Flufenamic acid (FFA) is a nonsteroidal antiinflammatory agent, commonly used to block nonselective cation channels. We previously reported that FFA potentiated, rather than inhibited, a cation current in Aplysia bag cell neurons. Prompted by this paradoxical result, the present study examined the effects of FFA on membrane currents and intracellular Ca2+ in cultured bag cell neurons. Under whole cell voltage clamp, FFA evoked either outward (I out) or inward (I in) currents. I out had a rapid onset, was inhibited by the K+ channel blocker, tetraethylammonium, and was associated with both an increase in membrane conductance and a negative shift in the whole cell current reversal potential. I in developed more slowly, was inhibited by the cation channel blocker, Gd3+, and was concomitant with both an increased conductance and positive shift in reversal potential. FFA also enhanced the use-dependent inactivation and caused a positive-shift in the activation curve of the voltage-dependent Ca2+ current. Furthermore, as measured by ratiometric imaging, FFA produced a rise in intracellular Ca2+ that persisted in the absence of extracellular Ca2+ and was reduced by depleting either the endoplasmic reticulum and/or mitochondrial stores. Ca2+ appeared to be involved in the activation of I in, as strong intracellular Ca2+ buffering effectively eliminated I in but did not alter I out. Finally, the effects of FFA were likely not due to block of cyclooxygenase given that the general cyclooxygenase inhibitor, indomethacin, failed to evoke either current. That FFA influences a number of neuronal properties needs to be taken into consideration when employing it as a cation channel antagonist.
Ion channel regulation is key to controlling neuronal excitability. However, the extent that modulators and gating factors interact to regulate channels is less clear. For Aplysia, a nonselective cation channel plays an essential role in reproduction by driving an afterdischarge in the bag cell neurons to elicit egg-laying hormone secretion. We examined the regulation of cation channel voltage and Ca2+ dependence by protein kinase C (PKC) and inositol trisphosphate (IP3)-two prominent afterdischarge signals. In excised, inside-out patches, the channel remained open longer and reopened more often with depolarization from -90 to +30 mV. As previously reported, PKC could closely associate with the channel and increase activity at -60 mV. We now show that, following the effects of PKC, voltage dependence was shifted to the left (essentially enhanced), particularly at more negative voltages. Conversely, the voltage dependence of channels lacking PKC was shifted to the right (essentially suppressed). Predictably, activity was increased at all Ca2+ concentrations following the effects of PKC; nevertheless, Ca2+ dependence was actually shifted to the right. Moreover, whereas IP3 did not alter activity at -60 mV, it drastically shifted Ca2+ dependence to the right-an outcome largely reversed by PKC. With respect to the afterdischarge, these data suggest PKC initially upregulates the channel by direct gating and shifting voltage dependence to the left. Subsequently, PKC and IP3 attenuate the channel by suppressing Ca2+ dependence. This ensures hormone delivery by allowing afterdischarge initiation and maintenance but also prevents interminable bursting. Similar regulatory interactions may be used by other neurons to achieve diverse outputs.
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