Limits...
Mechanism of arachidonic acid modulation of the T-type Ca2+ channel alpha1G.

Talavera K, Staes M, Janssens A, Droogmans G, Nilius B - J. Gen. Physiol. (2004)

Bottom Line: Here we analyze the effects of AA on the T-type Ca(2+) channel alpha(1G) heterologously expressed in HEK-293 cells.AA induced a slight increase of activation near the threshold and did not significantly change the deactivation kinetics or the rectification pattern.Model simulations indicate that AA inhibits T-type currents by switching the channels into a nonavailable conformation and by affecting transitions between inactivated states, which results in the negative shift of the inactivation curve.

View Article: PubMed Central - PubMed

Affiliation: Laboratorium voor Fysiologie, KU Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. karel.talavera@med.kuleuven.ac.be

ABSTRACT
Arachidonic acid (AA) modulates T-type Ca(2+) channels and is therefore a potential regulator of diverse cell functions, including neuronal and cardiac excitability. The underlying mechanism of modulation is unknown. Here we analyze the effects of AA on the T-type Ca(2+) channel alpha(1G) heterologously expressed in HEK-293 cells. AA inhibited alpha(1G) currents within a few minutes, regardless of preceding exposure to inhibitors of AA metabolism (ETYA and 17-ODYA). Current inhibition was also observed in cell-free inside-out patches, indicating a membrane-delimited interaction of AA with the channel. AA action was consistent with a decrease of the open probability without changes in the size of unitary currents. AA shifted the inactivation curve to more negative potentials, increased the speed of macroscopic inactivation, and decreased the extent of recovery from inactivation at -80 mV but not at -110 mV. AA induced a slight increase of activation near the threshold and did not significantly change the deactivation kinetics or the rectification pattern. We observed a tonic current inhibition, regardless of whether the channels were held in resting or inactivated states during AA perfusion, suggesting a state-independent interaction with the channel. Model simulations indicate that AA inhibits T-type currents by switching the channels into a nonavailable conformation and by affecting transitions between inactivated states, which results in the negative shift of the inactivation curve. Slow-inactivating alpha(1G) mutants showed an increased affinity for AA with respect to the wild type, indicating that the structural determinants of fast inactivation are involved in the AA-channel interaction.

Show MeSH

Related in: MedlinePlus

Effects of 3 μM AA on the kinetics of inactivation from closed states. (A and B) Example currents elicited by pulses to −20 mV, applied after steps of variable duration to −85 and −75 mV, respectively. The thick traces correspond to currents elicited from a holding potential of −110 mV. Thin and dashed traces correspond to currents recorded after 200 ms and 5 s–lasting prepulses (to −85 or −75 mV), respectively. For each condition, the traces were superimposed to allow better comparison. (C) Average time dependence of onset of inactivation at −85 (squares; n = 9) and −75 mV (circles; n = 7) in control (filled symbols) and in the presence of AA (open symbols). Continuous lines represent the fit with the kinetic model described in discussion. The inset shows the voltage protocol used. (D) The datasets shown in panel C were normalized to the values obtained at 5 s for −85 (left) and −75 mV (right).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2233885&req=5

fig5: Effects of 3 μM AA on the kinetics of inactivation from closed states. (A and B) Example currents elicited by pulses to −20 mV, applied after steps of variable duration to −85 and −75 mV, respectively. The thick traces correspond to currents elicited from a holding potential of −110 mV. Thin and dashed traces correspond to currents recorded after 200 ms and 5 s–lasting prepulses (to −85 or −75 mV), respectively. For each condition, the traces were superimposed to allow better comparison. (C) Average time dependence of onset of inactivation at −85 (squares; n = 9) and −75 mV (circles; n = 7) in control (filled symbols) and in the presence of AA (open symbols). Continuous lines represent the fit with the kinetic model described in discussion. The inset shows the voltage protocol used. (D) The datasets shown in panel C were normalized to the values obtained at 5 s for −85 (left) and −75 mV (right).

Mentions: The results described above indicate that AA speeds up the transition from the open to the inactivated state, but give no clues about the possible effect of AA on the rate of inactivation from closed states. We therefore studied the effects of AA on the time course of the onset of inactivation at potentials below the threshold of channel activation. This was achieved by measuring the amplitude of the currents elicited by pulses to −20 mV, applied after steps of variable duration to −85 or −75 mV (Fig. 5 C, inset). These amplitudes were normalized to that of the current elicited with a pulse to −20 mV from a holding potential of −110 mV. The data were then subtracted from 1 and plotted as a function of the time of sojourn at −85 or −75 mV. Fig. 5 shows that AA increased the steady-state level of inactivation at both −85 and −75 mV, in accordance with the hyperpolarizing shift of the inactivation curve. However, AA only slightly increased the rate of inactivation from closed states, as shown by the comparison of normalized data (Fig. 5 D).


Mechanism of arachidonic acid modulation of the T-type Ca2+ channel alpha1G.

Talavera K, Staes M, Janssens A, Droogmans G, Nilius B - J. Gen. Physiol. (2004)

Effects of 3 μM AA on the kinetics of inactivation from closed states. (A and B) Example currents elicited by pulses to −20 mV, applied after steps of variable duration to −85 and −75 mV, respectively. The thick traces correspond to currents elicited from a holding potential of −110 mV. Thin and dashed traces correspond to currents recorded after 200 ms and 5 s–lasting prepulses (to −85 or −75 mV), respectively. For each condition, the traces were superimposed to allow better comparison. (C) Average time dependence of onset of inactivation at −85 (squares; n = 9) and −75 mV (circles; n = 7) in control (filled symbols) and in the presence of AA (open symbols). Continuous lines represent the fit with the kinetic model described in discussion. The inset shows the voltage protocol used. (D) The datasets shown in panel C were normalized to the values obtained at 5 s for −85 (left) and −75 mV (right).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233885&req=5

fig5: Effects of 3 μM AA on the kinetics of inactivation from closed states. (A and B) Example currents elicited by pulses to −20 mV, applied after steps of variable duration to −85 and −75 mV, respectively. The thick traces correspond to currents elicited from a holding potential of −110 mV. Thin and dashed traces correspond to currents recorded after 200 ms and 5 s–lasting prepulses (to −85 or −75 mV), respectively. For each condition, the traces were superimposed to allow better comparison. (C) Average time dependence of onset of inactivation at −85 (squares; n = 9) and −75 mV (circles; n = 7) in control (filled symbols) and in the presence of AA (open symbols). Continuous lines represent the fit with the kinetic model described in discussion. The inset shows the voltage protocol used. (D) The datasets shown in panel C were normalized to the values obtained at 5 s for −85 (left) and −75 mV (right).
Mentions: The results described above indicate that AA speeds up the transition from the open to the inactivated state, but give no clues about the possible effect of AA on the rate of inactivation from closed states. We therefore studied the effects of AA on the time course of the onset of inactivation at potentials below the threshold of channel activation. This was achieved by measuring the amplitude of the currents elicited by pulses to −20 mV, applied after steps of variable duration to −85 or −75 mV (Fig. 5 C, inset). These amplitudes were normalized to that of the current elicited with a pulse to −20 mV from a holding potential of −110 mV. The data were then subtracted from 1 and plotted as a function of the time of sojourn at −85 or −75 mV. Fig. 5 shows that AA increased the steady-state level of inactivation at both −85 and −75 mV, in accordance with the hyperpolarizing shift of the inactivation curve. However, AA only slightly increased the rate of inactivation from closed states, as shown by the comparison of normalized data (Fig. 5 D).

Bottom Line: Here we analyze the effects of AA on the T-type Ca(2+) channel alpha(1G) heterologously expressed in HEK-293 cells.AA induced a slight increase of activation near the threshold and did not significantly change the deactivation kinetics or the rectification pattern.Model simulations indicate that AA inhibits T-type currents by switching the channels into a nonavailable conformation and by affecting transitions between inactivated states, which results in the negative shift of the inactivation curve.

View Article: PubMed Central - PubMed

Affiliation: Laboratorium voor Fysiologie, KU Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. karel.talavera@med.kuleuven.ac.be

ABSTRACT
Arachidonic acid (AA) modulates T-type Ca(2+) channels and is therefore a potential regulator of diverse cell functions, including neuronal and cardiac excitability. The underlying mechanism of modulation is unknown. Here we analyze the effects of AA on the T-type Ca(2+) channel alpha(1G) heterologously expressed in HEK-293 cells. AA inhibited alpha(1G) currents within a few minutes, regardless of preceding exposure to inhibitors of AA metabolism (ETYA and 17-ODYA). Current inhibition was also observed in cell-free inside-out patches, indicating a membrane-delimited interaction of AA with the channel. AA action was consistent with a decrease of the open probability without changes in the size of unitary currents. AA shifted the inactivation curve to more negative potentials, increased the speed of macroscopic inactivation, and decreased the extent of recovery from inactivation at -80 mV but not at -110 mV. AA induced a slight increase of activation near the threshold and did not significantly change the deactivation kinetics or the rectification pattern. We observed a tonic current inhibition, regardless of whether the channels were held in resting or inactivated states during AA perfusion, suggesting a state-independent interaction with the channel. Model simulations indicate that AA inhibits T-type currents by switching the channels into a nonavailable conformation and by affecting transitions between inactivated states, which results in the negative shift of the inactivation curve. Slow-inactivating alpha(1G) mutants showed an increased affinity for AA with respect to the wild type, indicating that the structural determinants of fast inactivation are involved in the AA-channel interaction.

Show MeSH
Related in: MedlinePlus