Decrease In Open Probability Research Articles

Exercise Training Reverses Unparallel Downregulation of MaxiK Channel - and 1-Subunit to Enhance Vascular Function in Aging Mesenteric Arteries

This study was designed to determine the effects of aerobic exercise training on aging-associated selective changes of the function and expression of the large-conductance Ca(2+)-activated K(+) (MaxiK) channels in mesenteric arteries. Male Wistar rats aged 19-21 months were randomly assigned to sedentary (O-SED) and exercise-trained groups (O-EX). Two-month-old rats were used as Young control. Addition of iberiotoxin (10(-8) M) increased the norepinephrine-induced arterial contraction in all three groups, with the greatest enhancement being in Young and the least in O-SED. Patch clamp study revealed the characteristics of aging on MaxiK channel function in mesenteric arteries, mainly including (a) decrease of iberiotoxin-sensitive whole-cell K(+) current, (b) decrease of open probability and Ca(2+)/voltage sensitivity of single MaxiK channel, and (c) reduction of tamoxifen-induced MaxiK activation. After exercise training, all of these changes were markedly inhibited. Western blotting revealed that the protein expression of MaxiK was significantly reduced with aging and the suppression of β1-subunit was larger than that of α-subunit, although exercise training diminished this alteration. Taken together, aerobic exercise training reverses the aging-related unparallel downregulation of MaxiK α- and β1-subunit expression on mesenteric arteries, which partly underlies the beneficial effect of exercise on restoring aging-associated reduction in mesenteric artery vasodilatory properties.

Inhibition of GluN2A-Containing N-Methyl-d-Aspartate Receptors by 2-Naphthoic Acid

N-Methyl-D-aspartate (NMDA) receptors mediate excitatory synaptic transmission in the central nervous system and play important roles in synaptic development and plasticity, but also mediate glutamate neurotoxicity. Recently, 2-naphthoic acid (NPA) and its derivatives have been identified as allosteric, noncompetitive NMDA receptor inhibitors. The selectivity of NPA derivatives among NMDA receptor subtypes was mapped structurally to the ligand-binding domain, and was proposed to be mediated by residues on the S1 segment. To delineate the kinetic mechanism by which NPA inhibits NMDA receptor activity, we examined its effects on the NMDA receptor gating reaction. Using whole-cell patch clamping on human embryonic kidney 293 cells expressing recombinant NMDA family of glutamate receptor subunits, GluN1/GluN2A, we found that NPA has a 50% inhibitory effect at 1.9 mM. Further, from one-channel current recordings, we found that 4 mM NPA caused a 62% decrease in open probability by decreasing mean open time 2.5-fold and by increasing mean closed time 2-fold. Kinetic modeling suggested that NPA binding stabilized NMDA receptor closed states and increased the energy barriers toward open states, causing NMDA receptors to dwell longer in pre-open states along the activation pathway. The reaction mechanisms we derived provide quantitative insight into the inhibitory mechanism of NPA and help anticipate its effects on GluN1/GluN2A receptors during both physiologic and pathologic activation modalities.

Inhibitory Effects of 2-Naphthoic Acid on the Gating of NMDA Receptors

N-methyl-D-aspartate (NMDA) receptors mediate excitatory synaptic transmission in central nervous system and play important roles in development and synaptic plasticity, but have also been found to mediate neurotoxicity. Recently, 2-naphthoic acid (NPA) and its derivatives have been identified as allosteric, noncompetitive NMDA receptors inhibitors. The inhibitory selectivity of NPA derivatives among NMDA receptor subtypes was mapped to the ligand-binding domain (LBD), and the binding site of NPA is proposed to be located in the LBD dimer interface. However, the mechanism by which NPA exerts inhibitory effect is still unclear. We examined how NPA affects the gating reaction of NMDA receptors. Whole-cell patch clamp on HEK 293 cells expressing recombinant NR1-1a/NR2A showed that NPA has 90% maximum inhibition with IC50 of 2.1 mM. Furthermore, by recording single-channel currents from NR1-1a/NR2A receptors with 4 mM NPA, we found a 62% decrease in open probability (Po), due to a 2.5-fold decrease in mean open time (MOT) and a 2-fold increase in mean closed time (MCT). Kinetic modeling suggests NPA increases energy barrier of gating and destabilizes the open state, thus making NRs accumulate in the closed states along the activation pathway. These results provide insight into the inhibitory mechanism of NPA, and help anticipate its effects on both physiological and pathological conditions.

Divalent Cations are Antagonists of the Sodium-Dependent Potassium Channel Slo2.2

We studied the effect of different divalent cations on the Na+-dependent potassium channel Slo2.2 (Slack) using inside-out patches from Xenopus oocytes and a Slack-HEK cell line heterologously expressing Slack channels. In the presence of intracellular sodium, the addition of divalent cations reduces significantly Slack currents recorded in macropatches. Among the divalent cations studied, the most effective in reducing channel activity was Cd++ followed by Ni++, Ca++ and Mg++. Several results suggest that this effect is not caused by blocking the pore of the channel. First, the decrease in currents is not voltage dependent as expected for a cation blocking the channel pore. Second, single channel recordings show a decrease in open probability but not a significant reduction in single channel conductance. Third, some of these cations activate rather than block the Ca++-dependent channel Slo1, which is likely to have a pore structure similar to Slo2.2. Outside-out patches have been used to show that divalent cations do not have an inhibitory effect when applied to the extracellular side of the channel. We propose that the divalent cations may be competing with Na+ for the Na+ binding site. To test this hypothesis, we will examine the effect of divalent cations on several channel mutants and chimeras. These experiments also may help to reveal the structure and localization of the Na+ binding site.

Constitutive Activation of the N-Methyl-d-aspartate Receptor via Cleft-spanning Disulfide Bonds

Although the N-methyl-D-aspartate (NMDA) receptor plays a critical role in the central nervous system, many questions remain regarding the relationship between its structure and functional properties. In particular, the involvement of ligand-binding domain closure in determining agonist efficacy, which has been reported in other glutamate receptor subtypes, remains unresolved. To address this question, we designed dual cysteine point mutations spanning the NR1 and NR2 ligand-binding clefts, aiming to stabilize these domains in closed cleft conformations. Two mutants, E522C/I691C in NR1 (EI) and K487C/N687C in NR2 (KN) were found to exhibit significant glycine- and glutamate-independent activation, respectively, and co-expression of the two subunits produced a constitutively active channel. However, both individual mutants could be activated above constitutive levels in a concentration-dependent manner, indicating that cleft closure does not completely prevent agonist association. Interestingly, whereas the NR2 KN disulfide was found to potentiate channel gating and M3 accessibility, NR1 EI exhibited the opposite phenotype, suggesting that the EI disulfide may trap the NR1 ligand-binding domain in a lower efficacy conformation. Furthermore, both mutants affected agonist sensitivity at the opposing subunit, suggesting that closed cleft stabilization may contribute to coupling between the subunits. These results support a correlation between cleft stability and receptor activation, providing compelling evidence for the Venus flytrap mechanism of glutamate receptor domain closure.

Abstract 174: Biophysical Basis of Protein Kinase C Inhibition of the Novel alpha1D Calcium Channel

The recently reported α 1D calcium channel in the heart is known to be regulated by protein kinase C (PKC) at the whole cell level and has been implicated in atrial fibrillation. The biophysical basis of this regulation at the single channel level is not known. Therefore, the effect of PKC activation was studied on α 1D calcium channel expressed in tsA201 cells using cell-attached method. Unitary currents were recorded in the presence of 70 mM Ba 2+ as the charge carrier. Unitary currents were evoked by 500 ms depolarizing pulses from a holding potential of −80 mV every 0.5 Hz. Under basal condition, channel activity was rare and infrequent, however Bay K 8644 (1 μM) induced channel openings with a conductance of 22.3 pS. Single channel analysis of open and closed time distributions were best fitted with a single exponential. PKC activation by PMA (10 nM), a phorbol ester derivative, resulted in a decrease in open probability and increase in closed-time without any significant effect on the conductance of the α 1D calcium channel. This is consistent with a decreased entry of α 1D Ca channel into open states in the presence of PMA. These data show, for the fist time, 1) the α 1D calcium channel activity at the single channel level and 2) the biophysical basis of by which PKC activation inhibits the α 1D calcium channel. The shortening of the open-time and the lengthening of the closed-time constants and the increase in blank sweeps may explain the inhibition of the α 1D Ca-channel activity and the reduction in whole-cell α 1D Ca current previously reported. Altogether, these data are relevant to the understanding of the patho-physiology of α 1D calcium channel and its regulation by the autonomics.

Protein kinase C activation inhibits α1D L-type Ca channel: A single-channel analysis

The recently reported alpha1D Ca channel in the heart is known to be regulated by protein kinase C (PKC) at the whole cell level and has been implicated in atrial fibrillation. The biophysical basis of this regulation at the single-channel level is not known. Therefore, the effect of PKC activation was studied on alpha1D Ca channel expressed in tsA201 cells using cell-attached configuration. Unitary currents were recorded in the presence of 70 mM Ba2+ as the charge carrier at room temperature. Under basal condition, channel activity was rare and infrequent; however, Bay K 8644 (1 microM) induced channel openings with a conductance of 22.3 pS. Single channel analysis of open and closed time distributions were best fitted with a single exponential. PKC activation by 4alpha-phorbol 12-myristate 13-acetate (PMA; 10 nM), a phorbol ester derivative, resulted in a decrease in open probability and increase in closed-time without any significant effect on the conductance of the alpha1D Ca channel. This is consistent with a decreased entry of alpha1D Ca channel into open states in the presence of PMA. PMA effects could not be reproduced by 4-alpha Phorbol, an inactive PMA analogue. These data show, for the first time, (1) the alpha1D Ca channel activity at the single-channel level and (2) the biophysical basis by which PKC activation inhibits the alpha1D Ca channel. The shortening of the open-time and the lengthening of the closed-time constants and the increase in blank sweeps may explain the inhibition of the previously reported whole-cell alpha1D Ca current. Altogether, these data are essential for understanding the complex role of alpha1D Ca channel not only in physiological settings but also in pathological settings such as atrial fibrillation.

Ca2+/calmodulin modulates TRPV1 activation by capsaicin.

TRPV1 ion channels mediate the response to painful heat, extracellular acidosis, and capsaicin, the pungent extract from plants in the Capsicum family (hot chili peppers) (Szallasi, A., and P.M. Blumberg. 1999. Pharmacol. Rev. 51:159–212; Caterina, M.J., and D. Julius. 2001. Annu. Rev. Neurosci. 24:487–517). The convergence of these stimuli on TRPV1 channels expressed in peripheral sensory nerves underlies the common perceptual experience of pain due to hot temperatures, tissue damage and exposure to capsaicin. TRPV1 channels are nonselective cation channels (Caterina, M.J., M.A. Schumacher, M. Tominaga, T.A. Rosen, J.D. Levine, and D. Julius. 1997. Nature. 389:816–824). When activated, they produce depolarization through the influx of Na+, but their high Ca2+ permeability is also important for mediating the response to pain. In particular, Ca2+ influx is thought to be required for the desensitization to painful sensations over time (Cholewinski, A., G.M. Burgess, and S. Bevan. 1993. Neuroscience. 55:1015–1023; Koplas, P.A., R.L. Rosenberg, and G.S. Oxford. 1997. J. Neurosci. 17:3525–3537). Here we show that in inside-out excised patches from TRPV1 expressed in Xenopus oocytes and HEK 293 cells, Ca2+/calmodulin decreased the capsaicin-activated current. This inhibition was not mimicked by Mg2+, reflected a decrease in open probability, and was slowly reversible. Furthermore, increasing the calmodulin concentration in our patches by coexpression of wild-type calmodulin with TRPV1 produced inhibition by Ca2+ alone. In contrast, patches excised from cells coexpressing TRPV1 with a mutant calmodulin did not respond to Ca2+. Using an in vitro calmodulin-binding assay, we found that TRPV1 in oocyte lysates bound calmodulin, although in a Ca2+-independent manner. Experiments with GST-fusion proteins corresponding to regions of the channel NH2-terminal domain demonstrated that a stretch of ∼30 amino acids adjacent to the first ankyrin repeat bound calmodulin in a Ca2+-dependent manner. The physiological response to pain involves an influx of Ca2+ through TRPV1. Our results indicate that this Ca2+ influx may feed back on the channels, inhibiting their gating. This type of feedback inhibition could play a role in the desensitization produced by capsaicin.

Contribution of Cytosolic Cysteine Residues to the Gating Properties of the Kir2.1 Inward Rectifier

The topological model proposed for the Kir2.1 inward rectifier predicts that seven of the channel 13 cysteine residues are distributed along the N- and C-terminus regions, with some of the residues comprised within highly conserved domains involved in channel gating. To determine if cytosolic cysteine residues contribute to the gating properties of Kir2.1, each of the N- and C-terminus cysteines was mutated into either a polar (S, D, N), an aliphatic (A,V, L), or an aromatic (W) residue. Our patch-clamp measurements show that with the exception of C76 and C311, the mutation of individual cytosolic cysteine to serine (S) did not significantly affect the single-channel conductance nor the channel open probability. However, mutating C76 to a charged or polar residue resulted either in an absence of channel activity or a decrease in open probability. In turn, the mutations C311S (polar), C311R (charged), and to a lesser degree C311A (aliphatic) led to an increase of the channel mean closed time due to the appearance of long closed time intervals ( T c ≥ 500 ms) and to a reduction of the reactivation by ATP of rundown Kir2.1 channels. These changes could be correlated with a weakening of the interaction between Kir2.1 and PIP 2, with C311R and C311S being more potent at modulating the Kir2.1-PIP 2 interaction than C311A. The present work supports, therefore, molecular models whereby the gating properties of Kir2.1 depend on the presence of nonpolar or neutral residues at positions 76 and 311, with C311 modulating the interaction between Kir2.1 and PIP 2.

Spermine block of the strong inward rectifier potassium channel Kir2.1: dual roles of surface charge screening and pore block.

Inward rectification in strong inward rectifiers such as Kir2.1 is attributed to voltage-dependent block by intracellular polyamines and Mg2+. Block by the polyamine spermine has a complex voltage dependence with shallow and steep components and complex concentration dependence. To understand the mechanism, we measured macroscopic Kir2.1 currents in excised inside-out giant patches from Xenopus oocytes expressing Kir2.1, and single channel currents in the inside-out patches from COS7 cells transfected with Kir2.1. We found that as spermine concentration or voltage increased, the shallow voltage-dependent component of spermine block at more negative voltages was caused by progressive reduction in the single channel current amplitude, without a decrease in open probability. We attributed this effect to spermine screening negative surface charges involving E224 and E299 near the inner vestibule of the channel, thereby reducing K ion permeation rate. This idea was further supported by experiments in which increasing ionic strength also decreased Kir2.1 single channel amplitude, and by mutagenesis experiments showing that this component of spermine block decreased when E224 and E299, but not D172, were neutralized. The steep voltage-dependent component of block at more depolarized voltages was attributed to spermine migrating deeper into the pore and causing fast open channel block. A quantitative model incorporating both features showed excellent agreement with the steady-state and kinetic data. In addition, this model accounts for previously described substate behavior induced by a variety of Kir2.1 channel blockers.

The low conductance K + channel in human colonic crypt cells has a voltage–dependent permeability not affected by Mg ++

The low conductance K + channel found in human colonocytes was investigated using the patch-clamp technique. The channel is Ca ++-dependent and is blocked by Ba ++ (5 mM) with a decrease in open probability from 0.42 to 0.19. At −40 mV the slope conductance was 29 pS (using intracellular solution in the pipette). In inside-out patches, inward rectification was seen both with KCl (pipette)/NaCl (bath) solutions as well as KCl/KCl solutions. The rectification could not be affected by omitting Mg ++ from the pipette or the bath solution, neither by exposing the patches to the polyamine spermine (1 mM). Using the Goldman–Hodgkin–Katz equation we show that the permeability decreased in a linear fashion from approximately 5.2 × 10 −14 cm 3/s to 1.8 × 10 −14 cm 3/s (−100 to +100 mV), both with and without Mg ++ in the solutions. There was no significant difference in the nominal values of permeability. This property of the K + channel may facilitate the hyperpolarization needed to sustain a chloride secretion.

Nitric oxide modulates cardiac Na(+) channel via protein kinase A and protein kinase G.

We directly tested the effects of nitric oxide (NO) on Na(+) channels in guinea pig and mouse ventricular myocytes using patch-clamp recordings. We have previously shown that NO donors have no observed effects on expressed Na(+) channels. In contrast, NO (half-blocking concentration of 523 nmol/L) significantly reduces peak whole-cell Na(+) current (I(Na)) in isolated ventricular myocytes. The inhibitory effect of NO on I(Na) was not associated with changes in activation, inactivation, or reactivation kinetics. At the single-channel level, the reduction in macroscopic current was mediated by a decrease in open probability and/or a decrease in the number of functional channels with no change in single-channel conductance. Application of cell permeable analogs of cGMP or cAMP mimics the inhibitory effects of NO. Furthermore, the effects of NO on I(Na) can only be blocked by inhibition of both cGMP and cAMP pathways. Sulfhydryl-reducing agent does not reverse the effect of NO. In summary, although NO exerts its action via the known guanylyl cyclase (GC)/cGMP pathway, our findings provide evidence that NO can mediate its function via a GC/cGMP-independent mechanism involving the activation of adynylyl cyclase (AC) and cAMP-dependent protein kinase.

Altered gating of opiate receptor-modulated K+ channels on amygdala neurons of morphine-dependent rats.

The molecular mechanism of tolerance to opiate drugs is poorly understood. We have used single-channel patch-clamp recordings to study opiate receptor effects on dissociated neurons from rat amygdala, a limbic region implicated in addiction processes. A 130-pS inwardly rectifying K(+)-preferring cation channel was activated by mu opioid receptors in a membrane-delimited manner. After chronic treatment of the rats with morphine, channel gating changed markedly, with an approximately 100-fold decrease in open probability at a given morphine concentration. The change in channel gating correlated both in time course and in dose of morphine treatment with the development of functional opiate dependence and appeared to arise at a step after G-protein activation and before channel permeation by K(+). This decreased receptor-channel coupling appears to be large enough to account quantitatively for opiate tolerance and may represent one of the mechanisms through which tolerance occurs.

Two types of BK channels in immature rat neocortical pyramidal neurons.

1. The properties of large conductance Ca(2+)-activated K+ channels (BK channels) were investigaed in neocortical infragranular pyramidal neurons by the use of inside-out patch recordings. Neurons were acutely isolated from slices of newborn to 28-day-old rats (P0-P28) by using minimal protease exposure followed by trituration with a vibrating glass probe. Two types of BK channels, slow-gating and fast-gating, were observed in immature neurons (P0-P5), whereas only slow-gating BK were found in more mature neurons. Fast-gating BK channels differed in conductance, voltage dependence, and kinetics from the slow-gating ones. 2. The properties of fast-gating channels included a conductance of 145 +/- 12.9 (SE) pS; frequent openings with short mean open times that were relatively voltage-independent, mean closed times that showed a voltage-dependent increase, a voltage-dependent decrease in open probability (Po). The properties of slow-gating channels contrasted with those of the fast-gating ones, in that the former had a conductance of 181 +/- 3.9 pS, longer mean open times that showed a voltage-dependent increase, mean closed times that showed a marked voltage-dependent decrease, a voltage-dependent increase in Po, and slight inward rectification. The significance of these developmental variations in channel properties is discussed.

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.

ATP-inhibitable Cl- channel in apical membranes of cultured rabbit cortical collecting duct cells.

Excised patches of apical membranes from immunodissected rabbit cortical collecting duct cells in primary culture were studied by the patch-clamp technique. Barium (1 mM) and tetraethylammonium chloride (5 mM) were added to all solutions to block potassium channel activity. A unique channel was observed that exhibited inward rectification under symmetrical ionic conditions with a measured chord conductance of 54.0 +/- 2.5 pS at -80 mV (n = 11) and 22.1 +/- 1.7 pS at +80 mV (n = 5). This channel was chloride selective, with a PNa:PCl of 0.16 (n = 3). Kinetic analysis revealed a voltage-independent open-time probability of 0.80 +/- 0.07 (n = 6). Open-time probability within bursts was 0.96 +/- 0.01. Addition of ATP to the cytosolic surface of the channel resulted in a dose-dependent decrease in open probability, with a threshold effect at 10(-4) M, due to a reduction in burst open time. The effect of ATP was immediate, rapidly reversible at room temperature, and mimicked by GTP, adenosine 5'-O-(3-thiotriphosphate), and guanosine 5'-O-(3-thiotriphosphate). This channel may link epithelial chloride permeability to cellular ATP content in the rabbit cortical collecting duct.

Trinitrophenyl-ATP blocks colonic Cl- channels in planar phospholipid bilayers. Evidence for two nucleotide binding sites.

Outwardly rectifying 30-50-pS Cl- channels mediate cell volume regulation and transepithelial transport. Several recent reports indicate that rectifying Cl- channels are blocked after addition of ATP to the extracellular bath (Alton, E. W. F. W., S. D. Manning, P. J. Schlatter, D. M. Geddes, and A. J. Williams. 1991. Journal of Physiology. 443:137-159; Paulmichl, M., Y. Li, K. Wickman, M. Ackerman, E. Peralta, and D. Clapham. 1992. Nature. 356:238-241). Therefore, we decided to conduct a more detailed study of the ATP binding site using a higher affinity probe. We tested the ATP derivative, 2',3',O-(2,4,6-trinitrocyclohexadienylidene) adenosine 5'-triphosphate (TNP-ATP), which has a high affinity for certain nucleotide binding sites. Here we report that TNP-ATP blocked colonic Cl- channels when added to either bath and that blockade was consistent with the closed-open-blocked kinetic model. The TNP-ATP concentration required for a 50% decrease in open probability was 0.27 microM from the extracellular (cis) side and 20 microM from the cytoplasmic (trans) side. Comparison of the off rate constants revealed that TNP-ATP remained bound 28 times longer when added to the extracellular side compared with the cytoplasmic side. We performed competition studies to determine if TNP-ATP binds to the same sites as ATP. Addition of ATP to the same bath containing TNP-ATP reduced channel amplitude and increased the time the channel spent in the open and fast-blocked states (i.e., burst duration). This is the result expected if TNP-ATP and ATP compete for block, presumably by binding to common sites. In contrast, addition of ATP to the bath opposite to the side containing TNP-ATP reduced amplitude but did not alter burst duration. This is the result expected if opposite-sided TNP-ATP and ATP bind to different sites. In summary, we have identified an ATP derivative that has a nearly 10-fold higher affinity for reconstituted rectifying colonic Cl- channels than any previously reported blocker (Singh, A. K., G. B. Afink, C. J. Venglarik, R. Wang, and R. J. Bridges. 1991. American Journal of Physiology. 260 [Cell Physiology. 30]:C51-C63). Thus, TNP-ATP should be useful in future studies of ion channel nucleotide binding sites and possibly in preliminary steps of ion channel protein purification. In addition, we have obtained good evidence that there are at least two nucleotide binding sites located on opposite sides of the colonic Cl- channel and that occupancy of either site produces a blocked state.

Regulation of high-conductance anion channels by G proteins and 5-HT1A receptors in CHO cells.

This study addresses the mechanisms responsible for regulation of high-conductance anion channels by GTP binding proteins in Chinese hamster ovary (CHO) cells. Single-channel currents were measured in inside-out membrane patches using patch-clamp techniques. Anion-selective channels with a unitary conductance of 381 +/- 8 pS activated spontaneously in 48% of excised patches. In patches with no spontaneous channel activity, addition of GppNHp, a nonhydrolyzable analogue of GTP, activated channels in 8 of 12 studies, and in patches with spontaneous channel activity, GppNHp increased open probability in 4 of 4 experiments. In contrast, GDP beta S, a nonhydrolyzable GDP analogue, inhibited both spontaneous and GppNHp-induced channel activity. In patches without spontaneous channel activity, addition of cholera toxin activated channels in five of eight studies. Interestingly, pertussis toxin had a similar effect, activating channels in five of seven previously quiescent patches. To further evaluate the possible role of inhibitory G proteins in channel regulation, activity was measured in cell-attached patches in cells transfected with the serotonin 5-HT1A receptor, which is coupled to effector mechanisms through a pertussis toxin-sensitive G protein. Stimulation of 5-HT1A-transfected cells with the receptor agonist (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin caused a transient decrease in open probability in either standard or high-potassium solutions. In aggregate, these findings suggest that both cholera and pertussis toxin-sensitive G proteins contribute to regulation of high-conductance anion channels in CHO cells.

Mechanism of heptanol-induced uncoupling of cardiac gap junctions: a perforated patch-clamp study.

The influence of heptanol on gap junctional and non-junctional membrane currents was studied in cultured neonatal rat heart cells using both the whole cell and perforated patch voltage-clamp method. With both methods, exposure to heptanol produced a dose-dependent decrease in the junctional current (dissociation constant = 0.54 and 1.20 mM for whole cell and perforated patch experiments, respectively). Heptanol-induced uncoupling was reversible. In the same concentration range, heptanol reduced all nonjunctional membrane ionic currents examined. This suggests that heptanol does not act specifically on gap junction channels but rather on the structure of the lipid membrane. This hypothesis is strengthened by the observation that in monolayer cultures of neonatal rat heart cells fluorescence steady-state anisotropy decreased proportional with increasing the heptanol concentration in the bath. Single-channel conductances (gamma j) were identical with both recording methods (21 and 40-45 pS); heptanol did not alter gamma j. Under conditions of reduced junctional coupling induced by heptanol, junctional conductance (gj) displayed voltage sensitivity at values of gj at which no voltage sensitivity could be observed under control conditions. These results suggest that heptanol-dependent uncoupling was caused by a decrease in open probability of the gap junction channels.

Sensitivity to flow of intrinsic gating in inwardly rectifying potassium channel from mammalian skeletal muscle.

1. Current through inwardly rectifying K+ channels was measured in inside-out patches from rat and human sarcolemmal vesicles and from dispersed rat flexor digitorum brevis muscle fibres. The patches were positioned so as to face the aperture of a large-diameter pipette from which solution of the same composition as the bath solution could be ejected. The solution within the patch pipette and the bath solution both contained principally 140 mM-KCl. 2. The kinetic behaviour of the inwardly rectifying channel was found to vary according to whether the patch was in static or flowing solution. At negative holding potentials, when the channel is open most of the time in static solution, flow produced a reversible and repeatable decrease in open probability. 3. In Mg2(+)-free solution the inwardly rectifying channel allows outward current to pass at positive holding potentials. This allows the kinetic behaviour of the channel in static and flowing solution to be compared over a wider voltage range. 4. In both static and flowing solution, the open probability-voltage relation is sigmoidal and can be fitted by a Boltzmann curve. As a result of flow, the maximum open probability at negative potentials is decreased and the mid-point of the relation is shifted to the right by more than 20 mV. 5. No evidence could be found for the existence of a local concentration gradient sensitive to flow. Application of suction to the patch pipette showed the inwardly rectifying channels not to be sensitive to membrane stretch.(ABSTRACT TRUNCATED AT 250 WORDS)