Steven N. Treistman

Ethanol inhibition of recombinant heteromeric NMDA channels in the presence and absence of modulators

Abstract: The NMDA receptor/channel has been shown to be inhibited by ethanol in the clinically relevant range 25–100 mM. We studied heteromeric assemblies (NR1b/NR2) of the NMDA receptor, expressed in oocytes, to address three questions regarding this inhibition, and discovered the following: (1) The inhibition was nearly equivalent when ethanol was coapplied with the agonist, and when ethanol was introduced after steady‐state current was established, suggesting that ethanol does not act by interfering with the activation process of the NMDA receptor. (2) The degree of inhibition was controlled by the NR2 subunit, with both NR2A and NR2B significantly more sensitive to ethanol than NR2C and NR2D. (3) Manipulation of the NMDA receptor with a number of agents that normally modulate it did not alter the degree of inhibition produced by ethanol. The presence of Mg2+ (3 and 12.5 µM), Zn2+ (1 and 10 µM), or the glycine antagonist 7‐chlorokynurenic acid (1.25 or 5 µM), did not alter the ethanol sensitivity of heteromeric (NR1b/NR2A, NR1b/NR2B, NR1b/NR2C) NMDA receptors. Redox modulation of the NMDA receptor by dithiothreitol (2 mM) or 5,5′‐dithiobis(2‐nitrobenzoic acid) (1 mM) also did not alter the degree to which ethanol inhibits NMDA receptors. Taken together, these results indicate that the ethanol sensitivity of native NMDA receptors, which likely exist in heteromeric form, results from actions at a site different from those of known modulators of the receptor.

Combinatorial RNA splicing alters the surface charge on the NMDA receptor

Transcripts encoding four NMDA receptor subunits, generated from the NMDAR1 gene by alternative RNA splicing, have been demonstrated in adult rat brain. RNA transcripts derived from cDNAs encoding each form direct the formation of functional NMDA receptors in Xenopus oocytes. The two amino acid cassettes of 21 and 37 amino acids found in the splice variants increase the positive extracellular surface charge on the subunits and may thereby modulate the functional properties of the receptor.

Rat supraoptic magnocellular neurones show distinct large conductance, Ca2+-activated K+ channel subtypes in cell bodies versus nerve endings

1 Large conductance, Ca2+‐activated K+ (BK) channels were identified in freshly dissociated rat supraoptic neurones using patch clamp techniques. 2 The single channel conductance of cell body BK channels, recorded from inside‐out patches in symmetric 145 mM K+, was 246.1 pS, compared with 213 pS in nerve ending BK channels (P < 0.01). 3 At low open probability (Po), the reciprocal of the slope in the ln(NPo)‐voltage relationship (N, number of available channels in the patch) for cell body and nerve ending channels were similar: 11 vs. 14 mVper e‐fold change in NPo, respectively. 4 At 40 mV, the [Ca2+]i producing half‐maximal activation was 273 nM, as opposed to > 1.53 μM for the neurohypophysial channel, indicating the higher Ca2+ sensitivity of the cell body isochannel. 5 Cell body BK channels showed fast kinetics (open time constant, 8.5 ms; fast closed time constant, 1.6 and slow closed time constant, 12.7 ms), identifying them as ‘type I’ isochannels, as opposed to the slow gating (type II) of neurohypophysial BK channels. 6 Cell body BK activity was reduced by 10 nM charybdotoxin (NPo, 37 % of control), or 10 nM iberiotoxin (NPo, 5 % of control), whereas neurohypophysial BK channels are insensitive to charybdotoxin at concentrations as high as 360 nM. 7 Whilst blockade of nerve ending BK channels markedly slowed the repolarization of evoked single spikes, blockade of cell body channels was without effect on repolarization of evoked single spikes. 8 Ethanol reversibly increased neurohypophysial BK channel activity (EC50, 22 mM; maximal effect, 100 mM). In contrast, ethanol (up to 100 mM) failed to increase cell body BK channel activity. 9 In conclusion, we have characterized BK channels in supraoptic neuronal cell bodies, and demonstrated that they display different electrophysiological and pharmacological properties from their counterparts in the nerve endings.

Two types of high-threshold calcium currents inhibited by omega-conotoxin in nerve terminals of rat neurohypophysis.

1. The neurohypophysis comprises the nerve terminals of hypothalamic neurosecretory cells, which contain arginine vasopressin (AVP) and oxytocin. The secretory terminals of rat neurohypophyses were acutely dissociated. The macroscopic calcium currents (ICa) of these isolated peptidergic terminals were studied using ‘whole‐cell’ patch‐clamp recording techniques. 2. There are two types (‘Nt’ (where the subscript ‘t’ denotes terminal) and ‘L’) of high‐threshold voltage‐activated ICa in the terminals, which can be distinguished by holding at different potentials i.e. ‐90 and ‐50 mV. Replacement of Ca2+ in the bathing solution by Ba2+ increased the amplitude of ICa, primarily due to an increase in the L‐type component. Both inward currents were eliminated by adding 50 microM‐Cd2+ or when in a Ca(2+)‐free bathing solution. 3. omega‐Conotoxin GVIA (omega‐CgTx) has been widely used as a Ca2+ channel blocker. However, whether this toxin can discriminate between different types of Ca2+ channels is still a subject of controversy. We applied omega‐CgTx over a wide range of concentrations (0.01‐2 microM) to examine its effects on both Nt‐ and L‐type ICa in these terminals. At a concentration of 30 nM, omega‐CgTx selectively reduced, by 48%, the amplitude of Nt‐type ICa. In contrast, a higher concentration (300 nM) of omega‐CgTx was necessary to inhibit the L‐type ICa. 4. omega‐CgTx inhibited both Nt‐ and L‐type ICa in a dose‐dependent manner, and the half‐maximum inhibition (IC50) of the ICa by the toxin was 50 and 513 nM, respectively, which was approximately a tenfold difference. The reduction in both types of currents did not result from any shift in their current‐voltage or steady‐state inactivation relationships. 5. In contrast, omega‐CgTx, at a concentration of 300 nM, had no effect on the tetrodotoxin‐sensitive sodium current (INa) of the isolated peptidergic nerve terminals. Furthermore, omega‐CgTx did not reduce the long‐lasting, non‐inactivating ICa in the isolated non‐neuronal secretory cells of the pars intermedia (PI) (intermediate lobe of the pituitary). 6. Our studies suggest that omega‐CgTx might exert specific blocking effects on both Nt‐ and L‐type Ca2+ channels, but that in the isolated peptidergic nerve terminals, the Nt‐type component is more susceptible to this toxin.

BK Channels: mediators and models for alcohol tolerance.

Enhanced acute tolerance predicts alcohol abuse. We describe work on the role of the calcium- and voltage-gated BK channel in alcohol tolerance, highlighting the lipid environment, BK protein isoform selection and auxiliary BK channel proteins. We show how ethanol, which had the reputation of a nonspecific membrane perturbant, is now being examined at realistic concentrations with cutting-edge techniques, providing novel molecular targets for therapeutic approaches to alcoholism. Addictive disorders impact our emotional, physical and financial status, and burden our healthcare system. Although alcohol is the focus of this review, it is highly probable, given the common neural and biochemical pathways used by drugs of abuse, that the findings described here will also apply to other drugs.

Identification of a BK channel auxiliary protein controlling molecular and behavioral tolerance to alcohol.

Tolerance, described as the loss of drug effectiveness over time, is an important component of addiction. The degree of acute behavioral tolerance to alcohol exhibited by a naïve subject can predict the likelihood of alcohol abuse. Thus, the determinants of acute tolerance are important to understand. Calcium- and voltage-gated (BK) potassium channels, consisting of pore forming α and modulatory β subunits, are targets of ethanol (EtOH) action. Here, we examine the role, at the molecular, cellular, and behavioral levels, of the BK β4 subunit in acute tolerance. Single channel recordings in HEK-293 cells show that, in the absence of β4, EtOH potentiation of activity exhibits acute tolerance, which is blocked by coexpressing the β4 subunit. BK channels in acutely isolated medium spiny neurons from WT mice (in which the β4 subunit is well-represented) exhibit little tolerance. In contrast, neuronal BK channels from β4 knockout (KO) mice do display acute tolerance. Brain slice recordings showed tolerance to EtOHs effects on spike patterning in KO but not in WT mice. In addition, β4 KO mice develop rapid tolerance to EtOHs locomotor effects, whereas WT mice do not. Finally, in a restricted access ethanol self-administration assay, β4 KO mice drink more than their WT counterparts. Taken together, these data indicate that the β4 subunit controls ethanol tolerance at the molecular, cellular, and behavioral levels, and could determine individual differences in alcohol abuse and alcoholism, as well as represent a therapeutic target for alcoholism.

Somatic Localization of a Specific Large-Conductance Calcium-Activated Potassium Channel Subtype Controls Compartmentalized Ethanol Sensitivity in the Nucleus Accumbens

Alcohol is an addictive drug that targets a variety of ion channels and receptors. To address whether the effects of alcohol are compartment specific (soma vs dendrite), we examined the effects of ethanol (EtOH) on large-conductance calcium-activated potassium channels (BK) in cell bodies and dendrites of freshly isolated neurons from the rat nucleus accumbens (NAcc), a region known to be critical for the development of addiction. Compartment-specific drug action was indeed observed. Clinically relevant concentrations of EtOH increased somatic but not dendritic BK channel open probability. Electrophysiological single-channel recordings and pharmacological analysis of the BK channel in excised patches from each region indicated a number of differences, suggestive of a compartment-specific expression of the β4 subunit of the BK channel, that might explain the differential alcohol sensitivity. These parameters included activation kinetics, calcium dependency, and toxin blockade. Reverse transcription-PCR showed that both BK channel β1 and β4 subunit mRNAs are found in the NAcc, although the signal for β1 is significantly weaker. Immunohistochemistry revealed that β1 subunits were found in both soma and dendrites, whereas β4 appeared restricted to the soma. These findings suggest that the β4 subunit may confer EtOH sensitivity to somatic BK channels, whereas the absence of β4 in the dendrite results in insensitivity to the drug. Consistent with this idea, acute EtOH potentiated αβ4 BK currents in transfected human embryonic kidney cells, whereas it failed to alter αβ1 BK channel-mediated currents. Finally, an EtOH concentration (50 mm) that increased BK channel open probability strongly decreased the duration of somatic-generated action potential in NAcc neurons.

Alcohol Tolerance in Large-Conductance, Calcium-Activated Potassium Channels of CNS Terminals Is Intrinsic and Includes Two Components: Decreased Ethanol Potentiation and Decreased Channel Density

Tolerance is an important element of drug addiction and provides a model for understanding neuronal plasticity. The hypothalamic–neurohypophysial system (HNS) is an established preparation in which to study the actions of alcohol. Acute application of alcohol to the rat neurohypophysis potentiates large-conductance calcium-sensitive potassium channels (BK), contributing to inhibition of hormone secretion. A cultured HNS explant from adult rat was used to explore the molecular mechanisms of BK tolerance after prolonged alcohol exposure. Ethanol tolerance was intrinsic to the HNS and consisted of: (1) decreased BK potentiation by ethanol, complete within 12 min of exposure, and (2) decreased current density, which was not complete until 24 hr after exposure, indicating that the two components of tolerance represent distinct processes. Single-channel properties were not affected by chronic exposure, suggesting that decreased current density resulted from downregulation of functional channels in the membrane. Indeed, we observed decreased immunolabeling against the BK α-subunit on the surface of tolerant terminals. Analysis using confocal microscopy revealed a reduction of BK channel clustering, likely associated with the internalization of the channel.

Ethanol reduces vasopressin release by inhibiting calcium currents in nerve terminals.

Ingestion of ethanol (EtOH) is known to result in a reduction of plasma arginine-vasopressin (AVP) levels in mammals. We examined the basis for this effect using a combination of biochemical and electrophysiological techniques. Release of AVP from nerve terminals isolated from the rat neurohypophysis was very sensitive to EtOH, with significant reductions in AVP release evident in 10 mM EtOH. However, EtOH did not affect the release of AVP from terminals which had been permeabilized with digitonin, suggesting that voltage-gated calcium channels might be the target of EtOHs actions. Patch clamping of these terminals indicated that both inactivating and long-lasting calcium currents were reduced in EtOH, but the long-lasting currents were more sensitive (significant reductions in 10 mM EtOH). EtOH-induced decreases in plasma AVP levels can be explained by EtOHs inhibition of calcium currents in the nerve terminals.

Regulation of the gating of BKCa channel by lipid bilayer thickness

Transmembrane segments of ion channels tend to match the hydrophobic thickness of lipid bilayers to minimize mismatch energy and to maintain their proper organization and function. To probe how ion channels respond to mismatch with lipid bilayers of different thicknesses, we examined the single channel activities of BKCa (hSlo α-subunit) channels in planar bilayers of binary mixtures of DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) with phosphatidylcholines (PCs) of varying chain lengths, including PC 14:1, PC 18:1, PC 22:1, PC 24:1, and with porcine brain sphingomyelin. Bilayer thickness and structure was measured with small angle x-ray diffraction and atomic force microscopy. The open probability (Po) of the BKCa channel was finely tuned by bilayer thickness, first decreasing with increases in bilayer thickness from PC 14:1 to PC 22:1 and then increasing from PC 22:1 to PC 24:1 and to porcine brain sphingomyelin. Single channel kinetic analyses revealed that the mean open time of the channel increased monotonically with bilayer thickness and, therefore, could not account for the biphasic changes in Po. The mean closed time increased with bilayer thickness from PC 14:1 up to PC 22:1 and then decreased with further increases in bilayer thickness to PC 24:1 and sphingomyelin, correlating with changes in Po. This is consistent with the proposition that bilayer thickness affects channel activity mainly through altering the stability of the closed state. We suggest a simple mechanical model that combines forces of lateral stress within the lipid bilayer with local hydrophobic mismatch between lipids and the protein to account for the biphasic modulation of BKCa gating.