| Abstract No.: | A-B1029 |
| Country: | Canada |
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| Title: | DYNAMIC MODULATION OF A CATION CHANNEL IN APLYSIA BAG CELL NEURONS. |
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| Authors/Affiliations: | 1 Kate Gardam*; 1 Neil Magoski;
1 Queens University, Kingston, ON, Canada
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| Content: | Objectives: Nonselective cation channels are found throughout the brain, where they determine excitability and activity by setting resting membrane potential. Because cation channels are present in many vertebrate and invertebrate neurons, it is possible to make universally applicable findings in a specific system. The activation of a cation channel in the bag cell neurons of Aplysia californica mediates the depolarization driving a 30-minute afterdischarge of action potential firing and egg-laying hormone secretion. Our objective was to determine the modulatory effects of inositol trisphosphate (IP3) and phosphorylation by protein kinase C (PKC), on channel voltage- and Ca2+-dependence, in order to establish the state of the channel during different phases of the afterdischarge.
Materials and Methods: Cultured bag cell neurons from adult Aplysia (150-300 g) were used after 1-3 d in vitro. Single-channel, excised, inside-out patch clamp recordings were obtained with a bath solution approximating intracellular saline and a pipette solution of artificial seawater. In experiments examining voltage-dependence, the free [Ca2+] of the intracellular saline was 10 uM. In experiments examining Ca2+-dependence, the cytosolic face of the patch was exposed to varying [Ca2+]. In all cases, current was low-pass filtered at 1 kHz and sampled at 10 kHz.
Results: It has previously been shown that PKC can directly associate with the channel, and PKC-mediated phosphorylation increases channel activity. Application of 1 mM ATP (as a phosphate source) to the cytoplasmic face of the patch when the channel was PKC-associated left-shifted the voltage-dependence, as indicated by a change in Vhalf of -3 mV. The shift was more pronounced at hyperpolarized potentials, i.e., the physiological range of channel activity (-30 to -60 mV). Interestingly, phosphorylation by PKC had the opposite effect on Ca2+-dependence, shifting the EC50 from 5 uM to 30 uM. Application of 5 uM IP3 to the cytoplasmic face of the channel drastically right-shifted the Ca2+-dependence of the channel. This effect was largely reversed by PKC-dependent phosphorylation. Thus, depending on phosphorylation state, IP3 may or may not be inhibitory. Furthermore, in a subset of animals examined during the reproductive off-season, voltage-dependence was right-shifted under standard conditions – suggesting reduced Ca2+-dependent modulation of the voltage-sensor and possible meta-regulation relating to seasonality.
Conclusion: Modulation of the cation channel by molecules that are present during different phases of the afterdischarge suggests its role changes throughout the phenomenon. During the initial phase of the afterdischarge, phosphorylation by PKC may act to increase channel activity by left-shifting voltage-dependence. Later in the afterdischarge, as IP3 builds up due to its synthesis by activated phospholipase C, the Ca2+-dependence of the cation channel is drastically right-shifted, acting to decrease channel activity, and possibly preventing an interminable afterdischarge that may be excitotoxic. The roles of these modulators overlap and oppose each other, creating a sophisticated mechanism to precisely control channel activity.
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