Increased Channel Open Probability Research Articles

Mechanisms of regulation of epithelial sodium channel by SGK1 in A6 cells.

The serum and glucocorticoid induced kinase 1 (SGK1) participates in the regulation of sodium reabsorption in the distal segment of the renal tubule, where it may modify the function of the epithelial sodium channel (ENaC). The molecular mechanism underlying SGK1 regulation of ENaC in renal epithelial cells remains controversial. We have addressed this issue in an A6 renal epithelial cell line that expresses SGK1 under the control of a tetracycline-inducible system. Expression of a constitutively active mutant of SGK1 (SGK1T S425D) induced a sixfold increase in amiloride-sensitive short-circuit current (I sc). Using noise analysis we demonstrate that SGK1 effect on I sc is due to a fourfold increase in the number of functional ENaCs in the membrane and a 43% increase in channel open probability. Impedance analysis indicated that SGK1T S425D increased the absolute value of cell equivalent capacitance by an average of 13.7%. SGK1T S425D also produced a 1.6–1.9-fold increase in total and plasma membrane subunit abundance, without changing the half-life of channels in the membrane. We conclude that in contrast to aldosterone, where stimulation of transport can be explained simply by an increase in channel synthesis, SGK1 effects are more complex and involve at least three actions: (1) increase of ENaC open probability; (2) increase of subunit abundance within apical membranes and intracellular compartments; and (3) activation of one or more pools of preexistent channels within the apical membranes and/or intracellular compartments.

Melatonin receptor signaling in pregnant and nonpregnant rat uterine myocytes as probed by large conductance Ca2+-activated K+ channel activity.

The mRNAs of MT1 and MT2 melatonin receptors are present in cells from nonpregnant (NPM) and pregnant (PM) rat myometrium. To investigate the coupling of melatonin receptors to Gq- and Gi-type of heterotrimeric G proteins, we analyzed the activity of large-conductance Ca2+-activated K+ (BKCa) channels, the expression of which in the uterus is confined to smooth muscle cells. The melatonin receptor agonist 2-iodomelatonin induced a pertussis toxin (PTX)-insensitive increase in channel open probability that was blocked by the nonselective antagonist luzindole. The 2-iodomelatonin effect on channel open probability was suppressed by overexpression of the Gqalpha-inactivating protein RGS16 and the phospholipase C inhibitor U-73122. The activity of BKCa channels is differentially regulated by protein kinase A (PKA) in NPM and PM cells. Thus, the beta-adrenoceptor agonist isoprenaline inhibited the BKCa channel conducted whole-cell outward current (Iout) in NPM cells and enhanced Iout in PM cells. Additional application of 2-iodomelatonin antagonized the isoprenaline effect on Iout in NPM cells but enhanced Iout in PM cells. All 2-iodomelatonin effects on Iout were sensitive to PTX treatment and the PKA inhibitor H-89. We therefore conclude that melatonin activates both the PTX-insensitive Gq/phospholipase C/Ca2+ and the PTX-sensitive Gi/cAMP/PKA signaling pathway in rat myometrium.

Increased open probability of single cardiac L-type calcium channels in patients with chronic atrial fibrillation. role of phosphatase 2A.

The L-type calcium channel (LCC) plays a crucial role in the electrical remodeling of atrial fibrillation (AF). AF is associated with reduction of L-type calcium current density, due to a transcriptional downregulation of the pore forming alpha(1c)-subunit of LCC. However, it is unclear, whether this current reduction is related to a decrease in channel number or to alterations in channel function. Hence, we performed a single LCC analysis to assess channel gating and function in human AF. We used the cell-attached patch-clamp technique in isolated atrial human cardiomyocytes of 25 patients with sinus rhythm (SR) and 15 patients with chronic AF. Protein expression of the pore-forming alpha(1c)-subunit of LCC was reduced by 40% in AF. Single channel peak average current was 1.7-fold higher in AF than in SR, due to a 3.1-fold higher open probability of LCC. Since phosphatase 2A (PP2A) is known to preferentially reduce LCC open probability via channel dephosphorylation, we assessed whether PP2A expression or activity is reduced in AF. Okadaic acid, an inhibitor of phosphatases, increased channel open probability in SR, but not in AF. However, Western blot analysis of atrial homogenates of the same patient population revealed unchanged expression of PP2A. Human AF is characterized by increased single LCC activity, due to an increase of channel open probability. The blunted effect of PP2A on LCC as shown by single channel analysis may be related to a reduction of cytosolic PP2A activity or impaired local interaction between PP2A and LCC in AF.

Diketopyridylryanodine Has Three Concentration-dependent Effects on the Cardiac Calcium-release Channel/Ryanodine Receptor

By interacting with more than one site, ryanoids induce multiple effects on calcium-release channels. To date, the kinetics of interaction of only one of these sites has been characterized. Using C(4),C(12)-diketopyridylryanodine in both [(3)H]ryanodine binding and single channel experiments we characterized another site on the cardiac ryanodine receptor (RyR2) with which ryanoids interact. Competitive binding of this ryanoid to RyR2 implied a minimal two-site binding model. At the single channel level, C(4),C(12)-diketopyridylryanodine induced three distinct effects. At nanomolar concentrations, it increased channel open probability severalfold without inducing a subconductance. This effect was independent of membrane holding potential. As for other ryanoids, low micromolar concentrations of C(4),C(12)-diketopyridylryanodine readily induced a subconductance state. The major subconductance had a current amplitude of 52% of fully open, it was reversible, and its time to induction and duration were voltage- and concentration-dependent, affording Hill slopes of >2. At higher micromolar concentrations C(4),C(12)-diketopyridylryanodine induced long lasting, yet reversible shut states. Using a pharmacological strategy we have discerned an additional ryanoid-binding site on RyR2 that triggers an increase in channel activity. This site likely resides outside the strict confines of the transmembrane conducting pathway.

Lanthanides Potentiate TRPC5 Currents by an Action at Extracellular Sites Close to the Pore Mouth

Mammalian members of the classical transient receptor potential channel (TRPC) subfamily (TRPC1-7) are Ca(2+)-permeable cation channels involved in receptor-mediated increases in intracellular Ca(2+). Unlike most other TRP-related channels, which are inhibited by La(3+) and Gd(3+), currents through TRPC4 and TRPC5 are potentiated by La(3+). Because these differential effects of lanthanides on TRPC subtypes may be useful for clarifying the role of different TRPCs in native tissues, we characterized the potentiating effect in detail and localized the molecular determinants of potentiation by mutagenesis. Whole cell currents through TRPC5 were reversibly potentiated by micromolar concentrations of La(3+) or Gd(3+), whereas millimolar concentrations were inhibitory. By comparison, TRPC6 was blocked to a similar extent by La(3+) or Gd(3+) at micromolar concentrations and showed no potentiation. Dual effects of lanthanides on TRPC5 were also observed in outside-out patches. Even at micromolar concentrations, the single channel conductance was reduced by La(3+), but reduction in conductance was accompanied by a dramatic increase in channel open probability, leading to larger integral currents. Neutralization of the negatively charged amino acids Glu(543) and Glu(595)/Glu(598), situated close to the extracellular mouth of the channel pore, resulted in a loss of potentiation, and, for Glu(595)/Glu(598) in a modification of channel inhibition. We conclude that in the micromolar range, the lanthanide ions La(3+) and Gd(3+) have opposite effects on whole cell currents through TRPC5 and TRPC6 channels. The potentiation of TRPC4 and TRPC5 by micromolar La(3+) at extracellular sites close to the pore mouth is a promising tool for identifying the involvement of these isoforms in receptor-operated cation conductances of native cells.

Protein tyrosine kinase-dependent modulation of isoflurane effects on cardiac sarcolemmal K(ATP) channel.

Cardiac adenosine triphosphate-sensitive potassium (K(ATP)) channels and protein tyrosine kinases (PTKs) are mediators of ischemic preconditioning, but the interaction of both and a role in myocardial protection afforded by volatile anesthetics have not been defined. Whole cell and single channel patch clamp techniques were used to investigate the effects of isoflurane and the PTK inhibitor genistein on the cardiac sarcolemmal K(ATP) channel in acutely dissociated guinea pig ventricular myocytes. At 0.5 mm internal ATP, genistein (50 microm) elicited whole cell K(ATP) current (22.5 +/- 7.9 pA/pF). Genistein effects were concentration-dependent, with an EC50 of 32.3 +/- 1.4 microm. Another PTK inhibitor, tyrphostin B42, had a similar effect. The inactive analog of genistein, daidzein (50 microm), did not elicit K(ATP) current. Isoflurane (0.5 mm) increased genistein (35 microm)-activated whole cell K(ATP) current from 14.5 +/- 3.1 to 32.5 +/- 6.6 pA/pF. Stimulation of receptor PTKs with epidermal growth factor, nerve growth factor, or insulin attenuated genistein and isoflurane effects, and the protein tyrosine phosphatase inhibitor orthovanadate (1 mm) prevented their actions on K(ATP) current. In excised inside-out membrane patches, and at fixed 0.2 mm internal ATP, genistein (50 microm) increased channel open probability from 0.053 +/- 0.016 to 0.183 +/- 0.039, but isoflurane failed to further increase open probability (0.162 +/- 0.051) of genistein-activated channels. However, applied in the presence of genistein and protein tyrosine phosphatase 1B (1 microg/ml), isoflurane significantly increased open probability to 0.473 +/- 0.114. These results suggest that the PTK-protein tyrosine phosphatase signaling pathway may be one of the regulators of cardiac sarcolemmal K(ATP) channel and may play a role in modulating its responsiveness to isoflurane. Relative importance of this modulation for cardioprotection by volatile anesthetics remains to be established.

Isoflurane-induced facilitation of the cardiac sarcolemmal K(ATP) channel.

Volatile anesthetics have cardioprotective effects that mimic ischemic preconditioning, including the involvement of adenosine triphosphate-sensitive potassium (K(ATP)) channels. However, evidence for a direct effect of volatile anesthetic on the K(ATP) channel is limited. In this study, the effects of isoflurane on the cardiac sarcolemmal K(ATP) channel were investigated. Single ventricular myocytes were enzymatically isolated from guinea pig hearts. Whole cell and single-channel configurations, specifically the cell-attached and inside-out patch mode, of the patch clamp technique were used to monitor sarcolemmal K(ATP) channel current. In the cell-attached patch configuration, 2,4-dinitrophenol (150 microm) opened the sarcolemmal K(ATP) channel. Isoflurane (0.5 mm) further increased channel open probability and the number of active channels in the patch. In contrast, in the inside-out patch experiments, isoflurane had no significant effect on the K(ATP) channel activated by low ATP (0.2-0.5 mm). In addition, isoflurane had no effect on the K(ATP) channel when activated by adenosine diphosphate, adenosine + guanosine triphosphate, bimakalim, and 2,4-dinitrophenol under inside-out patch configurations. When K(ATP) current was monitored in the whole cell mode, isoflurane alone was unable to elicit channel opening. However, during sustained protein kinase C activation by 12,13-dibutyrate, isoflurane activated the K(ATP) current that was sensitive to glibenclamide. In contrast, isoflurane had no effect on the K(ATP) channel activated by 12,13-dibutyrate in a cell-free environment. Isoflurane facilitated the opening of the sarcolemmal K(ATP) channel in the intact cell, but not in an excised, inside-out patch. The isoflurane effect was not due to a direct interaction with the K(ATP) channel protein, but required an intracellular component, likely including the translocation of specific protein kinase C isoforms. This suggests that the sarcolemmal K(ATP) channel may have a significant role in anesthetic-induced preconditioning.

Trek-like potassium channels in rat cardiac ventricular myocytes are activated by intracellular ATP.

Large (111 +/- 3.0 pS) K+ channels were recorded in membrane patches from adult rat ventricular myocytes using patch-clamp techniques. The channels were not blocked by 4-AP (5 mM), intracellular TEA (5 mM) or glybenclamide (100 mM). Applying stretch to the membrane (as pipette suction) increased channel open probability (Po) in both cell-attached and isolated patches (typically, Po approximately equals 0.005 with no pressure; approximately equals 0.328 with 90 cm H2O: Vm = 40 mV, pHi = 7.2). The channels were activated by a decrease in intracellular pH; decreasing pHi to 5.5 from 7.2 increased Po to 0.16 from approx. 0.005 (no suction, Vm held at 40 mV). These properties are consistent with those demonstrated for TREK-1, a member of the recently cloned tandem pore family. We confirmed, using RT-PCR, that TREK-1 is expressed in rat ventricle, suggesting that the channel being recorded is indeed TREK-1. However, we show also that the channels are activated by millimolar concentrations of intracellular ATP. At a pH of 6 with no ATP at the intracellular membrane face, Po was 0.048 +/-0.023, whereas Po increased to 0.22 +/- 0.1 with 1 mM ATP, and to 0.348 +/- 0.13 with 3 mM (n = 5; no membrane stretch applied). The rapid time course of the response and the fact that we see the effect in isolated patches appear to preclude phosphorylation. We conclude that intracellular ATP directly activates TREK-like channels, a property not previously described.

(-)-Isoproterenol modulation of maxi-K+ channel in nonpigmented ciliary epithelial cells through a G-protein gated pathway

Purpose. Adrenergic agents decrease intraocular pressure by reducing aqueous humor secretion from ciliary epithelial cells. Since the ionic concentration of aqueous humor contributes to intraocular pressure, we have investigated the effect of (-)-isoproterenol, a ß-adrenergic agonist on the maxi-K + channel in rabbit nonpigmented ciliary epithelial (NPE) cells. Methods. Single-channel currents were recorded from the basolateral surface of acutely isolated NPE cells using patch clamp techniques. Results. A calcium dependent maxi-K + channel was identified in 31% of cell-attached patches. In the excised condition the channel was activated in presence of calcium. In symmetrical K + solution a linear current-voltage relationship and unitary conductance of 158 ± 15pS was observed. Replacing K + with Na + the current-voltage curve shifted to the right and approached a reversal potential for K + (~–80 mV). Barium (2 mM) from the intracellular side or iberiotoxin (50 nM) from the extracellular side blocked the channel activity. In cell-attached patches, the ß-receptor agonist (-)-isoproterenol (2.5 µM) increased channel open probability (P o) only when applied directly through the patch pipette. ß 2 -adrenoceptorantagonists (ICI-118, 551, l-timolol) blocked the channel activity more efficiently than the ß 1 -adrenoceptor antagonist betaxolol. In excised patches, (-)-isoproterenol increased baseline P o 5-fold (0.5±0.13) when GTP (100 µM) and GTP?S (100 µM) were present at the cytosolic surface of the pipette (control; P o, 0.12±0.006). GTP augmented baseline channel activity (0.1 ± 0.004) 7-fold (0.7 ± 0.03) when (-)-isoproterenol was included in patch pipette. Conclusions. Rabbit NPE cells expressed maxi-K + channels on their basolateral surface. The adrenergic agonist (-)-isoproterenol activated these channels via a ß 2 -adrenoceptor that was modulated by a direct G-protein gated pathway.

TREK-1 regulation by nitric oxide and cGMP-dependent protein kinase. An essential role in smooth muscle inhibitory neurotransmission.

Potassium channels activated by membrane stretch may contribute to maintenance of relaxation of smooth muscle cells in visceral hollow organs. Previous work has identified K(+) channels in murine colon that are activated by stretch and further regulated by NO-dependent mechanisms. We have screened murine gastrointestinal, vascular, bladder, and uterine smooth muscles for the expression of TREK and TRAAK mRNA. Although TREK-1 was expressed in many of these smooth muscles, TREK-2 was expressed only in murine antrum and pulmonary artery. TRAAK was not expressed in any smooth muscle cells tested. Whole cell currents from TREK-1 expressed in mammalian COS cells were activated by stretch, and single channel recordings showed that the stretch-dependent conductance was due to 90 pS channels. Sodium nitroprusside (10(-6) or 10(-5) m) and 8-Br-cGMP (10(-4) or 10(-3) m) increased TREK-1 currents in perforated whole cell and single channel recordings. Mutation of the PKG consensus sequence at serine 351 blocked the stimulatory effects of sodium nitroprusside and 8-Br-cGMP on open probability without affecting the inhibitory effects of 8-Br-cAMP. TREK-1 encodes a component of the stretch-activated K(+) conductance in smooth muscles and may contribute to nitrergic inhibition of gastrointestinal muscles.

O(2) modulates large-conductance Ca(2+)-dependent K(+) channels of rat chemoreceptor cells by a membrane-restricted and CO-sensitive mechanism.

Hypoxic inhibition of large-conductance Ca(2+)-dependent K(+) channels (maxiK) of rat carotid body type I cells is a well-established fact. However, the molecular mechanisms of such inhibition and the role of these channels in the process of hypoxic transduction remain unclear. We have examined the mechanisms of interaction of O(2) with maxiK channels exploring the effect of hypoxia on maxiK currents recorded with the whole-cell and the inside-out configuration of the patch-clamp technique. Hypoxia inhibits channel activity both in whole-cell and in excised membrane patches. This effect is strongly voltage- and Ca(2+)-dependent, being maximal at low [Ca(2+)] and low membrane potential. The analysis of single-channel kinetics reveals a gating scheme comprising three open and five closed states. Hypoxia inhibits channel activity increasing the time the channel spends in the longest closed states, an effect that could be explained by a decrease in the Ca(2+) sensitivity of those closed states. Reducing maxiK channels with dithiothreitol (DTT) increases channel open probability, whereas oxidizing the channels with 2,2'-dithiopyridine (DTDP) has the opposite effect. These results suggest that hypoxic inhibition is not related with a reduction of channel thiol groups. However, CO, a competitive inhibitor of O(2) binding to hemoproteins, fully reverts hypoxic inhibition, both at the whole-cell and the single-channel level. We conclude that O(2) interaction with maxiK channels does not require cytoplasmic mediators. Such interaction could be mediated by a membrane hemoprotein that, as an O(2) sensor, would modulate channel activity.

Cell surface expression and turnover of the alpha-subunit of the epithelial sodium channel.

The renal epithelial cell line A6, derived from Xenopus laevis, expresses epithelial Na(+) channels (ENaCs) and serves as a model system to study hormonal regulation and turnover of ENaCs. Our previous studies suggest that the alpha-subunit of Xenopus ENaC (alpha-xENaC) is detectable as 150- and 180-kDa polypeptides, putative immature and mature alpha-subunit heterodimers. The 150- and 180-kDa alpha-xENaC were present in distinct fractions after sedimentation of A6 cell lysate through a sucrose density gradient. Two anti-alpha-xENaC antibodies directed against distinct domains demonstrated that only 180-kDa alpha-xENaC was expressed at the apical cell surface. The half-life of cell surface-expressed alpha-xENaC was 24-30 h, suggesting that once ENaC matures and is expressed at the plasma membrane, its turnover is similar to that reported for mature cystic fibrosis transmembrane conductance regulator. No significant changes in apical surface expression of alpha-xENaC were observed after treatment of A6 cells with aldosterone for 24 h, despite a 5.3-fold increase in short-circuit current. This lack of change in surface expression is consistent with previous observations in A6 cells and suggests that aldosterone regulates ENaC gating and increases channel open probability.

The effects of pH on the interaction between capsaicin and the vanilloid receptor in rat dorsal root ganglia neurons.

1. The vanilloid receptor of sensory neurons is a polymodal nociceptor sensitive to capsaicin, protons, heat and anandamide. Although it is known that interaction occurs between these different mediators the mechanism by which this occurs is poorly understood. In this study capsaicin elicited currents were recorded from vanilloid receptors found in adult rat isolated dorsal root ganglia (DRG) neurons under conditions of varying pH and the mechanism whereby protons can modulate this capsaicin response investigated. 2. Under whole-cell voltage clamp, modulating extracellular pH shifted the position of the capsaicin log(concentration)-response curve. Acidification from pH 9.0 to pH 5.5 lowered the EC50 values from 1150+/-250 nM to 5+/-2 nM with coincident change in the mean apparent slope factor from 2.3+/-0.3 to 0.9+/-0.2 and no change in maximal response. 3. The magnitude of the potentiation seen on reducing extracellular pH was not significantly affected by changes in extracellular calcium and magnesium concentration. 4. The response to capsaicin was not potentiated by a reduction in intracellular pH suggesting a site of action more accessible from the extracellular than the intracellular side of the membrane. 5. Potentiation by low pH was voltage independent indicating a site of action outside the membrane electric field. 6. At the single channel level, reducing extracellular pH increased channel open probability but had no significant effect on single channel conductance or open time. 7. These results are consistent with a model in which, on reducing extracellular pH, the vanilloid receptor in rat DRG neurons, changes from a state with low affinity for capsaicin to one with high affinity, coincident with a loss of cooperativity. This effect, presumed to be proton mediated, appears to involve one or more sites with pK(a) value 7.4-7.9, outside the membrane electrical field on an extracellularly exposed region of the receptor protein.

Epithelial Sodium Channel Pore Region: STRUCTURE AND ROLE IN GATING

Epithelial sodium channels (ENaC) have a crucial role in the regulation of extracellular fluid volume and blood pressure. To study the structure of the pore region of ENaC, the susceptibility of introduced cysteine residues to sulfhydryl-reactive methanethiosulfonate derivatives ((2-aminoethyl)methanethiosulfonate hydrobromide (MTSEA) and [(2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET)) and to Cd(2+) was determined. Selected mutants within the amino-terminal portion (alphaVal(569)-alphaTrp(582)) of the pore region responded to MTSEA, MTSET, or Cd(2+) with stimulation or inhibition of whole cell Na(+) current. The reactive residues were not contiguous but were separated by 2-3 residues where substituted cysteine residues did not respond to the reagents and line one face of an alpha-helix. The activation of alphaS580Cbetagamma mENaC by MTSET was associated with a large increase in channel open probability. Within the carboxyl-terminal portion (alphaSer(583)-alphaSer(592)) of the pore region, only one mutation (alphaS583C) conferred a rapid, nearly complete block by MTSEA, MTSET, and Cd(2+), whereas several other mutant channels were partially blocked by MTSEA or Cd(2+) but not by MTSET. Our data suggest that the outer pore of ENaC is formed by an alpha-helix, followed by an extended region that forms a selectivity filter. Furthermore, our data suggest that the pore region participates in ENaC gating.

Tetraethylammonium potentiates the activity of muscarinic potassium channels in guinea-pig atrial myocytes.

The modulation of native muscarinic potassium channels (KACh) by tetraethylammonium (TEA) was studied at 35 degrees C in cell-free patches from acutely dissociated guinea-pig atrial myocytes. The channels were identified unambiguously by their conductance, inward rectification, rapid gating kinetics and pharmacological responses to muscarinic agonists and GTPgammaS. Addition of 5 mM TEA to the cytoplasmic side of the patches almost doubled the open probability of KACh channels that had been activated maximally by GTPgammaS. In contrast even 30 mM TEA did not significantly potentiate the response to carbachol in whole-cell recordings. Unlike GTPgammaS, TEA alone did not activate KACh channels de novo, but in patches that showed spontaneous KACh activity, 5 mM TEA increased channel open probability fourfold in the absence of added sodium, ATP or guanine nucleotides. Furthermore, the effect of TEA was not blocked by 10 uM atropine or by 1 mM GDPbetaS, and subsequent addition of 0.1 mM GTPgammaS did not stimulate channel activity further in the presence of TEA. Phosphatidylinositol 4,5-bisphosphate (PIP2) also stimulates KACh channels under these conditions, but the kinetics of gating differ from channels stimulated by either TEA or GTP, which are very similar to one another. The effects of TEA were not mimicked by tetramethyl- or tetrapentylammonium or by sodium or spermine, and TEA did not potentiate the activity of other inwardly rectifying potassium (KATP) channels in patches from cardiac myocytes. We consider the possibility that TEA is mimicking the effect of an unidentified cellular factor, not sodium or PIP2, which normally occupies the TEA site on KACh channel proteins but which diffuses away when the patch is excised.

NMDA Channel Gating Is Influenced by a Tryptophan Residue in the M2 Domain but Calcium Permeation Is Not Altered

N-Methyl- d-aspartate (NMDA) receptors are susceptible to open-channel block by dizolcipine (MK-801), ketamine and Mg 2+ and are permeable to Ca 2+. It is thought that a tryptophan residue in the second membrane-associated domain (M2) may form part of the binding site for open-channel blockers and contribute to Ca 2+ permeability. We tested this hypothesis using recombinant wild-type and mutant NMDA receptors expressed in HEK-293 cells. The tryptophan was mutated to a leucine (W−5L) in both the NMDAR1 and NMDAR2A subunits. MK-801 and ketamine progressively inhibited currents evoked by glutamate, and the rate of inhibition was increased by the W−5L mutation. An increase in open channel probability accounted for the acceleration. Fluctuation analysis of the glutamate-evoked current revealed that the NMDAR1 W−5L mutation increased channel mean open time, providing further evidence for an alteration in gating. However, the equilibrium affinities of Mg 2+ and ketamine were largely unaffected by the W−5L mutation, and Ca 2+ permeability was not decreased. Therefore, the M2 tryptophan residue of the NMDA channel is not involved in Ca 2+ permeation or the binding of open-channel blockers, but plays an important role in channel gating.

Characterization and cADP-Ribose Activation of Reconstituted Ryanodine Receptors from Coronary Arterial Smooth Muscle

P144 Recent studies indicate that cyclic ADP-ribose (cADPR) serves as a second messenger to mobilize Ca 2+ from sarcoplasmic reticulum (SR) independent of IP 3 . However, the mechanism by which cADPR activates Ca 2+ mobilization is poorly understood. In the present study, ryanodine receptors from SR of coronary arterial smooth muscle cells (CASMCs) were reconstituted into planar lipid bilayer. A unitary Ca 2+ current through this receptor/channel complex was detected at membrane potentials from -40 to +40 mV. Mean slope conductance of this current was 245 ± 4.5 pS in a symmetric cesium methanesulfonate solution. Ryanodine at 0.1-10 μM produced a typical subconductance with increased channel open probability (NP O ), and 50 μM ryanodine, however, decreased the NP O of the channels. These ryanodine effects represent a unique feature of this SR Ca 2+ release channel confirmed in other tissues. Addition of Ca 2+ into bath solution (0.01-10 μM) significantly increased the NP O of these SR Ca 2+ channels. cADPR significantly increased the channel NP 0 in a concentration-dependent manner. A 9.7-fold increase in the NP O was observed at a concentration of cADPR of 1 μM. This cADPR-induced activation of the SR Ca 2+ channels was completely abolished by 50 μM ryanodine. IP 3 had no effect on the activity of these reconstituted SR Ca 2+ channels even at the highest concentration studied (10 μM). It is concluded that ryanodine receptor/Ca 2+ channels are present in CASMCs and can be reconstituted into planar lipid bilayer. cADPR stimulates Ca 2+ release through the activation of ryanodine receptors on the SR of CASMCs.

Characterization and cADP-Ribose Activation of Reconstituted Ryanodine Receptors from Coronary Arterial Smooth Muscle

P144 Recent studies indicate that cyclic ADP-ribose (cADPR) serves as a second messenger to mobilize Ca 2+ from sarcoplasmic reticulum (SR) independent of IP 3 . However, the mechanism by which cADPR activates Ca 2+ mobilization is poorly understood. In the present study, ryanodine receptors from SR of coronary arterial smooth muscle cells (CASMCs) were reconstituted into planar lipid bilayer. A unitary Ca 2+ current through this receptor/channel complex was detected at membrane potentials from -40 to +40 mV. Mean slope conductance of this current was 245 ± 4.5 pS in a symmetric cesium methanesulfonate solution. Ryanodine at 0.1-10 μM produced a typical subconductance with increased channel open probability (NP O ), and 50 μM ryanodine, however, decreased the NP O of the channels. These ryanodine effects represent a unique feature of this SR Ca 2+ release channel confirmed in other tissues. Addition of Ca 2+ into bath solution (0.01-10 μM) significantly increased the NP O of these SR Ca 2+ channels. cADPR significantly increased the channel NP 0 in a concentration-dependent manner. A 9.7-fold increase in the NP O was observed at a concentration of cADPR of 1 μM. This cADPR-induced activation of the SR Ca 2+ channels was completely abolished by 50 μM ryanodine. IP 3 had no effect on the activity of these reconstituted SR Ca 2+ channels even at the highest concentration studied (10 μM). It is concluded that ryanodine receptor/Ca 2+ channels are present in CASMCs and can be reconstituted into planar lipid bilayer. cADPR stimulates Ca 2+ release through the activation of ryanodine receptors on the SR of CASMCs.

Methylation Increases the Open Probability of the Epithelial Sodium Channel in A6 Epithelia

We used single channel methods on A6 renal cells to study the regulation by methylation reactions of epithelial sodium channels. 3-Deazaadenosine (3-DZA), a methyltransferase blocker, produced a 5-fold decrease in sodium transport and a 6-fold decrease in apical sodium channel activity by decreasing channel open probability (P(o)). 3-Deazaadenosine also blocked the increase in channel open probability associated with addition of aldosterone. Sodium channel activity in excised "inside-out" patches usually decreased within 1-2 min; in the presence of S-adenosyl-l-methionine (AdoMet), activity persisted for 5-8 min. Sodium channel mean time open (t(open)) before and after patch excision was higher in the presence of AdoMet than in untreated excised patches but less than t(open) in cell-attached patches. Sodium channel activity in excised patches exposed to both AdoMet and GTP usually remained stable for more than 10 min, and P(o) and the number of active channels per patch were close to values in cell-attached patches from untreated cells. These findings suggest that a methylation reaction contributes to the activity of epithelial sodium channels in A6 cells and is directed to some regulatory element closely connected with the channel, whose activity also depends on the presence of intracellular GTP.

Trophic regulation of synaptic plasticity

Trophins are now known to acutely regulate synaptic plasticity, in addition to exerting long-recognized actions on neuronal survival and development. To begin elucidating underlying mechanisms, cultured dissociated hippocampal neurons were examined by whole-cell patch-clamp recording. BDNF (brain derived neurotrophic factor) specifically and selectively increased the integrated synaptic current twofold within five minutes. The effect was mediated postsynaptically by the trkB receptor and intracellular phosphorylation-dependent mechanisms. Iontophoresis combined with patch-clamp recording indicated that BDNF increased activity of NMDA (N-methyl-D-aspartate) receptors. Single channel-channel recording indicated that BDNF increased channel open probability by increasing opening frequency. In parallel, the trophin specifically increased phosphorylation of NR1 and NR2B subunits of NMDA receptors in the postsynaptic density, providing potential mechanisms eliciting receptor activation. A model is proposed in which activity-driven experience activates specific BDNF gene promoters, leading to enhanced transcription, elevated trophin levels, postsynaptic NMDA receptor activation and increased synaptic transmission. These sequential mechanisms may contribute to enhanced long-term potentiation, a correlate of learning and memory. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 108–118, 1999