁻ than for K⁺, calculated from the reversal potential using the Goldman-Hodgkin-Katz equation. The channel was voltage dependent, with an open probability value ranging from 0.9 at −20 mV to 0.4 at +60 mV. In addition to its fully open state, the channel could also enter a flickering state, which appeared to involve rapid transitions to zero current level. Our results showed a decrease of the channel mean open time combined with an increase of the channel mean closed time at positive potentials. An analysis of the dwell time distributions for the open and closed intervals led to the conclusion that the observed fluctuation pattern was compatible with a kinetic scheme containing a single open state and a minimum of three closed states. The permeability sequence for test halides determined from reversal potentials was Br⁻ > Cl⁻ > I⁻≈ F⁻. The voltage dependence of the open probability was modified by the presence of halides in trans with a sequence reflecting the permeability sequence, suggesting that permeant anions such as Br⁻ and Cl⁻ have access to an internal site capable of controlling channel gating. Adding NPPB to the cis chamber inhibited the channel activity by increasing fast flickering and generating long silent periods, whereas channel activity was not affected by 50 μm DNDS in trans. The channel was reversibly inhibited by adding phosphate to the trans chamber. The inhibitory effect of phosphate was voltage-dependent and could be reversed by addition of Cl⁻. Our results suggest that channel block involves the interaction of HPO²⁻ ₄ with a site located at 70% of the membrane span. Received: 10 January 1997/Revised: 29 May 1997     

New properties of mitochondrial ATP-regulated potassium channels

The ATP-regulated potassium channel is present in the inner membrane of heart mitochondria. In this study, the activity of a single channel was measured after reconstituting the myocardium inner mitochondrial membrane into a planar lipid bilayer. We provide direct evidence of vectorial pH regulation of mitoK_ATP channels. When the matrix side was alkalized, this changed the channel conductance, the open probability, and the mean open and closed dwell time distributions. The conductance of the mitoK_ATP channel increased from about 110 ± 8 to 145 ± 5 pS upon changing the pH from 7.2 to 8.2. This effect was reversed by reverting the pH to the neutral value. The mitoK_ATP channel activity was not altered by alkalization of the cytosolic side of the planar lipid bilayer. We also observed that acidification from pH 7.2 to 6.2, in either the matrix or cytosolic compartments, decreased the open probability of the channel. This effect was reversed by perfusion with a pH 7.2 medium. Additionally, our results suggest that the mitoK_ATP channel is regulated by multiple phosphorylation events. The channel activity was inhibited by an ATP/Mg²⁺ complex, but not by ATP alone, nor by a non-hydrolysable ATP analog, e.g. AMP-PNP/Mg²⁺. The mitoK_ATP channel “run-down” was reversed by incubating with the ATP/Mg²⁺ complex on both sides of the planar lipid bilayer. We conclude that both pH and ATP play an important regulatory role for the cardiac mitoK_ATP channel with respect to the phenomenon of ischemia–reperfusion.