VGB is an anti-epileptic agent that is reported to be an inhibitor of gamma-aminobutyric acid breakdown. It has been approved for use as an adjunctive treatment for resistant epilepsy, and as a monotherapy for infantile spasms or West syndrome [2, 3]. In the current study, we found that VGB concentration-dependently reduced the probability of IKCa-channel openings, and that this reduction in channel activity is closely associated with an increase in mean closed time of the channel. The reduction of the channel open probability accounts mostly for its suppression in IKCa-channel activity, in spite of the failure to modify single-channel conductance of those IKCa channels. However, neither the activity of BKca nor that of Kir channels was perturbed by the presence of VGB. Therefore, in addition to the inhibition of GABA breakdown, this study revealed that VGB suppressed the activity of IKCa channels. This effect could be partly, if not entirely, responsible for its suppression of neoplastic cells [24]. Therefore, caution needs to be appropriately exercised when the effect of this compound is explained solely by its action on GABA-ergic dysregulation [16].
The biophysical properties of IKCa channels identified from human glioma cells (13-06-MG) in this study, including unitary currents displaying inward rectification and 32 pS in single-channel conductance, are consistent with most other studies [7, 14, 15, 25], but the single-channel conductance is apparently less than that of BKCa channels [26, 27]. VGB-mediated inhibition of IKCa-channel activity depends on membrane voltage and it is thought to occur via a direct interaction with the KCa3.1 channel protein in glioma cells.
In this study, the IC50 value required for VGB-induced inhibition of IKCa channels was 4.21 µM. There was noticed to be a wide range of serum/plasma concentrations (i.e., 0.8–36 mg/L) associated with successful epilepsy treatment [28]. The concentration in cerebrospinal fluid was found to be approximately 30–40% of plasma concentration, supporting that the IC50 value of VGB shown in the current study could be of clinical relevance. The presence of VGB inhibits IKCa channels in humans at these relatively low concentrations, and in contrast to other GABA compounds, it is lipophilic and able to cross the blood-brain barrier [29]. Therefore, findings from the present observations are novel and could be important in determining VGB’s in vivo anti-neoplastic mechanism.
Different types of kinetic behaviors perturbed by VGB might facilitate its inhibition of IKCa-channel activity. VGB has no apparent effect on IKCa single-channel conductance; therefore, the VGB molecule is most unlikely to act within the channel’s central pore. However, the mean closed time of the channel was noticeably lengthened in its presence. Therefore, it is likely that VGB-mediated inhibition of IKCa channels is characterized by a greater affinity for the IKCa channel in the closed (or resting) state. The activity of IKCa channels has been previously reported to regulate the proliferation of prostate cancer cells by controlling Ca2+ entry into these cells [11]. However, significant changes in neither BKCa- nor Kir-channel activity were observed. The effectiveness of VGB in inhibiting IKCa channels demonstrated here in glioma cells does not arise secondary to the reduction of intracellular Ca2+ [21]. In the present study, VGB inhibited IKCa-channel activity within a few minutes in the 13-06-MG cells. As the onset of inhibition was rapid, its action on channel activity was most unlikely to result from the binding to nuclear DNAs. The mechanism through which the VGB molecule binds to and then interact with IKCa channels tends to be direct and not in a genomic fashion, despite the detailed mechanism of VGB action remains to be further resolved.
Earlier studies, in which immunolabelling of KCa3.1 channels was performed, disclosed that IKCa channels have a tendency to be expressed differentially in either excitatory or inhibitory neurons [14, 25], where it plays a major role in a variety of cellular functions. Different isoforms of KCa3.1 might be present in various types of body tissue, including gliomas. Whether VGB is capable of modifying different subtypes of IKCa channels and the extent to which VGB-induced effects on glioma cells may result from direct inhibitory perturbations on the probability and gating kinetics of IKCa channels, are thus imperatively warranted.
Interestingly, one in vitro study implied that VGB should not be used for prophylaxis or the short-term treatment of epilepsy in glioblastoma [24]. However, another study suggested that blocking GABA flux into the TCA cycle, either through genetic depletion of GAD1 or pharmacological treatment with VGB, significantly suppressed aggressive metastatic outgrowth in the brain. Furthermore, it has been shown that VGB might bring an additional benefit of stabilizing tumor-induced seizures [17]. Moreover, our previous study on temozolomide, which demonstrated its inhibitory effect on IKCa accompanied by membrane depolarization, could account for an important underlying mechanism of temozolomide-induced antineoplastic actions [21].