| Abstract No.: | C-B3023 |
| Country: | Canada |
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| Title: | T-TYPE CALCIUM CHANNELS PROVIDE CALCIUM-DEPENDENT MODULATION OF A-TYPE POTASSIUM CHANNELS |
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| Authors/Affiliations: | 1 Mircea Iftinca*; 1 Dustin Anderson; 1 Renata Rehak; 1 Michael Molineux; 1 John McRory; 1 Gerald Zamponi; 1 Raymond Turner;
1 Hotchkiss Brain Institute, Calgary, AB, Canada
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| Content: | Objective: The timing of neuronal spike output reflects a delicate balance between inward and outward currents. We have shown that T-type (Cav3) calcium and A-type (Kv4) potassium channels in cerebellar stellate cells co-modulate first spike latency (FSL) following membrane hyperpolarizations. Kv4 channels exist as a complex with potassium channel interacting proteins (KChiPs), a calcium sensor protein that binds calcium, and dipeptidyl peptidase (DPP). While the kinetic properties of Kv4 channels have been well characterized for different combinations of Kv4 complex subunits, the source of calcium capable of modulating A-type current and the functional outcome has not been identified. The current project tested the Hypothesis: Cav3 T-type calcium channels provide the calcium influx necessary to modulate Kv4 potassium channel properties and FSL in cerebellar stellate cells.
Methods and Materials: Cerebellar slices were prepared from P16-20 rats to be maintained at 34°C in vitro and patch clamp recordings obtained from stellate cells. FSL was measured following a series of 500 ms hyperpolarizing steps from rest down to -100 mV. cDNA for Cav3, Kv4, KChiP and DPP channel subunits were co-expressed in tsA-201 (HEK) cells for voltage-clamp analysis.
Results: Stellate cells exhibit a non-monotonic FSL relationship to membrane hyperpolarizations, with an A-type mediated peak near -72 mV and shorter FSLs for more negative step potentials that is T-type mediated. The T-type channel blocker Mibefradil (0.5 µM) removed the short FSL evoked at negative potentials, but also substantially reduced the A-type mediated peak FSL from ~140 ms to 40 ms, suggesting that T-type calcium current normally increases A-type current magnitude. Voltage-clamp recordings of stellate cell Kv4 current revealed that Mibefradil shifted the Kv4 half-inactivation voltage from -74 ±1.92 to -83.5 ± 3.99 mV, increased the tau for inactivation. None of these effects were induced by Mibefradil on Kv4.2 or Kv4.3 current expressed in isolation in HEK cells. Immunocytochemistry revealed that stellate cells express Kv4.2, Kv4.3, KChiP2 / 3, and DPP10 subunits. Moreover, Cav3 channel co-immunoprecipitated with the Kv4 channel complex on Western Blots of brain homogenates, revealing a close association between Cav3 calcium and Kv4 potassium channels. Experiments co-expressing Cav3 and Kv4 complex subunits in HEK cells show that Cav3 calcium influx is sufficient to provide a calcium- and KChiP-dependent modulation of Kv4 channel properties.
Conclusion: Cav3 calcium channels form a close association with the Kv4 potassium channel complex to provide novel forms of calcium-dependent regulation of Kv4 channel properties and function.
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