BACKGROUND
gabapentin  and pregabalin -3-isobutyl gaba) were both originally designed as gaba mimetics , with the intention that they would be able to cross the blood-brain barrier and interact with gabaergic systems and enhance gaba mediated inhibition.
although gabapentin appears to have diverse therapeutic utility in the treatment of pain disorders  <cit> , psychiatric illnesses  <cit>  and epilepsy  <cit> , there is controversy regarding its molecular mechanisms of action.
whether the actions of gabapentin and pregabalin are mediated through gabaergic mechanisms or gaba receptors remains particularly contentious  <cit> .
however, high affinity binding sites for gabapentin and pregabalin on distinct α2δ subunits of voltage-activated calcium channels have been identified and characterised  <cit> .
for this reason voltage-activated ca2+ channels remain primary candidate sites of action for these novel anticonvulsant and antihyperalgesic drugs.
functional data from studies on gabapentin and pregabalin also support this contention.
specifically, both gabapentin and pregabalin inhibited hyperalgesia  <cit> , attenuated evoked ca2+ influx into brain slices and reduced evoked transmitter release  <cit> .
additionally, gabapentin and pregabalin inhibited multiple firing of action potentials evoked by  <dig> ms depolarising current commands in cultured sensory drg neurones  <cit> .
our previous studies have focused on the inhibitory effects of gabapentin  on whole cell voltage-activated ca2+ currents and k+ stimulated ca2+ entry measured with fura- <dig>  the cellular model systems used were primary cultures of dorsal root ganglion  neurones from 1– <dig> day old rats and differentiated f- <dig> cells   <cit> .
in this present study the effects of pregabalin and gabapentin on the electrophysiological properties of cultured neonatal rat drg neurones were measured with particular reference to ca2+ entry through high voltage-activated channels and enhancement of k+ conductances.
we had two specific aims for this project.
the first was to determine whether gabapentin and pregabalin have the same mechanisms of action, by examining whether saturating concentrations of gabapentin and pregabalin act in an additive manner to attenuate ca2+ influx.
the second aim was to further evaluate gaba receptors as target sites for these drugs in sensory neurones.
this latter element to the study was conducted because it has been proposed that specific gabab receptors with a gb1a-gb <dig> heterodimer composition are the sites of agonist activity of gabapentin.
these specific receptors can be coupled to an inwardly rectifying k+ channel and / or voltage-activated ca2+ channels to dampen neuronal electrical excitability  <cit> .
however, in certain situations gabapentin and the gabab receptor agonist, baclofen, have different actions.
weaver mutant mice  are insensitive to both gabapentin and baclofen, however in control littermates, weaver control mice , only baclofen evoked a k+ current  <cit> .
RESULTS
actions of pregabalin on voltage-activated calcium currents
the firing of multiple action potentials in response to a sustained depolarising current command is a property of a sub-population  of cultured drg neurones.
application for  <dig> minutes, of either pregabalin  or gabapentin  reduced the frequency of action potential spikes evoked by  <dig> ms depolarising current step commands applied every  <dig> s  <cit> .
gabapentin attenuated repetitive action potential firing as measured by a reduction in the mean number of evoked action potentials during  <dig> ms depolarisations from  <dig> to  <dig> .
pregabalin  reversibly reduced the number of action potentials during  <dig> ms depolarisations from  <dig> to  <dig> .
however, pregabalin and gabapentin did not significantly alter the properties  of single action potentials evoked by  <dig> ms depolarising current commands.
consistent with our previous work  <cit> , whole cell voltage-activated ca2+ currents  recorded from drg neurones were reversibly attenuated  by  <dig>  μm gabapentin.
similarly,  <dig> minutes application of pregabalin  inhibited the mean ca2+ current amplitude, measured at the peak of the inward current, and at the end of a  <dig> ms voltage step command to  <dig> mv.
the inhibitory actions of pregabalin were not accompanied by any shift in the voltage-dependence of activation for ica or by any change in holding or leak currents.
at least partial recovery of ica was observed  <dig> minutes after removal of the pregabalin-containing perfusion pipette .
a very low concentration of pregabalin  failed to produce significant inhibition of ica.
however, no clear dose-dependent relationship was established for the inhibitory actions of pregabalin, and a major part of the voltage-activated ca2+ current was insensitive to the drug.
it was also apparent that there was considerable variability in the sensitivity of the drg neurones to any given dose of pregabalin.
some neurones did not respond to  <dig>  μm pregabalin while in a few neurones this same concentration produced  <dig> % inhibition of ica .
***p <  <dig> ; **p <  <dig>  and *p <  <dig> .
each value is given ± s.e.m.
data shows inhibition but a lack of dose-dependent actions on ica of a wide range of pregabalin concentrations.
although varied responses were observed it was clear that a major component of ica was resistant to inhibition by pregabalin.
distinct voltage-activated ca2+ channels, defined by their alpha  <dig> subunit, have been suggested to be selective target sites for gabapentin and related drugs like pregabalin.
with this in mind, the  <dig> -dihydropyridine l-type ca2+ channel agonist bay k <dig> was used to determine whether pregabalin was inhibiting l-type channels.
previously, we found that in the continual presence of gabapentin, bay k <dig> enhanced ica  <cit> .
in this present study similar results were obtained.
after inhibiting part of the current with pregabalin , bay k <dig>  was applied with pregabalin and the enhanced ica was measured at its peak and at the end of a  <dig> ms voltage step command to  <dig> mv .
the percentage increases in current seen with bay k <dig> in the presence of either gabapentin or pregabalin were slightly less than those seen under control conditions in cultured drg neurones  <cit> .
this is consistent with some l-type ca2+ current modulation by pregabalin and gabapentin in drg neurones.
taken together these data suggest that neither gabapentin nor pregabalin are selectively inhibiting l-type ica in drg neurones.
this contrasts with the effects of gabapentin on cortical pyramidal neurones where inhibition of l-type ca2+ channels appears to be the predominant mechanism of action  <cit> .
actions of pregabalin on calcium influx through voltage-activated channels, measured using fura- <dig> imaging
given the variable and rather modest but reversible inhibitory actions of pregabalin on ica we tested the actions of pregabalin on k+-evoked ca2+ influx using fura- <dig> imaging.
an extracellular solution containing  <dig> mm k+ was used to depolarise the drg neurones and activate three consistent ca2+ transients  <cit> .
pregabalin  was applied during the second k+-evoked depolarisation and it produced a mixture of reversible effects on the ca2+ transients.
in  <dig> of  <dig> neurones the ca2+ flux was decreased by  <dig> ±  <dig> %  but in  <dig> neurones from the same cultures pregabalin evoked a mean increase in ca2+ flux to  <dig> ±  <dig> %  of the control.
raising the pregabalin concentration to  <dig> μm increased the proportion of inhibitory responses  but enhancement of ca2+ transients was still observed in the remaining  <dig> neurones .
however,  <dig> μm pregabalin caused an increase in k+-evoked ca2+ influx in all neurones studied .
although in some cells good recovery from pregabalin actions was observed  the inhibitory effect was often not associated with recovery.
this may reflect long lasting effects of pregabalin.
run-down in some cells can not be completely ruled out but under control conditions the mean level of run-down of three k+-evoked ca2+ transients was only  <dig>  ±  <dig>  % .
similarly, the mean control level of increase in k+-evoked ca2+ transients was only  <dig>  ±  <dig>  % .
to investigate the different responses to pregabalin, the sizes of cell somas were measured and compared.
although there is overlap, figure  <dig> shows that enhancement of k+-evoked ca2+ transients by pregabalin was mainly seen in neurones with larger cell somas and that in smaller neurones pregabalin produced inhibitory effects.
ca2+-dependent conductances of drg neurones are sensitive to ryanodine and ca2+-induced ca2+ release has been reported in these neurones.
modulation of either ca2+-induced ca2+ release and / or ca2+ homeostatic mechanisms might provide a mechanism by which pregabalin enhanced ca2+ transients in neurones with intermediate and large sized cell somas.
this was investigated in two ways.
firstly, the actions of pregabalin on caffeine-evoked ca2+ transients were evaluated in nominally ca2+-free extracellular conditions .
single caffeine  responses were obtained from drg neurones in either the absence or presence of  <dig> μm pregabalin.
no differences in either amplitudes or durations of ca2+ transients were seen when  <dig> control caffeine responses were compared with  <dig> caffeine responses obtained in the presence of pregabalin .
the second approach was to explore a possible role of na+ / ca2+ exchange by bathing cells in choline chloride-based medium containing only  <dig> mm na+.
under these conditions the contribution of the na+ / ca2+ exchanger to handling of intracellular ca2+ loads will be minimal.
in choline chloride-based medium pregabalin produced both enhancement and inhibition of total ca2+ flux in  <dig> and  <dig> neurones respectively.
enhancement in ca2+ flux by pregabalin under these experimental conditions, suggest that inhibition of na+ / ca2+ exchange is not the main mechanism by which pregabalin enhances k+-evoked ca2+ flux .
however, detailed analysis was made difficult by poor recovery of even the first ca2+ transient evoked in low extracellular na+ which does suggest that na+ / ca2+ exchange is an important homeostatic mechanism in drg neurones.
in conclusion these results indicate that modulation of ca2+-induced ca2+ release and ca2+ homeostatic mechanisms do not account for pregabalin-induced enhancement of k+-evoked ca2+ flux in a population of drg neurones.
inhibitory modulation of potassium conductances could result in increased k+-evoked ca2+ transients.
to test this alternative mechanism, a relatively low concentration,  <dig> mm, of tetraethylammonium  was applied with nacl-based extracellular medium and drg neurones were stimulated with  <dig> mm kcl.
attenuation of potassium conductances by tea markedly increased the k+-evoked ca2+ flux in all eight drg neurones studied, mimicking in part the action of pregabalin .
do pregabalin and gabapentin have additive effects on cultured dorsal root ganglion neurones?
pregabalin and gabapentin have related chemical structures and appear to have similar but usually modest inhibitory effects on ca2+ currents.
both pregabalin and gabapentin at a concentration of  <dig>  μm produced a maximum level of current inhibition.
simultaneous application of pregabalin  and gabapentin  produced modest but significant inhibition of ica at the peak of the current and at the end of the stimulus .
however, the percentage inhibition of ica produced was not significantly different for  <dig>  μm gabapentin alone ,  <dig>  μm pregabalin alone  and  <dig>  μm gabapentin and  <dig>  μm pregabalin applied together .
in spite of the added complexity of mixed responses with pregabalin, experiments were carried out using fura- <dig> to measure k+ evoked ca2+ flux and the effects of both ligands.
our previous experiments with gabapentin  had only identified inhibitory effects using this experimental approach  <cit> .
simultaneous application of pregabalin and gabapentin  reversibly reduced the k+ evoked ca2+ influx .
pregabalin  and gabapentin  together reduced the total ca2+ flux to  <dig> ±  <dig> %  of the control response to  <dig> mm k+.
consistent with the electrophysiological experiments, this level of inhibition was similar to the inhibitory effects of pregabalin and gabapentin applied separately.
the electrophysiological and fura- <dig> ca2+ imaging data show that no additive inhibitory effects were found during simultaneous application of saturating concentrations of gabapentin and pregabalin.
the data therefore support the contention that both drugs have a closely related or a common site of inhibitory action.
the data also indicate that in the modulation of ca2+ channels these drugs do not act in an additive manner even though a substantial proportion of current is resistant to both drugs.
it should be emphasized however that pregabalin can reversibly enhance k+-evoked ca2+ transients, an effect not seen with gabapentin.
therefore there may be other additional actions of pregabalin that can be identified in ca2+ imaging experiments when all membrane conductances are intact.
does gaba receptor modulation alter responses to pregabalin in cultured dorsal root ganglion neurones?
in this section of the study two strategies were used to evaluate the possible roles of gaba receptors in pregabalin responses in cultured drg neurones.
firstly, pregabalin actions were studied in the presence of a saturating concentration of gaba; this initially activated all types of gaba receptor and then caused desensitization of these receptors.
secondly, the effect of a potent and selective gabab receptor antagonist, cgp <dig>  <cit>  on pregabalin responses was evaluated.
gaba  evoked inward currents in a sub-population of cultured drg neurones and induced a transient rise in intracellular ca2+ in  <dig> of  <dig> drg neurones studied .
the inward currents were due to the activation of gabaa receptor cl- channels; with the equilibrium potential for cl- under our recording conditions being close to  <dig> mv the chloride conductance results in an inward current.
although in vivo the equilibrium potential for cl- in drg neurones is variable, it is predicted to be between - <dig> mv and - <dig> mv because of cl- loading into the intracellular environment .
therefore gabaa receptor cl- channel activation can produce a depolarisation of the resting membrane potential.
our data indicated that the gaba-evoked depolarisation was sufficient to result in voltage-activated ca2+ channel activity and produce a transient rise in intracellular ca2+.
in both neurones that responded to gaba and those that did not, subsequent application of pregabalin, produced either enhancement or inhibition of k+-evoked ca2+ influx .
these responses to pregabalin in the presence of gaba were no different in character to the pregabalin responses obtained in the absence of gaba.
in neurones that showed a ca2+ transient in response to a gaba-evoked depolarisation, there was an apparent increase  in the proportion of neurones that were inhibited by pregabalin.
it is not clear why this is but it may reflect the sensitivity of ca2+ transients evoked in drg neurones, which can only be evoked consistently in most drg neurones by three depolarising stimuli.
thus the gaba-evoked responses may influence ca2+ homeostatic mechanisms and subsequent stimulated ca2+ entry.
however, the critical observation from these experiments is that gaba receptor desensitisation does not prevent either ca2+ transient enhancement or inhibitory actions of pregabalin in neurones that were sensitive or insensitive to gaba.
responders were those neurones in which gaba evoked a transient rise in intracellular ca2+.
non-responder showed no change in fura- <dig> fluorescence ratio in response to gaba.
controls where neurones that pregabalin was applied to without gaba being present.
the amplitudes of the gaba responses did not vary with the inhibitory or enhancing responses to pregabalin.
the possibility of pregabalin-evoked inhibition of ica taking place through activation of g-protein coupled gabab receptors, was then assessed.
cgp <dig>  when applied alone had no effect on ica.
in the presence of the gabab receptor antagonist cgp <dig> , pregabalin  evoked a  <dig> ±  <dig> %  inhibition of the voltage-activated ca2+ current and complete recovery was observed  <dig> minutes after removal of the drug-containing pipette .
in ca2+ imaging experiments  <dig> μm cgp <dig> had no effect on the responses to  <dig> μm pregabalin.
in the presence of cgp <dig>   <dig> out of  <dig> neurones showed enhanced k+-evoked ca2+ influx  of control) in response to pregabalin and  <dig> neurones showed inhibition of ca2+ influx  of control) in response to pregabalin .
is there a role for g-proteins or g-protein coupled receptors in the responses to pregabalin in cultured dorsal root ganglion neurones?
previously, we found that the actions of gabapentin on ica were attenuated by pre-treating drg neurones with pertussis toxin, an effect that did not appear to involve metabotropic gabab receptors  <cit> .
in this study several different approaches were taken to examine the influence of receptor and g-protein function in pregabalin actions.
firstly, the effects of a novel thiadiazole compound, sch- <dig>  which inhibits ligand binding to a variety of g-protein coupled receptors  were investigated.
the selective and reversible action of sch- <dig> appears to involve allosteric modulation of both agonist and antagonist binding to g-protein coupled receptors  <cit> .
secondly, the potential influences of intracellular flash photolysis of caged gtp-γ-s and subsequent g-protein activation on pregabalin responses were also examined.
sch- <dig>  was applied to the intracellular environment via the patch pipette solution.
after  <dig> minutes equilibration the whole cell ca2+ current had a mean amplitude of - <dig>  ±  <dig>  na .
during this period there was a clear reduction in the inward current, although a steady state current level was reached.
however, subsequent application of pregabalin  resulted in a further significant reduction in ca2+ current amplitude to - <dig>  ±  <dig>  na .
this represents a mean inhibition of  <dig> ±  <dig> % by pregabalin, a value very similar to the percentage inhibition produced by pregabalin in the absence of sch- <dig> .
therefore sch- <dig> had no effect on the pregabalin responses, indicating that pregabalin was not acting through an sch-202676-sensitive g-protein coupled receptor .
preliminary ca2+ imaging experiments were also conducted to determine whether extracellular sch- <dig>  altered the effects of pregabalin  on k+-evoked ca2+ transients.
under these experimental conditions pregabalin was found to markedly enhance or inhibit k+-evoked ca2+ transients .
this data again suggests that pregabalin was not acting through mechanisms that were sensitive to sch- <dig>
caged gtp-γ-s  was applied to the intracellular environment via the patch pipette solution and after entering the whole cell recording configuration drg neurones were left for  <dig> minutes to equilibrate.
once a stable control ca2+ current was obtained, three  <dig> v flashes of intense near uv light were applied to the neurone to achieve intracellular flash photolysis of the caged gtp-γ-s. we estimate that approximately  <dig> μm gtp-γ-s was photoreleased by the three flashes.
as previously observed intracellular flash photolysis of caged gtp-γ-s reduced the amplitude and slowed the activation of ica  <cit> .
the effects of photoreleased gtp-γ-s were attenuated by applying a large depolarising pre-pulse  <cit> , which is consistent with voltage-dependent g-protein modulation of ca2+ channels .
no recovery from photoreleased gtp-γ-s was observed during the period of the experiment.
gtp-γ-s and pregabalin  had additive inhibitory effects on ica.
these additive actions of gtp-γ-s and pregabalin were apparent regardless of the order of application.
one set of experiments was performed in which gtp-γ-s was photoreleased in the intracellular environment and then after stabilisation of the response pregabalin was applied .
in another set of experiments pregabalin was applied first and after equilibration gtp-γ-s was photoreleased in the continual presence of pregabalin .
when pregabalin  was applied first it produced a mean inhibition in ica by  <dig> ±  <dig> % , when applied after photorelease of gtp-γ-s, pregabalin produced a  <dig> ±  <dig> %  inhibition of ica.
actions of pregabalin on voltage-activated potassium currents
it was clear from the ca2+ imaging experiments that not all the cellular actions of pregabalin could be explained by the inhibition of voltage-activated ca2+ channels because both inhibition and enhancement in k+ evoked ca2+ flux was observed.
to investigate the actions of pregabalin further, its' effects on voltage-activated k+ currents in drg neurones were studied.
previously, stefani and colleagues found that gabapentin modulated neuronal steady state non-inactivating k+ currents  <cit> .
three minutes application of pregabalin  had no significant effect on the voltage-activated k+ current activated from a holding potential of - <dig> mv by a  <dig> ms voltage step command to  <dig> mv .
however, raising the pregabalin concentration to  <dig> μm resulted in significant modulation of k+ currents in drg neurones.
pregabalin  applied for  <dig> to  <dig> minutes produced an enhanced k+ current in  <dig> out of  <dig> neurones but a modest inhibition of the outward current in  <dig> out of  <dig> neurones .
drg neurones are a heterogenous population of neurones and there is evidence that they express at least  <dig> diverse voltage-activated k+ channels and that this expression is in part determined by the type of drg neurone  <cit> .
to distinguish between the two responses experiments were carried out on drg neurones held at - <dig> mv to inactivate a proportion of the outward current.
under these conditions pregabalin inhibited the outward current in  <dig> out of  <dig> drg neurones but still produced enhancement in  <dig> neurones.
the level of inhibition increased at a holding potential of - <dig> mv to  <dig> ± 7%  compared to  <dig> ±  <dig> %  at - <dig> mv, .
interestingly, in apparent contrast to pregabalin,  <dig> μm gabapentin was initially only found to inhibit k+ currents in drg neurones .
however, when studies into long-term  actions of gabapentin were investigated slowly developing outward current enhancement was identified.
so similar to actions of pregabalin, biphasic responses to gabapentin were found with initial inhibition of k+ currents and then a delayed enhancement of the outward current ,  <cit> .
long-term modulation of k+ current by pregabalin was then investigated.
dramatic increases in outward current were observed after a delay.
this effect of pregabalin persisted even as pregabalin was removed from the extracellular environment, which may implicate a metabolic or intracellular signalling event in this response.
the mean k+ current amplitude at + <dig> mv increased from  <dig>  ±  <dig>  na to  <dig>  ±  <dig>  na   <dig> minutes after perfusion of  <dig> μm pregabalin.
figures 12a and 12b show an individual example record and trace of the delayed response to pregabalin.
these responses did reverse but this took about  <dig> minutes with the response developing 3– <dig> minutes after the start of pregabalin application.
no change in holding current or in the leak conductances were associated with the long-term effect of pregabalin and in the absence of pregabalin stable k+ currents were recorded from drg neurones for  <dig> minutes .
several pharmacological experimental approaches were taken to characterise the biphasic actions of pregabalin and to determine the possible mechanism of action associated with the long-term k+ current modulation.
apamin, a toxin from the honeybee, was used to block small conductance ca2+-activated k+ currents.
apamin  caused a reduction in outward current at + <dig> mv from  <dig>  ±  <dig>  na to  <dig>  ±  <dig>  na, subsequent application of  <dig> μm pregabalin enhanced the current to  <dig>  ±  <dig>  na .
no inhibitory effects were seen with pregabalin after treatment with apamin, suggesting that it is the apamin-sensitive ca2+-activated k+ channels that are inhibited by pregabalin .
gabapentin can be transported into cells via the l α-amino acid transporter and may achieve intracellular concentrations 10– <dig> times the levels in the extracellular environment  <cit> .
furthermore, actions through intracellular signalling pathways and specifically protein kinase a, have been proposed for gabapentin  <cit> .
to examine whether the long-term enhancement of k+ currents by pregabalin might involve intracellular sites of action, we applied pregabalin to the intracellular environments of drg neurones via the patch pipette solution.
intracellular pregabalin  evoked an increase in the k+ current that started to develop within the first minute of entering the whole cell recording configuration.
after an initial increase that could reflect equilibration with the kcl-based patch pipette solution containing pregabalin, a slow sustained significant increase in the outward k+ current continued to develop over a  <dig> minute period .
no similar change in k+ current was observed in control studies carried out in the absence of pregabalin .
pertussis toxin pre-treatment results in the uncoupling of sensitive g-proteins from effector mechanisms, including voltage-activated ca2+ and k+ channels.
additionally, pertussis toxin pre-treatment has previously been found to influence gabapentin actions on ica.
pertussis toxin pre-treatment  prevented pregabalin-induced long-term k+ current enhancement in cultured drg neurones .
the possible role of camp-dependent protein kinase a  in the pregabalin-induced enhancement of k+ current was then assessed using the inhibitor -camp  <cit> .
intracellular application of  <dig> μm -camp applied via the kcl-based patch pipette solution had no effect over a  <dig> minute equilibration period on voltage-activated k+ current .
this may indicate that pka has little or no basal or tonic activity on k+ currents in drg neurones in culture.
however, when pregabalin was applied to drg neurones loaded with -camp no long-term enhancement of the outward current was observed.
under these recording conditions pregabalin still produced some inhibition of the k+ current .
these data provide evidence that the long-term modulation of voltage-activated k+ channels by pregabalin is dependent on pka-mediated phosphorylation.
CONCLUSIONS
in conclusion, these results indicate that pregabalin acts via the same basic mechanisms as gabapentin to inhibit voltage-activated ca2+ channels and that these inhibitory actions are independent of gaba receptor activation.
some features of the actions of pregabalin on intermediate size and large drg neurones appear not to be seen with gabapentin.
however, these distinct responses involve enhanced k+-evoked ca2+ transients by pregabalin rather than inhibition of ca2+ channels.
alpha2δ subunits of voltage-activated ca2+ channels remain a possible site of action for both pregabalin and gabapentin.
dooley and colleagues showed that both gabapentin and pregabalin attenuated k+-evoked norepinephrine release from rat neocortical slices by inhibiting p/q-type ca2+ channels  <cit> .
in cortical pyramidal neurones gabapentin predominantly works through l-type ca2+ channels  <cit> .
our work with bay k <dig> indicates that in cultured drg neurones gabapentin  <cit>  and pregabalin act predominantly independently of l-type channels.
a recent study has also indicated that acting via g-protein coupled gabab receptors, gabapentin selectively inhibited n-type ca2+ channels in hippocampal pyramidal neurones  <cit> .
interestingly, this selectivity of gabapentin seen in the hippocampus is different from the ca2+ channel modulation seen with the gabab receptor agonist, baclofen.
our previous investigation using "toxityping" showed that gabapentin inhibited a variety of ca2+ channels in drg neurones  <cit> .
we conclude from all this work that gabapentin and pregabalin may have allosteric interactions with promiscuous α2δ ca2+ channel subunits.
these α2δ subunits are not specifically combined with distinct pore forming ca2+ channel subunits  in all neurones and may therefore inhibit pharmacologically diverse ca2+ channels depending on the expression of ca2+ channel subunits in different neurones.
however, transfection studies have shown that oocytes expressing ca2+ channels  containing β1b and α2δ- <dig> or α2δ- <dig> subunits are insensitive to acute application of  <dig> μm gabapentin  <cit> .
this work raises the possibility that other mechanisms independent of α2δ – ca2+ channel subunits are involved in the modulation of neuronal excitability by gabapentin.
additionally, the modulation of voltage-activated ca2+ channels by gabapentin and pregabalin acting through indirect mechanisms has been suggested.
candidates for such indirect mechanisms include the control of ca2+ channel functional expression  <cit>  and metabotropic mechanisms linked to pertussis toxin sensitivity and pka activation  <cit> .
in hippocampal pyramidal neurones, uncoupling g-proteins from metabotropic receptors with n-ethylmaleimide prevents modulation of k+ and ca2+ channels by gabapentin  <cit> .
however, in drg neurones it is not clear how pertussis toxin influences the actions of gabapentin and pregabalin but it may involve disruption of multi-protein complexes of g-proteins and ion channel subunits after adp-ribosylation of gα.
the role of any g-protein coupled receptors in either gabapentin or pregabalin responses in drg neurones are not supported by our investigations with sch- <dig>  furthermore, receptor and direct g-protein involvement in pregabalin effects on ca2+ channels also appears unlikely in drg neurones.
this is because in this study we found that intracellular flash photolysis of gtp-γ-s had no influence on pregabalin-evoked current inhibition and pregabalin did not alter responses to gtp-γ-s. our findings are in agreement with those made in a previous study on cells expressing gabab1a/b <dig> or gabab1b/b <dig> receptor subunits.
in this study gaba and the gabab receptor agonist baclofen evoked -gtp-γ-s binding responses but even at high concentrations both gabapentin and pregabalin did not  <cit> .
the experiments designed to assess potential roles of gaba receptors in the responses to pregabalin showed that gaba receptor desensitization or the blockade of gabab receptors did not attenuate pregabalin actions.
these findings add to the published studies that indicate that gabapentin and pregabalin effects are independent of gaba receptor activation at least in some preparations  <cit> .
specifically, in cultured drg neurones gabapentin was previously found not to activate a cl- conductance to alter membrane potential and input resistance and the gabab receptor antagonist saclofen did not influence the inhibitory action of gabapentin on ica  <cit> .
pregabalin has a higher affinity for α2δ subunits than gabapentin and in a number of studies is more effective.
when applied alone pregabalin produced enhancement in ca2+ flux in some neurones, an effect not seen with gabapentin.
so, it was surprising to see in the imaging experiments that when gabapentin and pregabalin were applied together they produced only inhibitory effects.
this may reflect the allosteric interactions between these drugs and α2δ subunits of ca2+ channels as well as indirect modulation of ca2+ dependent conductances and interactions with components of cell signalling.
there appear to be some inconsistencies in the pregabalin data when its actions on voltage-activated ca2+ currents are compared with effects on k+-evoked ca2+ transients.
this may in part be due to effects of pregabalin on membrane conductances in addition to voltage-activated ca2+ currents.
these effects may not be detected under voltage clamp recording conditions where na+ and k+ currents are blocked to isolate ca2+ currents.
in the imaging experiments all voltage-activated conductances are intact and may be modulated by pregabalin.
alternatively, pregabalin could potentially have a direct or indirect influence on ca2+-induced ca2+ release from intracellular stores and alter ca2+ homeostatic mechanisms.
these effects may be modulated differently in different sub-populations of drg neurones and so produce the mixed responses recorded in small, intermediate and large neurones in this study.
thus different actions of pregabalin may be detected using fura- <dig> fluorescence imaging but these additional mechanisms may not influence the measurements of ca2+ currents.
although mixed responses to gabapentin were not previously identified, neither were clear effects on the amplitude of k+-evoked ca2+ transients apparent in drg neurones  <cit> , although they were seen in differentiated f- <dig> cells  <cit> .
these apparent anomalies seen with pregabalin do not appear to be due to drug effects on ca2+-induced ca2+ release or ca2+ homeostatic mechanisms such as na+/ca2+ exchange.
this conclusion was reached because pregabalin did not influence ca2+ transients evoked by caffeine and still enhanced k+-evoked ca2+ transients in choline chloride-based  extracellular solution.
the mixed responses to pregabalin seen in the ca2+ imaging experiments prompted us to investigate the actions of pregabalin on voltage-activated k+ currents, which could be modulated to cause an increase in ca2+ influx.
this speculation was supported by the finding that application of the k+ channel inhibitor, tea, enhanced k+-evoked ca2+ transients.
stefani and colleagues have reported inhibition of outward k+ currents by gabapentin  <cit> .
additionally, in rat hippocampal and human neocortical brain slices gabapentin has been shown to inhibit k+-evoked -noradrenaline release.
the activation of katp channels is implicated in these responses because not only does glibenclamide, a katp channel antagonist, attenuate the gabapentin response but also pinacidil, a katp channel agonist, mimics the response and did not have additive effects with gabapentin  <cit> .
mixed or biphasic responses to gabapentin and pregabalin were seen, with both inhibition and enhancement of k+ currents observed.
the inhibitory actions of both gabapentin and pregabalin on k+ currents recorded from drg neurones may not reflect a direct action of these drugs on k+ channels.
the effects of apamin, which prevented the inhibition of outward current by pregabalin indicated that small conductance ca2+-activated k+ channels were involved in the response.
this is consistent with the effects of gabapentin seen in isolated cortical neurones  <cit> .
the pregabalin inhibitory action on voltage-activated ca2+ channels may underlie the inhibition of outward k+ current.
a reduction in ca2+ influx would lead to reduced activation of ca2+-activated k+ channels and therefore a smaller outward current.
if this indirect effect of pregabalin resulted in a disproportionate reduction in ca2+-activated k+ conductances this may result in prolonged depolarisation.
in turn a prolonged depolarisation may maintain activation of ca2+ channels that are insensitive to pregabalin and an increase in ca2+ influx as seen in a sub-population of larger drg neurones in the ca2+ imaging experiments.
it is not clear why gabapentin, which produced modest inhibition of k+ currents, did not also produce enhancement of k+-evoked ca2+ transients.
it may be explained by a balance between inhibition of ca2+ influx through voltage-activated channels and modulation of ca2+ flux through inhibition of k+ conductance.
consistent with this hypothesis, gabapentin appears to be less effective than pregabalin at modulating k+-evoked ca2+ transients in drg neurones.
this is supported by our previous study that showed that gabapentin did not significantly alter the peak ca2+ transients  <cit> .
the delayed enhancement of voltage-activated k+ currents by gabapentin and pregabalin is a powerful mechanism for reducing cell excitability.
the slow development of this response and its gradual decline suggested that intracellular signalling events were involved.
pregabalin produces enhancement of k+ currents when applied either inside or outside drg neurones.
the delay in the development of these responses is less with intracellular application of pregabalin, suggesting that it may have an intracellular site of action.
this effect may also depend on drug uptake into cells via l α-amino acid transporters and the presence and activity of these transporters may greatly influence cell sensitivity to gabapentin and pregabalin.
although it is not clear how gabapentin and pregabalin might activate pka, it is a candidate target site.
our previous work showed that inhibition of ca2+ currents by gabapentin was sensitive to camp analogues that activated or inhibited pka  <cit> .
similarly, in this present study -camp blocked enhancement of k+ currents by pregabalin.
in the literature there are a number of reports of pka altering neuronal excitability and modulating k+ conductances.
pka is involved in pain signalling, playing a role in prostaglandin-induced activation and sensitization of drg neurones  <cit> .
pka activity attenuates kv <dig>  channel currents  <cit>  and a-type current  <cit>  and is involved in the inhibition of k+ conductances by prostaglandin e- <dig>  <cit> .
our results are not explained by these findings but pka has also been implicated in presynaptic inhibition of gaba release  <cit> , large conductance calcium – and voltage-activated k+ channel activity  <cit>  and cannabinoid receptor mediated modulation of ion channels  <cit> .
in the context of our project, of particular interest is the biphasic modulation in retinal cones of voltage dependent k+ and ca2+ channels by a synthetic cannabinoid receptor agonist and the sensitivity of these responses to wiptide, a pka inhibitor  <cit> .
however, the g-protein pharmacology is different in the cone cells suggesting different pka activation pathways in drg neurones exposed to pregabalin.
there appears to be a considerable number of apparently conflicting effects mediated by pka, although a number of different preparations have been studied.
the concept of conditional protein phosphorylation such that initial phosphorylation state affects the sensitivity of different effector targets to subsequent activation  <cit>  could determine responses to gabapentin and pregabalin acting through pka.
a future challenge will be to determine how pka plays roles in activation and sensitization of drg neurones and also plays a role in gabapentin and pregabalin responses to dampen down electrical excitability in the same neurones.
perhaps answers will come when we have a better understanding of the changes in neuronal phenotype that develop with pain disorders?
an interesting feature of some actions of gabapentin and pregabalin is that their effects often outlast the period of drug delivery.
maintained intracellular signalling effects of these drugs and the delayed responses involving pka modulation of ca2+ and k+ conductances may underlie this characteristic.
gabapentin has previously been shown to attenuate high frequency action potential firing both in cns neurones  <cit>  and in cultured drg neurones  <cit> .
pregabalin produces the same effect, dramatically but reversibly reducing the number of action potentials fired in response to  <dig> ms depolarising current commands.
the underlying mechanisms involved in these reductions in electrical excitability could involve inhibition of voltage-activated ca2+ channels with resulting reduced activation of ca2+-activated k+ currents and effects on voltage-dependent ion channel availability.
although the delayed enhancement of voltage-activated k+ channels could contribute, this effect appears to develop over a longer period.
these actions may contribute both to anticonvulsant effects of gabapentin and pregabalin as well as their therapeutic actions in pain disorders.
it is particularly worth noting that the inhibition of ca2+ influx is most consistently seen in small diameter cultured drg neurones that are likely to be the pain fibres.
our data adds to previous work that has assessed the actions of gabapentin and pregabalin on k+-evoked neurotransmitter release in cns neurones  <cit>  and the pharmacology of pain in whole animal models  <cit> .
the present work and these previous studies suggest that gabapentin and pregabalin mostly act through the same basic mechanisms.
these mechanisms in cultured drg neurones appear to involve allosteric inhibitory modulation of ca2+ channels through drug interactions with α2δ subunits, reduced activation of ca2+-dependent conductances and modulation of both ca2+ and k+ channels through metabotropic mechanisms that involve pka.
interestingly, both gabapentin and pregabalin inhibit release of sensory peptides  this may provide a mechanism of action down-stream of ion channel modulation.
this effect is only observed after inflammation and may contribute to their analgesic properties of gabapentin and pregabalin  <cit> .
ca2+ channels in drg neurones are targets for antinociceptive agents but the pharmacology of these channels remains to be fully exploited in pain therapy  <cit> .
further studies on changes in ion channel subunit expression and alterations in intracellular signalling pathways in pain disorders may in the future open up new therapeutic opportunities.

 gabapentin and pregabalin -3-isobutyl gaba) were both originally designed as gaba mimetics, with the intention that they would be able to cross the blood-brain barrier and interact with gabaergic systems and enhance gaba mediated inhibition. although gabapentin appears to have diverse therapeutic utility in the treatment of pain disorders, psychiatric illnesses and epilepsy, there is controversy regarding its molecular mechanisms of action. whether the actions of gabapentin and pregabalin are mediated through gabaergic mechanisms or gaba receptors remains particularly contentious. however, high affinity binding sites for gabapentin and pregabalin on distinct α2δ subunits of voltage-activated calcium channels have been identified and characterised. for this reason voltage-activated ca2+ channels remain primary candidate sites of action for these novel anticonvulsant and antihyperalgesic drugs. functional data from studies on gabapentin and pregabalin also support this contention. specifically, both gabapentin and pregabalin inhibited hyperalgesia, attenuated evoked ca2+ influx into brain slices and reduced evoked transmitter release. additionally, gabapentin and pregabalin inhibited multiple firing of action potentials evoked by ms depolarising current commands in cultured sensory drg neurones. our previous studies have focused on the inhibitory effects of gabapentin on whole cell voltage-activated ca2+ currents and k+ stimulated ca2+ entry measured with fura- the cellular model systems used were primary cultures of dorsal root ganglion neurones from 1– day old rats and differentiated f- cells. in this present study the effects of pregabalin and gabapentin on the electrophysiological properties of cultured neonatal rat drg neurones were measured with particular reference to ca2+ entry through high voltage-activated channels and enhancement of k+ conductances. we had two specific aims for this project. the first was to determine whether gabapentin and pregabalin have the same mechanisms of action, by examining whether saturating concentrations of gabapentin and pregabalin act in an additive manner to attenuate ca2+ influx. the second aim was to further evaluate gaba receptors as target sites for these drugs in sensory neurones. this latter element to the study was conducted because it has been proposed that specific gabab receptors with a gb1a-gb heterodimer composition are the sites of agonist activity of gabapentin. these specific receptors can be coupled to an inwardly rectifying k+ channel and / or voltage-activated ca2+ channels to dampen neuronal electrical excitability. however, in certain situations gabapentin and the gabab receptor agonist, baclofen, have different actions. weaver mutant mice are insensitive to both gabapentin and baclofen, however in control littermates, weaver control mice, only baclofen evoked a k+ current.  actions of pregabalin on voltage-activated calcium currents the firing of multiple action potentials in response to a sustained depolarising current command is a property of a sub-population of cultured drg neurones. application for minutes, of either pregabalin or gabapentin reduced the frequency of action potential spikes evoked by ms depolarising current step commands applied every s. gabapentin attenuated repetitive action potential firing as measured by a reduction in the mean number of evoked action potentials during ms depolarisations from to. pregabalin reversibly reduced the number of action potentials during ms depolarisations from to. however, pregabalin and gabapentin did not significantly alter the properties of single action potentials evoked by ms depolarising current commands. consistent with our previous work, whole cell voltage-activated ca2+ currents recorded from drg neurones were reversibly attenuated by μm gabapentin. similarly, minutes application of pregabalin inhibited the mean ca2+ current amplitude, measured at the peak of the inward current, and at the end of a ms voltage step command to mv. the inhibitory actions of pregabalin were not accompanied by any shift in the voltage-dependence of activation for ica or by any change in holding or leak currents. at least partial recovery of ica was observed minutes after removal of the pregabalin-containing perfusion pipette. a very low concentration of pregabalin failed to produce significant inhibition of ica. however, no clear dose-dependent relationship was established for the inhibitory actions of pregabalin, and a major part of the voltage-activated ca2+ current was insensitive to the drug. it was also apparent that there was considerable variability in the sensitivity of the drg neurones to any given dose of pregabalin. some neurones did not respond to μm pregabalin while in a few neurones this same concentration produced % inhibition of ica. ***p <; **p < and *p <. each value is given ± s.e.m. data shows inhibition but a lack of dose-dependent actions on ica of a wide range of pregabalin concentrations. although varied responses were observed it was clear that a major component of ica was resistant to inhibition by pregabalin. distinct voltage-activated ca2+ channels, defined by their alpha subunit, have been suggested to be selective target sites for gabapentin and related drugs like pregabalin. with this in mind, the -dihydropyridine l-type ca2+ channel agonist bay k was used to determine whether pregabalin was inhibiting l-type channels. previously, we found that in the continual presence of gabapentin, bay k enhanced ica. in this present study similar results were obtained. after inhibiting part of the current with pregabalin, bay k was applied with pregabalin and the enhanced ica was measured at its peak and at the end of a ms voltage step command to mv. the percentage increases in current seen with bay k in the presence of either gabapentin or pregabalin were slightly less than those seen under control conditions in cultured drg neurones. this is consistent with some l-type ca2+ current modulation by pregabalin and gabapentin in drg neurones. taken together these data suggest that neither gabapentin nor pregabalin are selectively inhibiting l-type ica in drg neurones. this contrasts with the effects of gabapentin on cortical pyramidal neurones where inhibition of l-type ca2+ channels appears to be the predominant mechanism of action. actions of pregabalin on calcium influx through voltage-activated channels, measured using fura- imaging given the variable and rather modest but reversible inhibitory actions of pregabalin on ica we tested the actions of pregabalin on k+-evoked ca2+ influx using fura- imaging. an extracellular solution containing mm k+ was used to depolarise the drg neurones and activate three consistent ca2+ transients. pregabalin was applied during the second k+-evoked depolarisation and it produced a mixture of reversible effects on the ca2+ transients. in of neurones the ca2+ flux was decreased by ± % but in neurones from the same cultures pregabalin evoked a mean increase in ca2+ flux to ± % of the control. raising the pregabalin concentration to μm increased the proportion of inhibitory responses but enhancement of ca2+ transients was still observed in the remaining neurones. however, μm pregabalin caused an increase in k+-evoked ca2+ influx in all neurones studied. although in some cells good recovery from pregabalin actions was observed the inhibitory effect was often not associated with recovery. this may reflect long lasting effects of pregabalin. run-down in some cells can not be completely ruled out but under control conditions the mean level of run-down of three k+-evoked ca2+ transients was only ± %. similarly, the mean control level of increase in k+-evoked ca2+ transients was only ± %. to investigate the different responses to pregabalin, the sizes of cell somas were measured and compared. although there is overlap, figure shows that enhancement of k+-evoked ca2+ transients by pregabalin was mainly seen in neurones with larger cell somas and that in smaller neurones pregabalin produced inhibitory effects. ca2+-dependent conductances of drg neurones are sensitive to ryanodine and ca2+-induced ca2+ release has been reported in these neurones. modulation of either ca2+-induced ca2+ release and / or ca2+ homeostatic mechanisms might provide a mechanism by which pregabalin enhanced ca2+ transients in neurones with intermediate and large sized cell somas. this was investigated in two ways. firstly, the actions of pregabalin on caffeine-evoked ca2+ transients were evaluated in nominally ca2+-free extracellular conditions. single caffeine responses were obtained from drg neurones in either the absence or presence of μm pregabalin. no differences in either amplitudes or durations of ca2+ transients were seen when control caffeine responses were compared with caffeine responses obtained in the presence of pregabalin. the second approach was to explore a possible role of na+ / ca2+ exchange by bathing cells in choline chloride-based medium containing only mm na+. under these conditions the contribution of the na+ / ca2+ exchanger to handling of intracellular ca2+ loads will be minimal. in choline chloride-based medium pregabalin produced both enhancement and inhibition of total ca2+ flux in and neurones respectively. enhancement in ca2+ flux by pregabalin under these experimental conditions, suggest that inhibition of na+ / ca2+ exchange is not the main mechanism by which pregabalin enhances k+-evoked ca2+ flux. however, detailed analysis was made difficult by poor recovery of even the first ca2+ transient evoked in low extracellular na+ which does suggest that na+ / ca2+ exchange is an important homeostatic mechanism in drg neurones. in conclusion these results indicate that modulation of ca2+-induced ca2+ release and ca2+ homeostatic mechanisms do not account for pregabalin-induced enhancement of k+-evoked ca2+ flux in a population of drg neurones. inhibitory modulation of potassium conductances could result in increased k+-evoked ca2+ transients. to test this alternative mechanism, a relatively low concentration, mm, of tetraethylammonium was applied with nacl-based extracellular medium and drg neurones were stimulated with mm kcl. attenuation of potassium conductances by tea markedly increased the k+-evoked ca2+ flux in all eight drg neurones studied, mimicking in part the action of pregabalin. do pregabalin and gabapentin have additive effects on cultured dorsal root ganglion neurones? pregabalin and gabapentin have related chemical structures and appear to have similar but usually modest inhibitory effects on ca2+ currents. both pregabalin and gabapentin at a concentration of μm produced a maximum level of current inhibition. simultaneous application of pregabalin and gabapentin produced modest but significant inhibition of ica at the peak of the current and at the end of the stimulus. however, the percentage inhibition of ica produced was not significantly different for μm gabapentin alone, μm pregabalin alone and μm gabapentin and μm pregabalin applied together. in spite of the added complexity of mixed responses with pregabalin, experiments were carried out using fura- to measure k+ evoked ca2+ flux and the effects of both ligands. our previous experiments with gabapentin had only identified inhibitory effects using this experimental approach. simultaneous application of pregabalin and gabapentin reversibly reduced the k+ evoked ca2+ influx. pregabalin and gabapentin together reduced the total ca2+ flux to ± % of the control response to mm k+. consistent with the electrophysiological experiments, this level of inhibition was similar to the inhibitory effects of pregabalin and gabapentin applied separately. the electrophysiological and fura- ca2+ imaging data show that no additive inhibitory effects were found during simultaneous application of saturating concentrations of gabapentin and pregabalin. the data therefore support the contention that both drugs have a closely related or a common site of inhibitory action. the data also indicate that in the modulation of ca2+ channels these drugs do not act in an additive manner even though a substantial proportion of current is resistant to both drugs. it should be emphasized however that pregabalin can reversibly enhance k+-evoked ca2+ transients, an effect not seen with gabapentin. therefore there may be other additional actions of pregabalin that can be identified in ca2+ imaging experiments when all membrane conductances are intact. does gaba receptor modulation alter responses to pregabalin in cultured dorsal root ganglion neurones? in this section of the study two strategies were used to evaluate the possible roles of gaba receptors in pregabalin responses in cultured drg neurones. firstly, pregabalin actions were studied in the presence of a saturating concentration of gaba; this initially activated all types of gaba receptor and then caused desensitization of these receptors. secondly, the effect of a potent and selective gabab receptor antagonist, cgp on pregabalin responses was evaluated. gaba evoked inward currents in a sub-population of cultured drg neurones and induced a transient rise in intracellular ca2+ in of drg neurones studied. the inward currents were due to the activation of gabaa receptor cl- channels; with the equilibrium potential for cl- under our recording conditions being close to mv the chloride conductance results in an inward current. although in vivo the equilibrium potential for cl- in drg neurones is variable, it is predicted to be between - mv and - mv because of cl- loading into the intracellular environment. therefore gabaa receptor cl- channel activation can produce a depolarisation of the resting membrane potential. our data indicated that the gaba-evoked depolarisation was sufficient to result in voltage-activated ca2+ channel activity and produce a transient rise in intracellular ca2+. in both neurones that responded to gaba and those that did not, subsequent application of pregabalin, produced either enhancement or inhibition of k+-evoked ca2+ influx. these responses to pregabalin in the presence of gaba were no different in character to the pregabalin responses obtained in the absence of gaba. in neurones that showed a ca2+ transient in response to a gaba-evoked depolarisation, there was an apparent increase in the proportion of neurones that were inhibited by pregabalin. it is not clear why this is but it may reflect the sensitivity of ca2+ transients evoked in drg neurones, which can only be evoked consistently in most drg neurones by three depolarising stimuli. thus the gaba-evoked responses may influence ca2+ homeostatic mechanisms and subsequent stimulated ca2+ entry. however, the critical observation from these experiments is that gaba receptor desensitisation does not prevent either ca2+ transient enhancement or inhibitory actions of pregabalin in neurones that were sensitive or insensitive to gaba. responders were those neurones in which gaba evoked a transient rise in intracellular ca2+. non-responder showed no change in fura- fluorescence ratio in response to gaba. controls where neurones that pregabalin was applied to without gaba being present. the amplitudes of the gaba responses did not vary with the inhibitory or enhancing responses to pregabalin. the possibility of pregabalin-evoked inhibition of ica taking place through activation of g-protein coupled gabab receptors, was then assessed. cgp when applied alone had no effect on ica. in the presence of the gabab receptor antagonist cgp, pregabalin evoked a ± % inhibition of the voltage-activated ca2+ current and complete recovery was observed minutes after removal of the drug-containing pipette. in ca2+ imaging experiments μm cgp had no effect on the responses to μm pregabalin. in the presence of cgp out of neurones showed enhanced k+-evoked ca2+ influx of control) in response to pregabalin and neurones showed inhibition of ca2+ influx of control) in response to pregabalin. is there a role for g-proteins or g-protein coupled receptors in the responses to pregabalin in cultured dorsal root ganglion neurones? previously, we found that the actions of gabapentin on ica were attenuated by pre-treating drg neurones with pertussis toxin, an effect that did not appear to involve metabotropic gabab receptors. in this study several different approaches were taken to examine the influence of receptor and g-protein function in pregabalin actions. firstly, the effects of a novel thiadiazole compound, sch- which inhibits ligand binding to a variety of g-protein coupled receptors were investigated. the selective and reversible action of sch- appears to involve allosteric modulation of both agonist and antagonist binding to g-protein coupled receptors. secondly, the potential influences of intracellular flash photolysis of caged gtp-γ-s and subsequent g-protein activation on pregabalin responses were also examined. sch- was applied to the intracellular environment via the patch pipette solution. after minutes equilibration the whole cell ca2+ current had a mean amplitude of - ± na. during this period there was a clear reduction in the inward current, although a steady state current level was reached. however, subsequent application of pregabalin resulted in a further significant reduction in ca2+ current amplitude to - ± na. this represents a mean inhibition of ± % by pregabalin, a value very similar to the percentage inhibition produced by pregabalin in the absence of sch-. therefore sch- had no effect on the pregabalin responses, indicating that pregabalin was not acting through an sch-202676-sensitive g-protein coupled receptor. preliminary ca2+ imaging experiments were also conducted to determine whether extracellular sch- altered the effects of pregabalin on k+-evoked ca2+ transients. under these experimental conditions pregabalin was found to markedly enhance or inhibit k+-evoked ca2+ transients. this data again suggests that pregabalin was not acting through mechanisms that were sensitive to sch- caged gtp-γ-s was applied to the intracellular environment via the patch pipette solution and after entering the whole cell recording configuration drg neurones were left for minutes to equilibrate. once a stable control ca2+ current was obtained, three v flashes of intense near uv light were applied to the neurone to achieve intracellular flash photolysis of the caged gtp-γ-s. we estimate that approximately μm gtp-γ-s was photoreleased by the three flashes. as previously observed intracellular flash photolysis of caged gtp-γ-s reduced the amplitude and slowed the activation of ica. the effects of photoreleased gtp-γ-s were attenuated by applying a large depolarising pre-pulse, which is consistent with voltage-dependent g-protein modulation of ca2+ channels. no recovery from photoreleased gtp-γ-s was observed during the period of the experiment. gtp-γ-s and pregabalin had additive inhibitory effects on ica. these additive actions of gtp-γ-s and pregabalin were apparent regardless of the order of application. one set of experiments was performed in which gtp-γ-s was photoreleased in the intracellular environment and then after stabilisation of the response pregabalin was applied. in another set of experiments pregabalin was applied first and after equilibration gtp-γ-s was photoreleased in the continual presence of pregabalin. when pregabalin was applied first it produced a mean inhibition in ica by ± %, when applied after photorelease of gtp-γ-s, pregabalin produced a ± % inhibition of ica. actions of pregabalin on voltage-activated potassium currents it was clear from the ca2+ imaging experiments that not all the cellular actions of pregabalin could be explained by the inhibition of voltage-activated ca2+ channels because both inhibition and enhancement in k+ evoked ca2+ flux was observed. to investigate the actions of pregabalin further, its' effects on voltage-activated k+ currents in drg neurones were studied. previously, stefani and colleagues found that gabapentin modulated neuronal steady state non-inactivating k+ currents. three minutes application of pregabalin had no significant effect on the voltage-activated k+ current activated from a holding potential of - mv by a ms voltage step command to mv. however, raising the pregabalin concentration to μm resulted in significant modulation of k+ currents in drg neurones. pregabalin applied for to minutes produced an enhanced k+ current in out of neurones but a modest inhibition of the outward current in out of neurones. drg neurones are a heterogenous population of neurones and there is evidence that they express at least diverse voltage-activated k+ channels and that this expression is in part determined by the type of drg neurone. to distinguish between the two responses experiments were carried out on drg neurones held at - mv to inactivate a proportion of the outward current. under these conditions pregabalin inhibited the outward current in out of drg neurones but still produced enhancement in neurones. the level of inhibition increased at a holding potential of - mv to ± 7% compared to ± % at - mv,. interestingly, in apparent contrast to pregabalin, μm gabapentin was initially only found to inhibit k+ currents in drg neurones. however, when studies into long-term actions of gabapentin were investigated slowly developing outward current enhancement was identified. so similar to actions of pregabalin, biphasic responses to gabapentin were found with initial inhibition of k+ currents and then a delayed enhancement of the outward current,. long-term modulation of k+ current by pregabalin was then investigated. dramatic increases in outward current were observed after a delay. this effect of pregabalin persisted even as pregabalin was removed from the extracellular environment, which may implicate a metabolic or intracellular signalling event in this response. the mean k+ current amplitude at + mv increased from ± na to ± na minutes after perfusion of μm pregabalin. figures 12a and 12b show an individual example record and trace of the delayed response to pregabalin. these responses did reverse but this took about minutes with the response developing 3– minutes after the start of pregabalin application. no change in holding current or in the leak conductances were associated with the long-term effect of pregabalin and in the absence of pregabalin stable k+ currents were recorded from drg neurones for minutes. several pharmacological experimental approaches were taken to characterise the biphasic actions of pregabalin and to determine the possible mechanism of action associated with the long-term k+ current modulation. apamin, a toxin from the honeybee, was used to block small conductance ca2+-activated k+ currents. apamin caused a reduction in outward current at + mv from ± na to ± na, subsequent application of μm pregabalin enhanced the current to ± na. no inhibitory effects were seen with pregabalin after treatment with apamin, suggesting that it is the apamin-sensitive ca2+-activated k+ channels that are inhibited by pregabalin. gabapentin can be transported into cells via the l α-amino acid transporter and may achieve intracellular concentrations 10– times the levels in the extracellular environment. furthermore, actions through intracellular signalling pathways and specifically protein kinase a, have been proposed for gabapentin. to examine whether the long-term enhancement of k+ currents by pregabalin might involve intracellular sites of action, we applied pregabalin to the intracellular environments of drg neurones via the patch pipette solution. intracellular pregabalin evoked an increase in the k+ current that started to develop within the first minute of entering the whole cell recording configuration. after an initial increase that could reflect equilibration with the kcl-based patch pipette solution containing pregabalin, a slow sustained significant increase in the outward k+ current continued to develop over a minute period. no similar change in k+ current was observed in control studies carried out in the absence of pregabalin. pertussis toxin pre-treatment results in the uncoupling of sensitive g-proteins from effector mechanisms, including voltage-activated ca2+ and k+ channels. additionally, pertussis toxin pre-treatment has previously been found to influence gabapentin actions on ica. pertussis toxin pre-treatment prevented pregabalin-induced long-term k+ current enhancement in cultured drg neurones. the possible role of camp-dependent protein kinase a in the pregabalin-induced enhancement of k+ current was then assessed using the inhibitor -camp. intracellular application of μm -camp applied via the kcl-based patch pipette solution had no effect over a minute equilibration period on voltage-activated k+ current. this may indicate that pka has little or no basal or tonic activity on k+ currents in drg neurones in culture. however, when pregabalin was applied to drg neurones loaded with -camp no long-term enhancement of the outward current was observed. under these recording conditions pregabalin still produced some inhibition of the k+ current. these data provide evidence that the long-term modulation of voltage-activated k+ channels by pregabalin is dependent on pka-mediated phosphorylation.  in conclusion, these results indicate that pregabalin acts via the same basic mechanisms as gabapentin to inhibit voltage-activated ca2+ channels and that these inhibitory actions are independent of gaba receptor activation. some features of the actions of pregabalin on intermediate size and large drg neurones appear not to be seen with gabapentin. however, these distinct responses involve enhanced k+-evoked ca2+ transients by pregabalin rather than inhibition of ca2+ channels. alpha2δ subunits of voltage-activated ca2+ channels remain a possible site of action for both pregabalin and gabapentin. dooley and colleagues showed that both gabapentin and pregabalin attenuated k+-evoked norepinephrine release from rat neocortical slices by inhibiting p/q-type ca2+ channels. in cortical pyramidal neurones gabapentin predominantly works through l-type ca2+ channels. our work with bay k indicates that in cultured drg neurones gabapentin and pregabalin act predominantly independently of l-type channels. a recent study has also indicated that acting via g-protein coupled gabab receptors, gabapentin selectively inhibited n-type ca2+ channels in hippocampal pyramidal neurones. interestingly, this selectivity of gabapentin seen in the hippocampus is different from the ca2+ channel modulation seen with the gabab receptor agonist, baclofen. our previous investigation using "toxityping" showed that gabapentin inhibited a variety of ca2+ channels in drg neurones. we conclude from all this work that gabapentin and pregabalin may have allosteric interactions with promiscuous α2δ ca2+ channel subunits. these α2δ subunits are not specifically combined with distinct pore forming ca2+ channel subunits in all neurones and may therefore inhibit pharmacologically diverse ca2+ channels depending on the expression of ca2+ channel subunits in different neurones. however, transfection studies have shown that oocytes expressing ca2+ channels containing β1b and α2δ- or α2δ- subunits are insensitive to acute application of μm gabapentin. this work raises the possibility that other mechanisms independent of α2δ – ca2+ channel subunits are involved in the modulation of neuronal excitability by gabapentin. additionally, the modulation of voltage-activated ca2+ channels by gabapentin and pregabalin acting through indirect mechanisms has been suggested. candidates for such indirect mechanisms include the control of ca2+ channel functional expression and metabotropic mechanisms linked to pertussis toxin sensitivity and pka activation. in hippocampal pyramidal neurones, uncoupling g-proteins from metabotropic receptors with n-ethylmaleimide prevents modulation of k+ and ca2+ channels by gabapentin. however, in drg neurones it is not clear how pertussis toxin influences the actions of gabapentin and pregabalin but it may involve disruption of multi-protein complexes of g-proteins and ion channel subunits after adp-ribosylation of gα. the role of any g-protein coupled receptors in either gabapentin or pregabalin responses in drg neurones are not supported by our investigations with sch- furthermore, receptor and direct g-protein involvement in pregabalin effects on ca2+ channels also appears unlikely in drg neurones. this is because in this study we found that intracellular flash photolysis of gtp-γ-s had no influence on pregabalin-evoked current inhibition and pregabalin did not alter responses to gtp-γ-s. our findings are in agreement with those made in a previous study on cells expressing gabab1a/b or gabab1b/b receptor subunits. in this study gaba and the gabab receptor agonist baclofen evoked -gtp-γ-s binding responses but even at high concentrations both gabapentin and pregabalin did not. the experiments designed to assess potential roles of gaba receptors in the responses to pregabalin showed that gaba receptor desensitization or the blockade of gabab receptors did not attenuate pregabalin actions. these findings add to the published studies that indicate that gabapentin and pregabalin effects are independent of gaba receptor activation at least in some preparations. specifically, in cultured drg neurones gabapentin was previously found not to activate a cl- conductance to alter membrane potential and input resistance and the gabab receptor antagonist saclofen did not influence the inhibitory action of gabapentin on ica. pregabalin has a higher affinity for α2δ subunits than gabapentin and in a number of studies is more effective. when applied alone pregabalin produced enhancement in ca2+ flux in some neurones, an effect not seen with gabapentin. so, it was surprising to see in the imaging experiments that when gabapentin and pregabalin were applied together they produced only inhibitory effects. this may reflect the allosteric interactions between these drugs and α2δ subunits of ca2+ channels as well as indirect modulation of ca2+ dependent conductances and interactions with components of cell signalling. there appear to be some inconsistencies in the pregabalin data when its actions on voltage-activated ca2+ currents are compared with effects on k+-evoked ca2+ transients. this may in part be due to effects of pregabalin on membrane conductances in addition to voltage-activated ca2+ currents. these effects may not be detected under voltage clamp recording conditions where na+ and k+ currents are blocked to isolate ca2+ currents. in the imaging experiments all voltage-activated conductances are intact and may be modulated by pregabalin. alternatively, pregabalin could potentially have a direct or indirect influence on ca2+-induced ca2+ release from intracellular stores and alter ca2+ homeostatic mechanisms. these effects may be modulated differently in different sub-populations of drg neurones and so produce the mixed responses recorded in small, intermediate and large neurones in this study. thus different actions of pregabalin may be detected using fura- fluorescence imaging but these additional mechanisms may not influence the measurements of ca2+ currents. although mixed responses to gabapentin were not previously identified, neither were clear effects on the amplitude of k+-evoked ca2+ transients apparent in drg neurones, although they were seen in differentiated f- cells. these apparent anomalies seen with pregabalin do not appear to be due to drug effects on ca2+-induced ca2+ release or ca2+ homeostatic mechanisms such as na+/ca2+ exchange. this conclusion was reached because pregabalin did not influence ca2+ transients evoked by caffeine and still enhanced k+-evoked ca2+ transients in choline chloride-based extracellular solution. the mixed responses to pregabalin seen in the ca2+ imaging experiments prompted us to investigate the actions of pregabalin on voltage-activated k+ currents, which could be modulated to cause an increase in ca2+ influx. this speculation was supported by the finding that application of the k+ channel inhibitor, tea, enhanced k+-evoked ca2+ transients. stefani and colleagues have reported inhibition of outward k+ currents by gabapentin. additionally, in rat hippocampal and human neocortical brain slices gabapentin has been shown to inhibit k+-evoked -noradrenaline release. the activation of katp channels is implicated in these responses because not only does glibenclamide, a katp channel antagonist, attenuate the gabapentin response but also pinacidil, a katp channel agonist, mimics the response and did not have additive effects with gabapentin. mixed or biphasic responses to gabapentin and pregabalin were seen, with both inhibition and enhancement of k+ currents observed. the inhibitory actions of both gabapentin and pregabalin on k+ currents recorded from drg neurones may not reflect a direct action of these drugs on k+ channels. the effects of apamin, which prevented the inhibition of outward current by pregabalin indicated that small conductance ca2+-activated k+ channels were involved in the response. this is consistent with the effects of gabapentin seen in isolated cortical neurones. the pregabalin inhibitory action on voltage-activated ca2+ channels may underlie the inhibition of outward k+ current. a reduction in ca2+ influx would lead to reduced activation of ca2+-activated k+ channels and therefore a smaller outward current. if this indirect effect of pregabalin resulted in a disproportionate reduction in ca2+-activated k+ conductances this may result in prolonged depolarisation. in turn a prolonged depolarisation may maintain activation of ca2+ channels that are insensitive to pregabalin and an increase in ca2+ influx as seen in a sub-population of larger drg neurones in the ca2+ imaging experiments. it is not clear why gabapentin, which produced modest inhibition of k+ currents, did not also produce enhancement of k+-evoked ca2+ transients. it may be explained by a balance between inhibition of ca2+ influx through voltage-activated channels and modulation of ca2+ flux through inhibition of k+ conductance. consistent with this hypothesis, gabapentin appears to be less effective than pregabalin at modulating k+-evoked ca2+ transients in drg neurones. this is supported by our previous study that showed that gabapentin did not significantly alter the peak ca2+ transients. the delayed enhancement of voltage-activated k+ currents by gabapentin and pregabalin is a powerful mechanism for reducing cell excitability. the slow development of this response and its gradual decline suggested that intracellular signalling events were involved. pregabalin produces enhancement of k+ currents when applied either inside or outside drg neurones. the delay in the development of these responses is less with intracellular application of pregabalin, suggesting that it may have an intracellular site of action. this effect may also depend on drug uptake into cells via l α-amino acid transporters and the presence and activity of these transporters may greatly influence cell sensitivity to gabapentin and pregabalin. although it is not clear how gabapentin and pregabalin might activate pka, it is a candidate target site. our previous work showed that inhibition of ca2+ currents by gabapentin was sensitive to camp analogues that activated or inhibited pka. similarly, in this present study -camp blocked enhancement of k+ currents by pregabalin. in the literature there are a number of reports of pka altering neuronal excitability and modulating k+ conductances. pka is involved in pain signalling, playing a role in prostaglandin-induced activation and sensitization of drg neurones. pka activity attenuates kv channel currents and a-type current and is involved in the inhibition of k+ conductances by prostaglandin e-. our results are not explained by these findings but pka has also been implicated in presynaptic inhibition of gaba release, large conductance calcium – and voltage-activated k+ channel activity and cannabinoid receptor mediated modulation of ion channels. in the context of our project, of particular interest is the biphasic modulation in retinal cones of voltage dependent k+ and ca2+ channels by a synthetic cannabinoid receptor agonist and the sensitivity of these responses to wiptide, a pka inhibitor. however, the g-protein pharmacology is different in the cone cells suggesting different pka activation pathways in drg neurones exposed to pregabalin. there appears to be a considerable number of apparently conflicting effects mediated by pka, although a number of different preparations have been studied. the concept of conditional protein phosphorylation such that initial phosphorylation state affects the sensitivity of different effector targets to subsequent activation could determine responses to gabapentin and pregabalin acting through pka. a future challenge will be to determine how pka plays roles in activation and sensitization of drg neurones and also plays a role in gabapentin and pregabalin responses to dampen down electrical excitability in the same neurones. perhaps answers will come when we have a better understanding of the changes in neuronal phenotype that develop with pain disorders? an interesting feature of some actions of gabapentin and pregabalin is that their effects often outlast the period of drug delivery. maintained intracellular signalling effects of these drugs and the delayed responses involving pka modulation of ca2+ and k+ conductances may underlie this characteristic. gabapentin has previously been shown to attenuate high frequency action potential firing both in cns neurones and in cultured drg neurones. pregabalin produces the same effect, dramatically but reversibly reducing the number of action potentials fired in response to ms depolarising current commands. the underlying mechanisms involved in these reductions in electrical excitability could involve inhibition of voltage-activated ca2+ channels with resulting reduced activation of ca2+-activated k+ currents and effects on voltage-dependent ion channel availability. although the delayed enhancement of voltage-activated k+ channels could contribute, this effect appears to develop over a longer period. these actions may contribute both to anticonvulsant effects of gabapentin and pregabalin as well as their therapeutic actions in pain disorders. it is particularly worth noting that the inhibition of ca2+ influx is most consistently seen in small diameter cultured drg neurones that are likely to be the pain fibres. our data adds to previous work that has assessed the actions of gabapentin and pregabalin on k+-evoked neurotransmitter release in cns neurones and the pharmacology of pain in whole animal models. the present work and these previous studies suggest that gabapentin and pregabalin mostly act through the same basic mechanisms. these mechanisms in cultured drg neurones appear to involve allosteric inhibitory modulation of ca2+ channels through drug interactions with α2δ subunits, reduced activation of ca2+-dependent conductances and modulation of both ca2+ and k+ channels through metabotropic mechanisms that involve pka. interestingly, both gabapentin and pregabalin inhibit release of sensory peptides this may provide a mechanism of action down-stream of ion channel modulation. this effect is only observed after inflammation and may contribute to their analgesic properties of gabapentin and pregabalin. ca2+ channels in drg neurones are targets for antinociceptive agents but the pharmacology of these channels remains to be fully exploited in pain therapy. further studies on changes in ion channel subunit expression and alterations in intracellular signalling pathways in pain disorders may in the future open up new therapeutic opportunities. 
gabapentin, pregablin and gaba can all modulate voltage-activated ca2+ channels. the aims of this study were to determine whether pregabalin and gabapentin had additive inhibitory effects on high voltage-activated ca2+ channels, evaluate whether the actions of pregabalin were dependent on gaba receptors and characterise the actions of pregabalin on voltage-activated potassium currents. the residual ca2+ current recorded in the presence of pregabalin was sensitive to the l-type ca2+ channel modulator, bay k saturating concentrations of gabapentin failed to have additive effects when applied with pregabalin, indicating that these two compounds act on the same type of voltage-activated ca2+ channels but the majority of ca2+ current was resistant to both drugs. pregabalin produced biphasic effects on voltage-activated k+ currents, the inhibitory effect of pregabalin was prevented with apamin. the delayed enhancement of k+ currents was attenuated by pertussis toxin and by intracellular application of a -analogue of camp. the actions of pregabalin may involve both extracellular and intracellular drug target sites and modulation of a variety of neuronal conductances, by direct interactions, and through intracellular signalling involving protein kinase a. the enhanced ca2+ influx evoked by pregabalin was mimicked by partial block of k+ conductances with tetraethylammonium. pregabalin reduces excitatory properties of cultured drg neurones by modulating voltage-activated ca2+ and k+ channels. pregabalin inhibited 20–30% of the high voltage-activated ca2+ current in cultured drg neurones. in this study we have used whole cell patch clamp recording and fura- ca2+ imaging to characterise the actions of pregabalin on the electrophysiological properties of cultured dorsal root ganglion neurones from neonatal rats. the continual application of gaba, the gabab receptor antagonist cgp or intracellular photorelease of gtp-γ-s had no effect on pregabalin-induced inhibition of ca2+ currents.
 gabapentin and pregabalin have wide-ranging therapeutic actions, and are structurally related to the inhibitory neurotransmitter gaba. gabapentin, pregablin and gaba can all modulate voltage-activated ca2+ channels. in this study we have used whole cell patch clamp recording and fura- ca2+ imaging to characterise the actions of pregabalin on the electrophysiological properties of cultured dorsal root ganglion neurones from neonatal rats. the aims of this study were to determine whether pregabalin and gabapentin had additive inhibitory effects on high voltage-activated ca2+ channels, evaluate whether the actions of pregabalin were dependent on gaba receptors and characterise the actions of pregabalin on voltage-activated potassium currents.  pregabalin inhibited 20–30% of the high voltage-activated ca2+ current in cultured drg neurones. the residual ca2+ current recorded in the presence of pregabalin was sensitive to the l-type ca2+ channel modulator, bay k saturating concentrations of gabapentin failed to have additive effects when applied with pregabalin, indicating that these two compounds act on the same type of voltage-activated ca2+ channels but the majority of ca2+ current was resistant to both drugs. the continual application of gaba, the gabab receptor antagonist cgp or intracellular photorelease of gtp-γ-s had no effect on pregabalin-induced inhibition of ca2+ currents. although clear inhibition of ca2+ influx was produced by pregabalin in a population of small neurones, a significant population of larger neurones showed enhanced ca2+ influx in response to pregabalin. the enhanced ca2+ influx evoked by pregabalin was mimicked by partial block of k+ conductances with tetraethylammonium. pregabalin produced biphasic effects on voltage-activated k+ currents, the inhibitory effect of pregabalin was prevented with apamin. the delayed enhancement of k+ currents was attenuated by pertussis toxin and by intracellular application of a -analogue of camp.  pregabalin reduces excitatory properties of cultured drg neurones by modulating voltage-activated ca2+ and k+ channels. the pharmacological activity of pregabalin is similar but not identical to that of gabapentin. the actions of pregabalin may involve both extracellular and intracellular drug target sites and modulation of a variety of neuronal conductances, by direct interactions, and through intracellular signalling involving protein kinase a. 