Distinction between Steady-State Inactivation and Voltage-Dependent Facilitation in L-Type Ca^2＋ Channel α_1c and α_1c/β Subunits

Abstract

The L-type Ca2+ channel has a unique kinetic property known as voltage-dependent facilitation. Many researchers have repeatedly investigated the mechanism in response to the voltage-dependent facilitation since the first observation by Fenwick et al. in 1982. Electrophysiological evaluations of voltage-dependent facilitation, however, remain inconsistent, partially because of its unclear definition. Some scientists understand it as a current augmentation by a conditioning prepulse prior to the test pulse, and others understand it as a result of the U-shape steady-state inactivation curve. We therefore investigated to identify the distinction between the voltage-dependent facilitation and the steady-state inactivation, by use of Ba2+ as the charge in order to avoid the other inactivation mechanism or the Ca(2+)-dependent inactivation upon this analysis. Conventional whole-cell mode patch clamp technique was applied to chinese hamster fibroblast (CHW) cells that express the alpha1c subunit alone or the alpha1c subunit with the beta subunit (alpha1c/beta) derived from rabbit heart to investigate the voltage-dependent facilitation depending on the composition of the subunits. Coexpression of the beta subunit augmented alpha1 subunit channel current and shifted current-voltage relation towards hyperpolarized direction. In the experiment using conventional double pulse protocol to investigate steady-state inactivation, alpha1c subunit channel current and alpha1c/beta subunit channel current were not fully inactivated. Subtraction of the steady-state inactivation component from whole recovered current enabled us to identify the voltage-dependent facilitation component of the L-type Ca2+ channel. The voltage-dependent facilitation of the alpha1 subunit current and the alpha1c/beta subunit current were identical in kinetics, and could be generated at 0 mV or depolarized potentials partially overlapped with the potential range for the steady-state inactivation of the current. These results suggest that the voltage-dependent facilitation of the L-type Ca2+ channel could be formed by the alpha1c subunit without interaction with the beta subunit, and that the range for the voltage-dependent facilitation and the steady-state inactivation overlap each other at 0 mV or more depolarized potentials up to approximately + 100 mV.

Methods of activation and the role of calcium/calmodulin-dependent protein kinase II in the regulation of acetylcholine secretion in the motor synapses of mice

“…At present, it is universally recognized that regard less of the neuron or terminal type, L type calcium channels are the target of various phosphatase-kinase and voltage dependent regulations [27,[32][33][34], although, in motor terminals, inhibitory phosphatase actions dominate resulting in the inhibition of L type calcium channels [10,20]. However, in cases where L type calcium channels are disinhibited, CaMKII activity is generally present in the cell; quite often, it is a form of positive feedback for the maintenance of the functioning of a channel itself [21,30,35].…”

“…However, in cases where L type calcium channels are disinhibited, CaMKII activity is generally present in the cell; quite often, it is a form of positive feedback for the maintenance of the functioning of a channel itself [21,30,35]. This sce nario is well known for cardiomyocytes, where cal cium channels that possess several described sites for phosphorylation by calmodulin kinase are a target of CaMKII activated by calcium influx via L type cal cium channels [30,32]. It is possible that the studied motor synapses also have a similar positive feedback between partially disinhibited L type calcium chan nels and CaMKII activity, which shifts the balance of regulatory actions on L type channels towards ampli fication and maintenance of their activity and thus potentiates calcium influx and the following calcium dependent ACh release.…”

Participation of PLA2 and PLC in DhL-induced activation ofRhinella arenarumoocytes

“…Several observations suggest that phosphoinositide metabolism is a vital intracellular signalling system involved in many cellular functions including fertilization and embryogenesis. PLCs are key enzymes within this system, hydrolyzing the membrane phospholipid substrate, phosphatidylinositol 4,5-bisphosphate (PIP 2 ) (Halet et al , 2003; Lee et al , 2006; Whitaker, 2006) to trigger cytoplasmic Ca 2+ oscillations via the inositol 1,4,5-trisphosphate (IP 3 ) intracellular Ca 2+ signalling pathway (Berridge, 1993).…”

Physiology and pharmacology of flatworm muscle

“…Channel regulation may be possible due to the intrinsic property of α 1 subunit, or may require involvement of other subunits. Voltage-dependent facilitation in case of smooth and cardiac muscle α 1C has been reported without the requirement of β subunits (Kleppisch et al, 1994;Lee et al, 2006), while in some cases their coexpression is obligatory for the proper channel regulation (Bourinet et al, 1994;Kamp et al, 2000;Platano et al, 2000;Cens et al, 1996). Out of four subunits (β1, β2, β3 and β4), only β2 is not permissive for Ca 2+ channel facilitation (Cens et al, 1996;Carbone et al, 2001).…”