The presence of Ca(2+) channel beta subunit is required for mitogen-activated protein kinase (MAPK)-dependent modulation of alpha1B Ca(2+) channels in COS-7 cells.

摘要

In rat sensory neurones, voltage-dependent calcium channels (VDCCs), including the N-type, are tonically up-regulated via Ras/mitogen-activated protein kinase (MAPK) signalling. To determine whether VDCC beta subunit is involved in this process, the role of the four neuronal betas (beta1b, beta2a, beta3, beta4) in MAPK-dependent modulation of alpha1B (Ca(v)2.2, N-type) Ca(2+) channels has been examined in COS-7 cells. MAPK is exclusively activated by MAPK kinase (MEK), and here, acute application of a MEK-specific inhibitor UO126, significantly inhibited peak alpha1B Ca(2+) channel current (I(max)) within a period of 5-10 min, regardless of which beta subunit was co-expressed (25-50 %, P < 0.01). With beta2a however, the percentage inhibition of I(max) was less than that observed with any other beta (ANOVA: F(3,34) = 6.48, P < 0.01). UO126 also caused a hyperpolarising shift (6 +/- 1 mV, P < 0.001) in the voltage dependence of beta2a current activation, such that inhibition occurred only at depolarisedpotentials (> +5 mV) whereas at more negative potentials the current amplitude was enhanced. A marked change in beta2a current kinetics, perceived either as decreased activation or increased inactivation, was also associated with UO126 application. A similar effect of UO126 on beta4 current kinetics was also observed. The beta2a-specific effects of UO126 on current inhibition and voltage dependence of activation were abolished when alpha1B was co-expressed with de-palmitoylated beta2a(C3,4S), in which amino terminal cysteines 3 and 4 had been mutated to serines. In the absence of beta subunit, UO126 had no effect on alpha1B Ca(2+) channel current. Together, these data suggest an absolute requirement for beta in MAPK-dependent modulation of these channels. Since beta subunits vary both in their temporal expression and localisation within neurones, beta subunit-dependent modulation of N-type Ca(2+) channels via MAPK could provide an important new mechanism by which to fine-tune neurotransmitter release.