Amir Paydar
Rhodes College
Memphis, TN
Faculty Mentor: Dr. Michelle Mynlieff

The GABA-B receptor pathway has been conjectured to regulate neurotransmitter release by modulating calcium currents (ICa2+) in both excitatory and inhibitory neurons located in the superior region of the hippocampus. Upon activation, this metabotropic pathway controls calcium influx through presynaptic high voltage activated (HVA) calcium channels (chiefly N- and L-type). The GABA-B receptor pathway is known to distinctively attenuate calcium conductance through N-type channels and facilitate, or increase, the currents that permeate through L-type channels. This study investigated the specific intracellular pathways through which GABA-B receptor activation inhibits and facilitates N- and L-type calcium channels respectively, in cultured hippocampal neurons. Many intracellular mechanisms for both N-type inhibition and L-type facilitation of ICa2+ have been observed and proposed in different neuronal systems, all of which involve transmembrane G-protein activation. Different subtypes of G-proteins are speculated to mediate this pathway either by direct allosteric coupling with the calcium channels or, alternatively, by amplification or depression of second messenger molecules (e.g. cAMP and DAG), possibly resulting in an increase in kinase (e.g. PKA and PKC) activity.

Whole cell patch clamp recording in the voltage clamp mode was carried out on cultured hippocampal neurons of 5 to 8 day old rat pups. Calcium currents were elicited by a series of 300ms depolarizing pulses from –50mV to +50mV in 10mV increments from a holding potential of –80mV. Baclofen was perfused to specifically activate the GABA-B receptors during a control experiment. Subsequently, various pharmacological compounds were used to address the baclofen-activated GABA-B intracellular second messenger system that modulates the N- and L-type ICa2+. Former investigations of GABA-B modulation of ICa2+ in heterogeneous hippocampal cell populations identified 15% of cells being non-responsive to baclofen, 54% showing inhibition, and 30% exhibiting facilitation under normal physiological conditions. These different effects might reflect the complement of Ca2+ channels in different cell types. Results from similar control conditions carried out in this study included 2 cells showing no statistically significant response to the application of baclofen (-3.33%), 6 cells exhibiting inhibition (-27.60% ± 2.16), and 1 cell displaying facilitation of calcium currents (31.73%). Subsequently, 8 cells were incubated overnight with pertussis toxin (PTX), which permanently inactivates the Go/Gi _-subunit. Six cells exhibited no response to baclofen (-5.25% ± 2.42), 0 cells showed inhibition, and 2 cells displayed facilitation (29.33%). In a separate study, RP-cAMP (PKA inhibitor) was applied to 9 cells by intracellular dialysis to test PKA’s involvement in the GABA-B receptor pathway. Two of these cells showed no response to baclofen (-2.26%), 4 cells showed inhibition (-24.21% ± 5.28), and 3 cells displayed facilitation (26.08% ± 2.51).

The data suggest that N-type inhibition of ICa2+ was hindered by PTX’s inactivating Go_/Gi__protein subunits, verifying that N-type inhibition is probably mediated by Gi__or Go_ rather than by Gs_, Gq_, or G____subunits. L-type facilitation of ICa2+, however, was not affected by PTX application, suggesting that facilitation does not occur through a PTX-sensitive Gi/Go subtype. Moreover, neither inhibition nor facilitation was affected by RP-cAMP’s PKA blocking capacity. Thus, according to these data, it is likely that kinase activity by PKA in particular, does not play a major role in intracellular modulation of ICa2+ by GABA-B receptors in hippocampal neurons. In conclusion, it can be postulated that inhibition of ICa2+ by activated GABA-B receptors is most likely through direct allosteric coupling of Go proteins with N-type calcium channels. In addition, GABA-B ICa2+ facilitation may be primarily linked to PKC kinase activity that leads to activation of L-type channels through internal phosphorylation. More studies need to be performed to confirm these results. Furthermore, other pharmacological manipulations need to be carried out to eliminate other possibilities of how the GABA-B receptor modulates these HVA ICa2+ in hippocampal neurons.