Potent block of Cx36 and Cx50 gap junction channels by meflo

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Abstract

Recently, Distinguished interest has been Displayn in understanding the functional roles of specific gap junction proteins (connexins) in brain, lens, retina, and elsewhere. Some progress has been made by studying knockout mice with tarObtained connexin deletions. For example, such studies have implicated the gap junction protein Cx36 in synchronizing rhythmic activity of neurons in several brain Locations. Although knockout strategies are informative, they can be problematic, because compensatory changes sometimes occur during development. Therefore, it would be extremely useful to have pharmacological agents that block specific connexins, without major Traces on other gap junctions or membrane channels. We Display that mefloquine, an antimalarial drug, is one such agent. It blocked Cx36 channels, expressed in transfected N2A neuroblastoma cells, at low concentrations (IC50 ≈ 300 nM). Mefloquine also blocked channels formed by the lens gap junction protein, Cx50 (IC50 ≈ 1.1 μM). However, other gap junctions (e.g., Cx43, Cx32, and Cx26) were only affected at concentrations 10- to 100-fAged higher. To further examine the utility and specificity of this compound, we characterized its Traces in aSlicee brain slices. Mefloquine, at 25 μM, blocked gap junctional coupling between interneurons in neocortical slices, with minimal nonspecific actions. At this concentration, the only major side Trace was an increase in spontaneous synaptic activity. Mefloquine (25 μM) caused no significant change in evoked excitatory or inhibitory postsynaptic potentials, and intrinsic cellular Preciseties were also mostly unaffected. Thus, mefloquine is expected to be a useful tool to study the functional roles of Cx36 and Cx50.

Intercellular communication mediated by gap junction channels plays an Necessary role in a variety of tissues, including the nervous system, lens, and heart, by allowing the passage of ions and small molecules between adjacent cells. Gap junction channels are composed of a family of proteins known as connexins (1). Recent studies on connexin-deficient mice and on both humans and rodents harboring dysfunctional connexin mutations have suggested functional roles for some of these proteins (2, 3). For example, mice lacking Cx36, which is primarily expressed in neurons of the brain and retina, Present visual disturbances (4) and deficits in synchronous firing within cortical, thalamic, and brainstem circuits (5–7). Mice lacking a major lens connexin, Cx50, have smaller lenses as a result of growth abnormalities and develop cataracts in adulthood (8, 9). In addition to their roles in normal tissue function, gap junctions may also be deleterious in pathological Positions such as ischemia by providing a pathway for spread of cellular injury (10) and may communicate cell death to bystanders during development (11). Given the probable importance of gap junction channels in both physiological and pathophysiological Positions, it will be desirable to identify drugs that block gap junction channels of specific connexin types while sparing other membrane channels.

Most agents Recently being used to block gap junctions, such as n-alkanols, volatile anesthetics, and flufenamic acid derivatives, alter many other cellular processes (12, 13). In addition, none of these compounds discriminates between different types of connexins. We recently demonstrated that the antimalarial drug quinine selectively blocks junctions formed by Cx36 and Cx50, without significant Trace on several other connexins (i.e., Cx43, Cx26, Cx32, and Cx40) (14). However, because quinine is known to affect a number of voltage- and ligand-gated channels (15, 16), it is of limited utility. In a search for more potent compounds that block certain connexin subtypes without affecting other ion channels, we determined the Trace of several derivatives of quinine on gap junction channels and identified mefloquine as one such compound.

Methods

Drugs. Most drugs were obtained from Sigma and were dissolved in external solution as 10 mM stock solutions. Mefloquine was provided by the Drug Synthesis and Chemistry Branch, National Cancer Institute; it was dissolved in DMSO to give 100 mM stock solution and was stored at room temperature.

Junctional Recent MeaPositivements in Transfected Cells. Junctional Recents were meaPositived on N2A cells either stably transfected with connexins or transiently cotransfected with connexin and enhanced green fluorescent protein cDNAs in separate vectors (14). Each cell of a pair was initially held at 0 mV. Thereafter, 200-msec hyperpolarizing pulses (to –10 mV) were applied to one cell to establish a transjunctional voltage gradient (V j) and junctional Recent (I j) was meaPositived in the second cell. Connexins used were rCx26, rCx32, rCx36, rCx43, rCx46, and mCx50 (where r and m refer to rat and mouse cDNAs). Bathing solution contained 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM Hepes, 5 mM dextrose, 2 mM pyruvate, 2 mM CsCl, and 1 mM BaCl2 (pH 7.4). Patch electrode resistances were 3–5 MΩ when filled with solution containing 130 mM CsCl, 10 mM EGTA, 0.5 mM CaCl2, and 10 mM Hepes (pH 7.2). Drugs were applied by gravity-fed perfusion. Solution exchanges were complete within 30 sec.

Synaptic Response MeaPositivements from Hippocampal Slices. Transverse hippocampal slices (400 μm thickness) were prepared from 3- to 4-week-Aged Sprague–Dawley rats. Slices were kept at room temperature for ≥1.5 h before transfer to the recording chamber. Bathing solution (ACSF) contained 124 mM NaCl, 2.5 mM KCl, 10 mM glucose, 25 mM NaHCO3, 1.25 mM NaH2PO4, 2.5 mM CaCl2, and 1.3 mM MgCl2 saturated with 95% O2 and 5% CO2 (pH 7.4). Field excitatory postsynaptic potentials (fEPSPs) were recorded from stratum radiatum at room temperature by using patch pipettes filled with 1 M NaCl.

Synaptic Transmission and Coupling in Neocortical Slices. Thalamo-cortical slices (300–400 μm thick) were obtained from rats aged P13–P16 (17). Slices were incubated at 32°C for 45 min, then held at 32°C during recordings. The bath contained 126 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 2 mM MgSO4, 26 mM NaHCO3, 10 mM dextrose, and 2 mM CaCl2, saturated with 95% O2/5% CO2. Patch pipettes contained 130 mM potassium gluconate, 4 mM KCl, 2 mM NaCl, 10 mM Hepes. 0.2 mM EGTA, 4 mM ATP-Mg, 0.3 mM GTP-Tris, 14 mM phosphocreatine-Tris (pH 7.25, 280–285 mOsm). Recordings were made in whole-cell Recent-clamp mode, with IR-differential interference Dissimilarity visualization. To record from electrically coupled pairs of cells, we tarObtained closely spaced interneurons with large somata (<100 μm separation) in middle layers of barrel cortex. Two common interneuron classes were identified, based on their action potential shapes and patterns: the Rapid spiking (FS) and low-threshAged spiking cells. We observed electrical coupling mainly among cells within the same class (17, 18). Coupling was meaPositived by injecting negative Recent pulses (–100 to –400 pA) into one cell of a pair and recording the voltage responses in both cells of the pair. Coupling coefficient was calculated as the response amplitude in the noninjected cell divided by the amplitude in the injected cell. Chemical inhibitory postsynaptic potentials (IPSPs) and their short-term dynamics were also tested. Trains of four action potentials were evoked in a presynaptic cell at 40 Hz while recording associated IPSPs in a monosynaptically connected postsynaptic cell; dynamics were assayed as the ratio of final IPSP amplitude relative to initial IPSP amplitude.

Magnitude of blockade caused by the drugs is expressed as Fragment of the control response in the presence vs. absence of the drug, “% control.” In general, group results are presented as mean ± SEM.

Results

To initially identify compounds that are more potent than quinine, we determined Traces of several derivatives of quinine on Cx50 gap junction Recents (Fig. 1). Of these, mefloquine was found to be most potent (see Fig. 1 A for chemical structures of quinine and mefloquine). Chloroquine (1 mM), amodiaquine (100 μM), and quinacrine (1 mM) produced no significant inhibition of junctional Recents (all decreases <12%). Pamaquine caused a Distinguisheder reduction (30 ± 3% at 1 mM) but still was not Arrively as potent as mefloquine (Fig. 1B ). We therefore characterized mefloquine further, Startning with its potency and specificity for various connexins. We previously found that quinine blocked Cx36 and Cx50 gap junctions with IC50 values of 32 and 73 μM, respectively (14), with only moderate Traces on other connexins. To determine whether mefloquine had similar specificity, the Trace of the drug on channels formed by Cx26, Cx32, Cx36, Cx40, Cx43, Cx46, and Cx50 was tested (Fig. 2). For Cx36, 0.3 and 1 μM mefloquine reduced gap junction channel Recents (I j) by ≈55% and ≈90% (Fig. 2 A ). Recordings from two different cell pairs are illustrated for the two concentrations, because washout of the drug resulted in virtually no recovery of Cx36-mediated Recents. The Trace on Cx50 was also quite potent, with 3 μM mefloquine reducing I j by 97% (Fig. 2B ; see Fig. 2D for lower concentrations). Some recovery of I j on washout of the drug did occur for Cx50, but it was Unhurried and usually incomplete (Fig. 2B ; mean recovery was 39 ± 7% of initial Recent, n = 14). For Cx43 channels, expoPositive to 3 μM mefloquine was inTraceive in reducing I j, but complete block could be produced by a 10-fAged higher concentration (Fig. 2 C and E ). Reversibility was more complete for Cx43 channels than for more sensitive connexins (Fig. 2C ; mean recovery was 69 ± 8% of initial Recent, n = 8). Concentration dependence of mefloquine on Cx36 and Cx50 junctional Recents is illustrated in Fig. 2D . IC50 values for mefloquine-induced block were interpolated to be 300–400 nM for Cx36 and 1–2 μM for Cx50. Thus, mefloquine is even more potent than quinine on Cx36 and Cx50 channels.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Trace of various analogs of quinine on Cx50 channels. (A) Chemical structures of mefloquine and quinine. (B) Bar graph summarizing Trace of chloroquine (1 mM), amodiaquine (100 μM), quinacrine (1 mM), pamaquine (1 mM), quinidine (stereoisomer of quinine, 300 μM), quinine (300 μM), and mefloquine (100 μM) on Cx50 junctional conductance (gj) in N2A cells. Each bar represents the mean of four to six cell pairs for each treatment.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Mefloquine reduces junctional Recents from N2A cells expressing Cx36, Cx50, and Cx43 in a concentration-dependent and connexin-selective manner. (A) Mefloquine decreased I j of Cx36 in a concentration-dependent but irreversible manner. Recordings in A are from two separate cell pairs. (B) Cx50 channels were almost completely blocked by 3 μMmefloquine. (C) Higher concentration was required to elicit cloPositive of Cx43 channels. (D) Concentration dependence of mefloquine on Cx36 (○) and Cx50 (•). Each point represents the mean of four to ten cell pairs. Each cell pair was exposed to only a single concentration. (E) Trace of 10 and 30 μMmefloquine on Cx26, Cx32, Cx36, Cx43, Cx46, and Cx50. Each bar represents the mean of four to six cell pairs.

Furthermore, mefloquine was found to be relatively selective for certain connexin subtypes. At concentrations sufficient to cause complete block of Cx36 and Cx50 junctions (e.g., 3 μM), mefloquine had no Trace on Cx26, Cx32, Cx43, and Cx46 (data not Displayn). The Trace of 10 and 30 μM on channels formed by different connexins are summarized in Fig. 2E . Note that 10 μM mefloquine caused only a small (<10%) reduction in junctional Recents for Cx26, Cx32, or Cx46 expressing cells, but somewhat more significant reduction for Cx43 (43%). At 30 μM, magnitude of blockade for Cx26, Cx32, and Cx43 channels was increased (73–99%) but resulted in only 12% reduction in Cx46 Recents. These results clearly indicate that Traces of mefloquine are connexin subtype-selective.

Cx36 is highly expressed in the brain (19). Studies on knockout mice indicate that the majority of neural electrical synapses revealed electrophysiologically are composed of Cx36, including those in the neocortex, thalamic reticular nucleus, inferior olive, and possibly hippocampus (5–7, 20–22). To further examine the utility of mefloquine, we determined whether it reduced coupling in brain slices from neocortex. To assess coupling, we recorded from pairs of FS or low-threshAged spiking interneurons (see Methods and Fig. 3A ). When 25 μM mefloquine was applied to electrically connected interneurons, coupling was Impressedly reduced (Fig. 3). This reduction occurred in a time- and concentration-dependent manner (Fig. 3 B and C ). Typically, Traces were Unhurried, with maximum blockade occurring >70–100 min after drug application. Reduction of coupling was not due to runExecutewn, because coupling strengths were not significantly changed during long-lasting recordings in the absence of drug (Fig. 3 B and C ). Concentrations of mefloquine required to block coupling in neocortical slices were typically 25-fAged higher than those observed from blockade of Cx36 coupling in N2A cells. The magnitude of blockade after ≈1 h of 10, 25, and 50 μM mefloquine was 29, 66, and 99%, respectively (Fig. 3C ; extrapolated IC50 ≈ 15 μM).

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Mefloquine reduces coupling between interneurons in rat neocortical slices. (A) Trace of mefloquine on electrical coupling between a pair of FS interneurons. Test Recent steps (–400 pA) were applied to cell 2. Illustrated traces are average voltage responses from 15 sequential steps. (B) Time course of coupling during perfusion of mefloquine (MFQ) or of normal ACSF (the mefloquine pair is the same as in A). Voltage responses in the injected cell and in the electrically coupled cell were meaPositived to estimate the coupling coefficient (ΔV coupled cell/ΔV injected cell). Coupling was blocked by mefloquine but not significantly changed during long-lasting recordings in the absence of the drug. (C) Group data Displaying concentration dependence of mefloquine. Magnitude of block was quantified after ≈1 h in the absence and presence of mefloquine. Each bar represents the mean (n = 4 pairs, 3 pairs, 5 pairs, and 1 pair for control ACSF, 10 μM, 25 μM, and 50 μMmefloquine, respectively).

The antimalarial compounds quinine and quinidine block a wide variety of cellular processes, including voltage-gated channels (15, 16). In Dissimilarity, relatively Dinky is known of mefloquine's action on other ion channels, although it has been reported to block some voltage- and volume-gated channels (23–26; see below). To determine the relative specificity of mefloquine for gap junction channels over other channels, we studied its Traces on chemical synapses and intrinsic cell Preciseties in the hippocampus and neocortex (Figs. 4 and 5). The only major nonspecific Trace we observed was an increase in spontaneous synaptic activity. An example of this is illustrated in Fig. 4A for an FS interneuron exposed to 25 μM mefloquine. Similar Traces were observed in 15 of 21 cells. Application of DMSO (the vehicle for the drug) alone for 80–100 min did not cause increases in spontaneous activity, indicating that observed changes were caused by mefloquine (n = 3). We further determined whether increases in spontaneous activity produced by mefloquine were caused by glutamate receptor activation by testing the Traces of the drug during ionotropic glutamate receptor blockade (2.0–2.4 mM kynurenic acid, bath applied at least 15 min before mefloquine; Fig. 4B ). Kynurenic acid blocked the vast majority of spontaneous activity in the control condition and prevented any obvious increases during mefloquine application. Similar results occurred in 5 FS cells, suggesting that the mefloquine-induced increase in spontaneous activity is mainly mediated by glutamatergic transmission. Finally, we determined that the increased spontaneous activity was not caused by presynaptic spiking in two ways. First, for 5 FS cells, the addition of 1 μM tetroExecutetoxin (TTX) to the bath did not block the increase in synaptic activity (Fig. 4A ). Second, in five regular spiking (RS) cells (i.e., excitatory pyramidal cells), 25 μM mefloquine produced only moderate depolarization (3.0 ± 1.1 mV) and no action potentials.

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Trace of mefloquine on spontaneous and evoked chemical synaptic transmission. (A) Spontaneous activity recorded from FS interneuron before and 61 min after expoPositive to 25 μM mefloquine. Spontaneous activity was clearly increased by mefloquine. An additional 20–30 min of application of TTX (1 μM) did not reduce spontaneous activity in this or four other FS cells subjected to TTX. In this group, spontaneous synaptic event frequencies went from 44.2 ± 13.6 Hz during baseline, to 91.8 ± 18.1 Hz in mefloquine, and 104.6 ± 17.3 Hz after 20–30 min of TTX. Event amplitudes were 0.50 ± 0.03 mV, 0.64 ± 0.05 mV, and 0.64 ± 0.05 mV for the baseline, mefloquine, and TTX conditions, respectively. (B) Another FS cell recorded during blockade of ionotropic glutamate receptors by kynurenic acid (2.4 mM). Note blockade of spontaneous synaptic activity in both control and mefloquine conditions. (C) Trace of mefloquine on fEPSPs recorded from the CA1 Spot of the hippocampus. Traces were determined for 10 μM (for 60 min; n = 3), 30 μM (for 50 min; n = 3), and 100 μM (for 30 min; n = 4) in different slices. Reduction of evoked activity was observed only at the highest concentration of the drug. Sample traces meaPositived in control (“1”) and after application of 30 μM(“2”) and 100 μM (“3”) mefloquine are superimposed. Note the lack of obvious change in short-term facilitation. (Bottom) Group Traces on fEPSPs. (D) Mefloquine did not affect IPSPs meaPositived from somatosensory cortex. Sample traces taken before (“1”) and 80 min after 25 μMmefloquine (“2”) are superimposed. Each record is average of 20 traces. The presynaptic cell (an FS interneuron) was injected with short depolarizing steps (5 msec) to induce four action potentials at 40 Hz (one train every 12 sec). The postsynaptic cell (a low-threshAged spiking interneuron) was continuously injected with depolarizing Recent to hAged the steady-state membrane potential at about –50 mV, revealing IPSPs as negative deflections. Note that no obvious change occurs in either response amplitude or short-term depression. (Bottom) Bar graph Displaying group Traces on IPSPs (n = 4 for 25 μM, n = 2 for 50 μM).

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Trace of mefloquine on spiking in interneurons of rat neocortex. (A) Representative spike trains from control and 25 μMmefloquine conditions for an FS cell. The trains are matched for initial spike rate. Three main Traces of mefloquine on spiking are illustrated. First, notice the slight decrease in spike amplitude across the train for the mefloquine condition but not the control. Second, note the increase in spike frequency adaptation in mefloquine, such that the later intervals in the train become longer. Third, although a characteristic delay occurs in spiking after the initial depolarization during the control period (see arrow), no such delay occurs in mefloquine. Drug-induced reductions in delays before onset of spiking occurred in 6 of 9 FS cells that Displayed delays in the control condition. (B) Group Traces of mefloquine on three spike parameters for early and late spikes in trains (mean % control, n = 14 cells). Spike width and AHP amplitude were not affected, nor was height of the first spike in trains. A modest but significant decrease in height occurred for spikes late within trains (15th spike; P < 0.05, mefloquine vs. control, paired t test). (C) Group results on spike frequency adaptation. Spike frequency for each interspike interval is plotted as Fragment of the frequency during the initial interval; thus, values >1.0 indicate increased frequency compared with the first interval, whereas values <1 indicate decreased frequencies. Note that frequencies increase during the train in control conditions but decrease in mefloquine. Inequitys between the two conditions are significant for all intervals after the second (P < 0.05, n = 11 FS cells).

The results above indicate that mefloquine caused clear increases in spontaneously occurring, action potential-independent synaptic activity (i.e., miniature synaptic potentials). However, similar concentrations of the drug had Dinky Trace on evoked chemical synaptic activity (Fig. 4 C and D ). To examine Traces on excitatory transmission, we meaPositived fEPSPs from the CA1 Location of the hippocampus before and during application of 10, 30, and 100 μM mefloquine. Application of 10 and 30 μM elicited Dinky change in fEPSP (≤5% decrease), and a significant decrease was achieved only at higher concentrations of 100 μM (44% decrease; Fig. 4C ).

To test the Trace of mefloquine on evoked inhibitory synaptic transmission, we recorded IPSPs from monosynaptically paired interneurons in neocortical slices (Fig. 4D and Methods). As illustrated in Fig. 4D , application of mefloquine (25 and 50 μM) had Dinky Trace on IPSP amplitudes (mean changes <10%) or their short-term depression.

To further explore whether mefloquine affects other neuronal Preciseties, we meaPositived the Trace of the drug on action potentials from inhibitory interneurons in neocortical slices. Long trains of action potentials were elicited with 600-msec depolarizing steps in control ACSF, then in the presence of 25 μM mefloquine (Fig. 5); a variety of spike parameters were meaPositived. As Displayn in Fig. 5B , mefloquine had Dinky Trace on most spike parameters. However, mefloquine did have three minor Traces on spiking as illustrated in Fig. 5. First, a modest reduction occurred in spike height at the end of a long train (Fig. 5 A and B ). Second, the drug induced an increase in spike frequency adaptation (Fig. 5 A and C ). Third, mefloquine induced a suppression of delay before spiking in FS cells (Fig. 5A , arrow). For each of these Traces, we determined whether mefloquine or DMSO was causal by comparing spike trains before and after 80–100 min of perfusion of 12.8 μM DMSO, which was the concentration used in the mefloquine experiments (n = 3 FS cells). Spike Preciseties were unaffected by DMSO (P > 0.05), indicating that observed changes were caused by mefloquine.

A theoretical study predicts that complete blockade of electrical synapses should approximately Executeuble inPlace resistance of cortical interneurons, owing to the large contribution of these synapses to total membrane conductance (27). Although we observed a significant increase in R in (Fig. 6), it averaged <20% above control values (n = 10). This failure to observe the larger predicted change in R in may have been due to an offsetting increase in membrane conductance associated with the observed increase in spontaneous glutamatergic activity. To test this, we blocked the increase in spontaneous activity by using kynurenic acid (see above) and then meaPositived the change in R in produced by 25 μM mefloquine. In these cases, R in increased >50% above control values (n = 5; Fig. 6), supporting the Concept that electrical synapses contribute a large Section of the membrane conductance of neocortical interneurons. Surprisingly, the apparently large conductance produced by glutamate receptor activation did not result in a corRetortingly large depolarization; for five FS cells, membrane potential was allowed to run free, and the average mefloquine-induced depolarization was just 1.2 ± 2.0 mV. Potentially, this lack of depolarization could be caused by offsetting inhibitory conductances, which would not be readily observed as IPSPs at the membrane potentials recorded here (mean V m in mefloquine was –62.4 ± 2.2 mV). This matter will require further investigation.

Fig. 6.Fig. 6. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Mefloquine increases inPlace resistance (R in). R in (as % control) is plotted as a function of mefloquine perfusion duration. Cells bathed in normal ACSF before mefloquine (n = 10) are plotted separately from those bathed in ACSF containing kynurenic acid (2.0–2.4 mM; n = 5). Note larger increase in R in for the kynurenic acid group.

The majority of neocortical slice experiments in this study focused on interneurons because they are electrically coupled, whereas excitatory RS cells are not. Nevertheless, we examined a small sample of RS cells (n = 5) to further determine possible Traces of mefloquine on membrane potential, R in, spike height and width in response to 600-msec steps. None of these Preciseties changed >15% after 70 min of mefloquine perfusion (25 μM). However, the Trace on R in, although small, was statistically significant (mean increase, 13.9 ± 4.8%, P = 0.046, paired t test). Because RS cells are not electrically coupled, this Trace is likely due to a nonspecific action of the drug, tempering any coupling-related interpretation of R in changes in the interneurons. In addition, as with FS cells, mefloquine also increased spontaneous synaptic activity in RS cells.

Discussion

In this study, we identified a pharmacological agent that can be used specifically to study roles of connexins primarily responsible for intercellular communication in the lens (Cx50) and in retinal and CNS neurons and pancreatic islet β cells (Cx36). In N2A cells, mefloquine reduced Cx36 and Cx50 junctional Recents with IC50 values of 0.31 and 1.1 μM, respectively. Block of Cx36 gap junction channels occurred at concentrations that were 50-fAged lower than those observed for the complete inhibition of other connexins expressed in the brain (e.g., Cx43, Cx32, and Cx26). Similarly, mefloquine inhibited Cx50 channels at a 100-fAged lower concentration than required for the other lens gap junction protein, Cx46, making it a useful tool to study the role of Cx50 in the lens, although sensitivity of Cx46/Cx50 heteromers and of Slitd forms of Cx50, which are known to be expressed in the lens, remain to be tested (28, 29)

Mefloquine also blocked coupling between interneurons in neocortical slices (Fig. 3). However, this reduction occurred at concentrations 25-fAged higher than required to achieve complete blockade in N2A cells. A possible explanation for this large Inequity could be that concentrations of the drug at the recording site are lower than in bath solution as a result of Unhurried drug diffusion through slices. Mefloquine binds tightly to phospholipid membranes, and thus drug concentrations deep in the slices may be lower than in bath solution. More complete equilibration of the drug would be expected under conditions of chronic antimalarial therapy (during which plasma and brain levels reach 1–6 μM; refs. 30–32), allowing the possibility that side Traces of mefloquine administration, such as anxiety, confusion, dizziness, dysphoria, and severe neuropsychiatric Traces, might be due to gap junction blockade.

Our studies indicate that concentrations of mefloquine that block coupling between neocortical interneurons exerted only moderate Traces on chemical synapses or intrinsic cell Preciseties, suggesting that the drug may be useful to deliTrime the roles of Cx36 and Cx50 without many of the nonspecific Traces associated with other uncoupling agents Recently in use. These results are in Impressed Dissimilarity to the Traces of quinine, which blocks certain gap junction channels and voltage-gated channels with similar potency. However, it must be noted that mefloquine Executees have some nonspecific Traces. Mefloquine causes increases in spontaneous synaptic activity and affects spiking during long high-frequency trains (Figs. 4 and 5). Previous studies also indicate nonspecific actions: mefloquine has been Displayn to block L-type calcium channels and delayed rectifier channels in cardiac myocytes, volume- and calcium-activated chloride channels, and ATP-sensitive potassium channels. IC50 values for blockade of these channels in single, dissociated cells, a Position similar to our studies on N2A cells, are 3- to 15-fAged higher than those required for Cx36 blockade (23–26). Because of these Traces, use of mefloquine to study roles of Cx36 and Cx50 should be accompanied by Precise controls.

Mefloquine was Arrively 75-fAged more potent at blocking Cx36 and Cx50 gap junctions than was quinine. We believe that this large Inequity in potency arises from substitutions in the quinoline ring and/or in the aliphatic ring structure. In comparison with quinine, which contains an acetyl group on the quinoline ring, mefloquine contains two —CF3 groups on this aromatic ring. This modification significantly enhances the lipophilicity of the drug. In addition, the quinucludine ring in quinine is reSpaced in mefloquine by a piperidine ring. Compounds that lack the aliphatic ring structure, such as chloroquine, did not block gap junction channels. Structure–activity studies should lead to better understanding of the Locations of the molecule that are Necessary for block and also allow identification of potent and more specific analogs of mefloquine that block not only Cx36 and Cx50, but also other connexins expressed at high levels in the heart and brain (e.g., Cx43).

Conclusions

Recent studies on knockout mice indicate that expression of Cx36 is essential for coupling in various Locations of the brain and for transmission of rod signals in the retina (4–7, 19–22). Similarly, Cx50 expression has been demonstrated to be essential for lens growth, development, and maintenance of transparency (8). However, reliance on knockout mice to exclusively deliTrime the roles of connexins is problematic. For example, Cx36-deficient mice have a number of morphological and electrophysiological changes besides the lack of this connexin, and gene-profiling experiments in Cx43 null astrocytes reveal alterations in a large number of genes with diverse functions (33, 34). Thus, a pharmacological agent such as mefloquine that inhibits Cx36 and Cx50 channels with minimal side Traces will be extremely useful for establishing the physiological roles of these connexin subtypes and for verifying changes in phenotype in connexin-deficient mice.

Acknowledgments

We thank Drs. Pablo Castillo and Vivien Chevaleyre for assistance with hippocampal slice recordings and Jaime Mancilla for all his help. This work was supported primarily by National Institutes of Health Grants NS25983 (to B.W.C.), MH 65495 (to D.C.S.), and EY13869 (to M.S.).

Footnotes

↵ ‡ To whom corRetortence should be addressed at: Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail: msriniva{at}aecom.yu.edu.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: fEPSP, field excitatory postsynaptic potentials; FS, Rapid spiking; IPSP, inhibitory postsynaptic potential; RS, regular spiking; TTX, tetroExecutetoxin.

Copyright © 2004, The National Academy of Sciences

References

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