Effects of Chlorotrifluoroethylene Oligomer Fatty Acids on Recombinant GABA Receptors Expressed in Xenopus Oocytes

GABA-activated Cl⁻ current was expressed in Xenopus oocytes after injecting cRNA that had been transcribed in vitro from complementary DNA (cDNA) coding for a single GABA ρ_i-subunit cloned from human retina. The expressed current was insensitive to 100 μm bicuculline, but was activated by the GABA analogue trans-4-aminocrontonic acid (TACA). Anion-selective permeability of the expressed ρ₁-subunit was determined by isotonically replacing the extracellular Cl⁻ with different anions. The anion permeability was very similar to the native GABA_A receptor/channel following a sequence of SCN⁻ > I⁻ > NO₃ ⁻ > Br⁻≥ Cl⁻. Halogenated fatty acids, such as chlorotrifluoroethylene (CTFE) and perfluorinated oligomer acids inhibited the GABA-induced current in oocytes expressing the human retinal GABA ρ₁-subunit or rat brain GABA_A receptor α₁,β₂,γ₂ subunits. The inhibitory effect of halogenated fatty acids demonstrated a carbon chain length-dependent manner of: C₁₀ > C₈ > C₆ > C₄. Perfluorinated C₈-oligomer acid (PFOA) was less effective at blocking this channel than the C₈-CTFE oligomer acid. Radiolabeled GABA binding assay indicated that CTFE oligomer acids do not interfere at the GABA binding site of the receptor. Furthermore, the C₈-CTFE oligomer fatty acid did not compete with picrotoxin for binding sites within the pore of the channel. These studies demonstrated that the heterologous expression system is useful for studying the molecular interaction between potential neurotoxic agents and neuroreceptors. Our results provide detailed information that should contribute to our understanding of the structure and function of retinal GABA receptors. Received: 12 June 1995/Revised: 21 September 1995     

Separate Domains for Desensitization of GABA ρ1 and β2 Subunits Expressed in Xenopus Oocytes

Desensitization of ligand-gated receptor channels is an intrinsic feedback mechanism and prevents the receptor/channels from becoming overly activated thereby maintaining biological function of the nervous system. Desensitization also plays an important role in neuronal plasticity. By taking advantage of biophysical and pharmacological diversities of GABA β₂ subunits from the brain and ρ₁ subunits from the retina, structural determinants that confer agonist-induced desensitization were identified. A synthetic chimeric receptor/channel was created from the β₂ and ρ₁ subunits for this investigation. The chimera was constructed from the extracellular N-domain of the β₂ subunit, extending from the amino terminus to the beginning region of the M1 transmembrane segment, and from the C-domain of the ρ₁ subunit extending from the M1 transmembrane segment to the carboxyl terminus. The C-domain region included the M1 to M4 transmembrane regions and the large intracellular loop between the M3 and M4 transmembrane segments. Homo-oligomeric GABA β₂, ρ₁, and β₂/ρ₁ chimeric receptor/channels were individually expressed in Xenopus oocytes, and the desensitization characteristics attributable to each type of subunit were compared. Results from the present study reveal that motifs in the amino-terminal and carboxyl-terminal domains of the β₂ subunit conferred the agonist-induced desensitization; chloroform modulation was linked to specific phases of the GABA-activated current decay. Received: 2 April 1997/Revised: 27 March 1998     

Identification of Major Phylogenetic Branches of Inhibitory Ligand-Gated Channel Receptors

The gene superfamily of ligand-gated ion channel (LGIC) receptors is composed of members of excitatory LGIC receptors (ELGIC) and inhibitory LGIC receptors (ILGIC), all using amino acids as ligands. The ILGICs, including GABA_A, Gly, and GluCl receptors, conduct Cl⁻ when the ligand is bound. To evaluate the phylogenetic relationships among ILGIC members, 90 protein sequences were analyzed by both maximum-parsimony and distance matrix-based methods. The strength of the resulting phylogenetic trees was evaluated by means of bootstrap. Four major phylogenetic branches are recognized. Branch I, called BZ, for the majority of the members are known to be related to benzodiazepine binding, is subdivided into IA, composed of all GABA_A receptor α subunits, and IB, composed of the γ and ε subunits, which are shown to be tightly linked. Branch II, named NB for non–benzodiazepine binding, and consisting of GABA_A receptor β, δ, π, and ρ subunits, is further subdivided into IIA, containing β subunits; IIB, containing δ, and π subunits; and IIC, containing ρ subunits. Branch IIIA, composed of vertebrate Gly receptors, is loosely clustered with Branch IIIB, composed of invertebrate GluCl receptors, to form Branch III, which is designated NA for being non–GABA responsive. Branch IV is called UD for being undefined in specificity. The existence of primitive forms of GABA_A receptor non-β subunits in invertebrates is first suggested by the present analysis, and the identities of sequences p25123 from Drosophila melanogaster, s34469 from Lymnaea stagnalis, and u14635 and p41849 from C. aenorhabditis elegans are determined to be different from their previously given annotations. The proposed branching classification of ILGICs provides a phylogenetic map, based on protein sequences, for tracing the evolutionary pathways of ILGIC receptor subunits and determining the identities of newly discovered subunits on the basis of their protein sequences. Received: 15 April 1997 / Accepted: 11 March 1998     

Subunit Regulation of the Human Brain α1E Calcium Channel

The α₁ subunit coding for the human brain type E calcium channel (Schneider et al., 1994) was expressed in Xenopus oocytes in the absence, and in combination with auxiliary α₂δ and β subunits. α_1E channels directed with the expression of Ba²⁺ whole-cell currents that completely inactivated after a 2-sec membrane pulse. Coexpression of α_1E with α_2bδ shifted the peak current by +10 mV but had no significant effect on whole-cell current inactivation. Coexpression of α_1E with β_2a shifted the peak current relationship by −10 mV, and strongly reduced Ba²⁺ current inactivation. This slower rate of inactivation explains that a sizable fraction (40 ± 10%, n= 8) of the Ba²⁺ current failed to inactivate completely after a 5-sec prepulse. Coinjection with both the cardiac/brain β_2a and the neuronal α_2bδ subunits increased by ≈10-fold whole-cell Ba²⁺ currents although coinjection with either β_2a or α_2bδ alone failed to significantly increase α_1E peak currents. Coexpression with β_2a and α_2bδ yielded Ba²⁺ currents with inactivation kinetics similar to the β_2a induced currents, indicating that the neuronal α_2bδ subunit has little effect on α_1E inactivation kinetics. The subunit specificity of the changes in current properties were analyzed for all four β subunit genes. The slower inactivation was unique to α_1E/β_2a currents. Coexpression with β_1a, β_1b, β₃, and β₄, yielded faster-inactivating Ba²⁺ currents than currents recorded from the α_1E subunit alone. Furthermore, α_1E/α_2bδ/β_1a; α_1E/α_2bδ/β_1b; α_1E/α_2bδ/β₃; α_1E/α_2bδ/β₄ channels elicited whole-cell currents with steady-state inactivation curves shifted in the hyperpolarized direction. The β subunit-induced changes in the properties of α_1E channel were comparable to modulation effects reported for α_1C and α_1A channels with β₃≈β_1b > β_1a≈β₄≫β_2a inducing fastest to slowest rate of whole-cell inactivation. Received: 27 March 1997/Revised: 10 July 1997     

Expression of Cyclic Nucleotide-Gated Cation Channels in Airway Epithelial Cells

Using the whole-cell patch-clamp technique, the selectivity and pharmacology of 8-Br-cGMP-stimulated currents in the human alveolar cell line A549 was compared to 8-Br-cGMP-stimulated currents in HK293 cells transfected with hαCNC1. Whole cell currents stimulated by 8-Br-cGMP in HK293 cells transfected with hαCNC1 or A549 cells are carried by inward sodium and outward potassium with nearly the same selectivity. The whole-cell inward currents that are stimulated by 8-Br-cGMP in HK293 cells transfected with hαCNC1 are inhibited by l-cis-diltiazem with an IC₅₀ of 154 μm, by 2′,4′-dichlorobenzamil with an IC₅₀ of 50 μm and by amiloride with an IC₅₀ of 133 μm. The whole-cell inward currents in A549 cells that are stimulated by 8-Br-cGMP, are inhibited by l-cis-diltiazem with an IC₅₀ of 87 μm, by 2′4′-dichlorobenzamil with an IC₅₀ of 38 μm and by amiloride with an IC₅₀ of 32 μm suggesting that these airway cells contain cyclic nucleotide-gated cation channels. RT-PCR data suggest that mRNA of both αCNC1 and βCNC subunits are present in A549 cells and the presence of the βCNC subunit, may as previously reported, increase the affinity of these channel blockers compared to the hαCNC1 subunit alone. The mRNA of two other isoforms of this channel, CNC2 and CNC3, are also expressed in the A549 cell line. This study documents the IC₅₀ of externally applied channel blockers that can be used for in vitro or in vivo experiments to document sodium absorption via cyclic nucleotide-gated cation channels in airway cells. Received: 24 February/Revised: 28 May 1999     

Effects of Mutating Leucine to Threonine in the M2 Segment of α1 and β1 Subunits of GABAAα1β1 Receptors

The conserved leucine residues at the 9′ positions in the M2 segments of α₁ (L264) and β₁ (L259) subunits of the human GABA_A receptor were replaced with threonine. Normal or mutant α₁ subunits were co-expressed with normal or mutant β₁ subunits in Sf9 cells using the baculovirus/Sf9 expression system. Cells in which one or both subunits were mutated had a higher ``resting' chloride conductance than cells expressing wild-type α₁β₁ receptors. This chloride conductance was blocked by 10 mm penicillin, a recognized blocker of GABA_A channels, but not by bicuculline (100 μm) or picrotoxin (100 μm) which normally inhibit the chloride current activated by GABA: nor was it potentiated by pentobarbitone (100 μm). In cells expressing wild-type β₁ with mutated α₁ subunits, an additional chloride current could be elicited by GABA but the rise time and decay were slower than for wild-type α₁β₁ receptors. In cells expressing mutated β₁ subunits with wild-type or mutated α₁ subunits (αβ(L9′T) and α(L9′T)β(L9′T)), no response to GABA could be elicited: this was not due to an absence of GABA_A receptors in the plasmalemma because the cells bound [³H]-muscimol. It was concluded that in GABA_A channels containing the L9′T mutation in the β₁ subunit, GABA-binding does not cause opening of channels, and that the L9′T mutation in either or both subunits gives an open-channel state of the GABA_A receptor in the absence of ligand. Received: 17 April 1996/Revised: 5 July 1996     

Evolutionary Relationship of the Ligand-Gated Ion Channels and the Avermectin-Sensitive,Glutamate-Gated Chloride Channels

Two cDNAs, GluClα and GluClβ, encoding glutamate-gated chloride channel subunits that represent targets of the avermectin class of antiparasitic compounds, have recently been cloned from Caenorhabditis elegans (Cully et al., Nature, 371, 707–711, 1994). Expression studies in Xenopus oocytes showed that GluClα and GluClβ have pharmacological profiles distinct from the glutamate-gated cation channels as well as the γ-aminobutyric acid (GABA)- and glycine-gated chloride channels. Establishing the evolutionary relationship of related proteins can clarify properties and lead to predictions about their structure and function. We have cloned and determined the nucleotide sequence of the GluClα and GluClβ genes. In an attempt to understand the evolutionary relationship of these channels with the members of the ligand-gated ion channel superfamily, we have performed gene structure comparisons and phylogenetic analyses of their nucleotide and predicted amino acid sequences. Gene structure comparisons reveal the presence of several intron positions that are not found in the ligand-gated ion channel superfamily, outlining their distinct evolutionary position. Phylogenetic analyses indicate that GluClα and GluClβ form a monophyletic subbranch in the ligand-gated ion channel superfamily and are related to vertebrate glycine channels/receptors. Glutamate-gated chloride channels, with electrophysiological properties similar to GluClα and GluClβ, have been described in insects and crustaceans, suggesting that the glutamate-gated chloride channel family may be conserved in other invertebrate species. The gene structure and phylogenetic analyses in combination with the distinct pharmacological properties demonstrate that GluClα and GluClβ belong to a discrete ligand-gated ion channel family that may represent genes orthologous to the vertebrate glycine channels. Received: 30 September 1996 / Accepted: 15 November 1996     

Functional Expression and Characterization of G-protein-gated Inwardly Rectifying K+ Channels Containing GIRK3

The G-protein-gated inwardly rectifying K ⁺(GIRK) family of ion channels form functional Gβγ-sensitive channels as heteromultimers of GIRK1 and either the GIRK2 or GIRK4 subunits. However, the homologous mouse brain GIRK3 clone failed to express in the earliest reported functional experiments in Xenopus oocytes. We recloned the GIRK3 subunit from mouse brain and found that the new clone differed significantly from that originally reported. The functional aspects of GIRK3 were reinvestigated by expression in CHO cells. The single channel properties of GIRK1/GIRK3 were characterized and compared to those of the GIRK1/GIRK2 and GIRK1/GIRK4 channels. All three GIRK1/GIRKx combinations produced channels with nearly indistinguishable conductances and kinetics. The response of GIRK1/GIRK3 to Gβγ in the 1–47 nm range was examined and found to be indistinguishable from that of GIRK1/GIRK4 channels. We conclude that GIRK1, with either GIRK2, 3, or 4, gives rise to heteromultimeric channels with virtually identical conductances, kinetics, and Gβγ sensitivities. Received: 13 January 1999/Revised: 2 March 1999     

Nickel Block of a Family of Neuronal Calcium Channels: Subtype- and Subunit-Dependent Action at Multiple Sites

Nickel ions have been reported to exhibit differential effects on distinct subtypes of voltage-activated calcium channels. To more precisely determine the effects of nickel, we have investigated the action of nickel on four classes of cloned neuronal calcium channels (α_1A, α_1B, α_1C, and α_1E) transiently expressed in Xenopus oocytes. Nickel caused two major effects: (i) block detected as a reduction of the maximum slope conductance and (ii) a shift in the current-voltage relation towards more depolarized potentials which was paralleled by a decrease in the slope of the activation-curve. Block followed 1:1 kinetics and was most pronounced for α_1C, followed by α_1E > α_1A > α_1B channels. In contrast, the change in activation-gating was most dramatic with α_1E, with the remaining channel subtypes significantly less affected. The current-voltage shift was well described by a simple model in which nickel binding to a saturable site resulted in altered gating behavior. The affinity for both the blocking site and the putative gating site were reduced with increasing concentration of external permeant ion. Replacement of barium with calcium reduced both the degree of nickel block and the maximal effect on gating for α_1A channels, but increased the nickel blocking affinity for α_1E channels. The coexpression of Ca channel β subunits was found to differentially influence nickel effects on α_1A, as coexpression with β_2a or with β₄ resulted in larger current-voltage shifts than those observed in the presence of β_1b, while elimination of the β subunit almost completely abolished the gating shifts. In contrast, block was similar for the three β subunits tested, while complete removal of the β subunit resulted in an increase in blocking affinity. Our data suggest that the effect of nickel on calcium channels is complex, cannot be described by a single site of action, and differs qualitatively and quantitatively among individual subtypes and subunit combinations. Received: 12 October 1995/Revised: 17 January 1996     

Cytoplasmic Determinants of Piperidine Blocking Affinity for N-type Calcium Channels

Piperidines are a relatively novel class of calcium channel blockers which act at a unique receptor site associated with the calcium channel α₁ subunit. Calcium channel blocking affinities ranging from subnanomolar to several hundred micromolar have been reported in the literature, suggesting that piperidine block is highly sensitive to the cellular environment experienced by the channel. Here, I have investigated some of the cytoplasmic determinants of haloperidol block of N-type calcium channels expressed in human embryonic kidney cells. In perforated patch clamp recordings, haloperidol blocks N-type calcium channels with an inhibition constant of 120 μM. Upon internal dialysis with chloride containing pipette solution, the blocking affinity increases by 40-fold. This effect could be attributed in part to the presence of internal chloride ions, as replacement of intracellular chloride with methanesulfonate reduced haloperidol blocking affinity by almost one order of magnitude. Tonic inhibition of N-type channels by G_βγ subunits further enhanced the blocking effects of haloperidol, suggesting the possibility of direct effects of G_βγ binding on the local environment of the piperidine receptor site. Overall, depending on the cytoplasmic environment experienced by the channel, the blocking affinity of N-type calcium channels for haloperidol may vary by more than two orders of magnitude. Thus, absolute blocking affinities at the piperidine receptor site must be interpreted cautiously and in the context of the particular experimental setting. Received: 23 July 1998/Revised: 19 October 1998     

Extracellular ATP activates a P2 receptor in necturus erythrocytes during hypotonic swelling

We recently reported that ATP is released from Necturus erythrocytes via a conductive pathway during hypotonic swelling and that extracellular ATP potentiates regulatory volume decrease (RVD). This study was designed to determine whether extracellular ATP exerts its effect via a purinoceptor. This was accomplished using three different experimental approaches: 1) hemolysis studies to examine osmotic fragility, 2) a Coulter counter to assess RVD, and 3) the whole-cell patch-clamp technique to measure membrane currents. We found extracellular ATP and ATPγS, two P2 agonists, decreased osmotic fragility, enhanced cell volume recovery in response to hypotonic shock, and increased whole-cell currents. In addition, 2-methylthio-ATP potentiated RVD. In contrast, UTP, α,β-methylene-ATP, and 2′-& 3′-O-(4-benzoyl-benzoyl) adenosine 5′-triphosphate and the P1 agonist adenosine had no effect regardless of experimental approach. Furthermore, the P2 antagonist suramin increased osmotic fragility, inhibited RVD, and reduced whole-cell conductance in swollen cells. Consistent with a previous study that indicated cell swelling activates a K⁺ conductance, suramin had no effect in the presence of gramicidin (a cationophore used to maintain a high K⁺ permeability). We also found the P2 antagonist pyridoxal-5-phosphate-6-azophenyl-2′4-disulfonic acid (PPADS) increased osmotic fragility; however, reactive blue 2 and the P1 antagonists caffeine and theophylline had no effect. Our results show that extracellular ATP activated a P2 receptor in Necturus erythrocytes during hypotonic swelling, which in turn potentiated RVD by stimulating K⁺ efflux. Pharmacological evidence suggested the presence of a P2X receptor subtype. Received: 6 January 2001/Revised: 17 April 2001     

Chloride Conductance and P i Transport are Separate Functions Induced by the Expression of NaPi-1 in Xenopus Oocytes

Expression of the protein NaPi-1 in Xenopus oocytes has previously been shown to induce an outwardly rectifying Cl⁻ conductance (G_Cl), organic anion transport and Na⁺-dependent P _i -uptake. In the present study we investigated the relation between the NaPi-1 induced G_Cl and P _i -induced currents and transport. NaPi-1 expression induced P _i -transport, which was not different at 1–20 ng/oocyte NaPi-1 cRNA injection and was already maximal at 1–2 days after cRNA injection. In contrast, G_Cl was augmented at increased amounts of cRNA injection (1–20 ng/oocyte) and over a five day expression period. Subsequently all experiments were performed on oocytes injected with 20 ng/oocytes cRNA. P _i -induced currents (Ip) could be observed in NaPi-1 expressing oocytes at high concentrations of P _i (≥ 1 mm P _i ). The amplitudes of Ip correlated well with G_Cl. Ip was blocked by the Cl⁻ channel blocker NPPB, partially Na⁺-dependent and completely abolished in Cl⁻ free solution. In contrast, P _i -transport in NaPi-1 expressing oocytes was not NPPB sensitive, stronger depending on extracellular Na⁺ and weakly affected by Cl⁻ substitution. Endogenous P _i -uptake in water-injected oocytes amounted in all experiments to 30–50% of the Na⁺-dependent P _i -transport observed in NaPi-1 expressing oocytes. The properties of the endogenous P _i -uptake system (K _m for P _i > 1 mm; partial Na⁺- and Cl⁻-dependence; lack of NPPB block) were similar to the NaPi-1 induced P _i -uptake, but no Ip could be recorded at P _i -concentrations ≤3 mm. In summary, the present data suggest that Ip does not reflect charge transfer related to P _i -uptake, but a P _i -mediated modulation of G_Cl. Received: 22 October 1997/Revised: 24 March 1998     

Expression of the Avian Na,K-ATPase Subunits in Dictyostelium discoideum

This study explored whether Dictyostelium discoideum can be used to express the avian Na,K-ATPase, a heterodimeric membrane protein. Dictyostelium was able to express mRNAs encoding the avian Na,K-ATPase subunits. However, Dictyostelium expressed avian Na,K-ATPase protein when only when a Dictyostelium consensus ribosomal binding sequence, AAAATAAA, was inserted in front of the open reading frames of the α₁- and β₁-subunit cDNAs and the first eight codons following the start-translation codons were changed to Dictyostelium preferred codons. These modified mRNAs appeared to be much less stable than the forms that were not readily translated. Dictyostelium could express the avian β-subunit alone but only expressed the α₁-subunit when the β₁-subunit was co-expressed. Subunit assembly occurred in cells expressing both α₁- and β₁-subunits. The bulk of the exogenously expressed sodium pump subunits remained in an intracellular compartment, presumed to be the endoplasmic reticulum. Dictyostelium exported little or no Na,K-ATPase or free β-subunit to the plasma membrane. Received: 7 July 1998/Revised: 8 October 1998     

Characterization of the Stretch-activated Chloride Channel in Isolated Human Atrial Myocytes

Macroscopic and unitary currents through stretch-activated Cl⁻ channels were examined in isolated human atrial myocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K⁺ and Ca²⁺ conductances were blocked and the intracellular Ca²⁺ concentration ([Ca²⁺] _i ) was reduced, application of positive pressure via the pipette activated membrane currents under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by 60 mV per 10-fold change in the external Cl⁻ concentration, indicating that the current was Cl⁻ selective. The current was inhibited by bath application of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and 9-anthracenecarboxylic acid (9-AC). β-Adrenergic stimulation failed to activate a Cl⁻ current. In single channel recordings from outside-out patches, positive pressure in the pipette activated the unitary current with half-maximal activation of 14.7 mm Hg at +40 mV. The current-voltage relationship of single channel activity obtained in inside-out patches was linear in symmetrical Cl⁻ solution with the averaged slope conductance of 8.6 ± 0.7 pS (mean ±sd, n= 10). The reversal potential shift of the channel by changing Cl⁻ concentration was consistent with a Cl⁻ selective channel. The open time distribution was best described by a single exponential function with mean open lifetime of 80.4 ± 9.6 msec (n= 9), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 11.5 ± 2.2 msec (n= 9) and that for the slow component of 170.2 ± 21.8 msec (n= 9). Major changes in the single channel activity in response to pressure were caused by changes in the interburst interval. Single channel activity was inhibited by DIDS and 9-AC in a manner similar to whole-cell configuration. These results suggest that membrane stretch induced by applying pressure via the pipette activated a Cl⁻ current in human atrial myocytes. The current was sensitive to Cl⁻ channel blockers and exhibited membrane voltage-independent bursting opening without sensitive to β-adrenergic stimulation. Received: 21 October 1996/Revised: 17 December 1997     

Effects of 8-cpt-cAMP on the epithelial sodium channel expressed in Xenopus oocytes

Vasopressin stimulates the activity of the epithelial Na channel (ENaC) through the cAMP/PKA pathway in the cortical collecting tubule, or in similar amphibian epithelia, but the mechanism of this regulation is not yet understood. This stimulation by cAMP could not be reproduced with the rat or Xenopus ENaC expressed in Xenopus oocyte. Recently, it was shown that the α-subunit cloned from the guinea-pig colon (αgp) could confer the ability to be activated by the membrane-permeant cAMP analogue 8-chlorophenyl-thio-cAMP (cpt-cAMP) to channels produced by expression of αgp, βrat and γrat ENaC subunits. In this study we investigate the mechanism of this activation. Forskolin treatment, endogenous production of cAMP by activation of coexpressed β adrenergic receptors, or intracellular perfusion with cAMP did not increase the amiloride-sensitive Na current, even though these maneuvers stimulated CFTR (cystic fibrosis transmembrane conductance regulator)-mediated Cl currents. In contrast, extracellular 8-cpt-cAMP increased αgp, βrat and γrat ENaC activity but had no effect on CFTR. Swapping intracellular domains between the cpt-cAMP-sensitive αgp and the cpt-cAMP-resistant αrat-subunit showed that neither the N-terminal nor the C-terminal of α ENaC was responsible for the effect of cpt-cAMP. The mechanisms of activation of ENaC by cpt-cAMP and of CFTR by the cAMP/PKA pathway are clearly different. cpt-cAMP seems to increase the activity of ENaC formed by αgp and βγrat by interacting with the extracellular part of the protein. Received: 19 January 2001/Revised: 27 April 2001     

GABA Concentration Sets the Conductance of Delayed GABAA Channels in Outside-out Patches from Rat Hippocampal Neurons

GABA_A channels were activated by GABA in outside-out patches from rat cultured hippocampal neurons. They were blocked by bicuculline and potentiated by diazepam. In 109 of 190 outside-out patches, no channels were active before exposure to GABA (silent patches). The other 81 patches showed spontaneous channel activity. In patches containing spontaneous channel activity, rapid application of GABA rapidly activated channels. In 93 of the silent patches, channels could be activated by GABA but only after a delay that was sometimes as long as 10 minutes. The maximum channel conductance of the channels activated after a delay increased with GABA concentration from less than 10 pS (0.5 μm GABA) to more than 100 pS (10 mm GABA). Fitting the data with a Hill-type equation gave an EC ₅₀ value of 33 μm and a Hill coefficient of 0.6. The channels showed outward rectification and were chloride selective. In the presence of 1 μm diazepam, the GABA EC ₅₀ decreased to 0.2 μm but the maximum conductance was unchanged. Diazepam decreased the average latency for channel opening. Bicuculline, a GABA antagonist, caused a concentration-dependent decrease in channel conductance. In channels activated with 100 μm GABA the bicuculline IC ₅₀ was 19 μm. The effect of GABA on channel conductance shows that the role of the ligand in GABA_A receptor channel function is more complex than previously thought. Received: 23 October 2000/Revised: 27 February 2001     

A Structural Determinant of Desensitization and Allosteric Regulation by Pentobarbitone of the GABAA Receptor

Functional properties of the α₁β₁ GABA_A receptor changes in a subunit-specific manner when a threonine residue in the M2 region at the 12′ position was mutated to glutamine. The rate and extent of desensitization increased in all mutants but the rate of activation was faster in the β₁ mutants. A negligible plateau current and abolition of potentiation by pentobarbitone of the GABA-activated current depended on the Thr 12′ Gln mutation being present in the β₁ subunit. The Hill coefficient of the peak current response to GABA was reduced to less than one also in a β₁ subunit-specific manner. It was concluded that the β₁ subunit dominated conformational changes activated by GABA. Received: 18 July 1996/Revised: 30 September 1996     

Alpha and beta subunits of F1-ATPase are required for survival of petite mutants in Saccharomyces cerevisiae

Although Saccharomyces cerevisiae can form petite mutants with deletions in mitochondrial DNA (mtDNA) (ρ⁻) and can survive complete loss of the organellar genome (ρ^o), the genetic factor(s) that permit(s) survival of ρ⁻ and ρ^o mutants remain(s) unknown. In this report we show that a function associated with the F₁-ATPase, which is distinct from its role in energy transduction, is required for the petite-positive phenotype of S. cerevisiae. Inactivation of either the α or β subunit, but not the γ, δ, or ɛ subunit of F₁, renders cells petite-negative. The F₁ complex, or a subcomplex composed of the α and β subunits only, is essential for survival of ρ^o cells and those impaired in electron transport. The activity of F₁ that suppresses ρ^o lethality is independent of the membrane F_o complex, but is associated with an intrinsic ATPase activity. A further demonstration of the ability of F₁ subunits to suppress ρ^o lethality has been achieved by simultaneous expression of S. cerevisiae F₁α and γ subunit genes in Kluyveromyces lactis– which allows this petite-negative yeast to survive the loss of its mtDNA. Consequently, ATP1 and ATP2, in addition to the previously identified AAC2, YME1 and PEL1/PGS1 genes, are required for establishment of ρ⁻ or ρ^o mutations in S. cerevisiae. Received: 20 March 1999 / Accepted: 18 July 1999     

Tryptophan substitutions at lipid-exposed positions of the gamma M3 transmembrane domain increase the macroscopic ionic current response of the Torpedo californica nicotinic acetylcholine receptor

Our previous amino-acid substitutions at the postulated lipid-exposed transmembrane segment M4 of the Torpedo californica acetylcholine receptor (AChR) focused on the alpha subunit. In this study we have extended the mutagenesis analysis using single tryptophan replacements in seven positions (I288, M291, F292, S294, L296, M299 and N300) near the center of the third transmembrane domain of the gamma subunit (γM3). All the tryptophan substitution mutants were expressed in Xenopus laevis oocytes following mRNA injections at levels close to wild type. The functional response of these mutants was evaluated using macroscopic current analysis in voltage-clamped oocytes. For all the substitutions the concentration for half-maximal activation, EC ₅₀, is similar to wild type using acetylcholine. For F292W, L296W and M299W the normalized macroscopic responses are 2- to 3-fold higher than for wild type. Previous photolabeling studies demonstrated that these three positions were in contact with membrane lipids. Each of these M3 mutations was co-injected with the previously characterized αC418W mutant to examine possible synergistic effects of single lipid-exposed mutations on two different subunits. For the γM3/αM4 double mutants, the EC ₅₀s were similar to those measured for the αC418W mutant alone. Tryptophan substitutions at positions that presumably face the interior of the protein (S294 and M291) or neighboring helices (I288) did not cause significant inhibition of channel function or surface expression of AChRs. Received: 29 January 2001/Revised: 14 May 2001