The presence of ca2+ channel β subunit is required for mitogen‐activated protein kinase (mapk)‐dependent modulation of α1b ca2+ channels in cos‐7 cells

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Abstract
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 β subunit is involved in this process, the role of the four neuronal βs (β1b, β2a, β3, β4) in MAPK-dependent modulation of α1B (Cav2.2, N-type) Ca2+ 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 α1B Ca2+ channel current (Imax) within a period of 5–10 min, regardless of which β subunit was co-expressed (25-50%, P < 0.01). With β2a however, the percentage inhibition of Imax was less than that observed with any other β (ANOVA: F3,34 = 6.48, P < 0.01). UO126 also caused a hyperpolarising shift (6 ± 1 mV, P < 0.001) in the voltage dependence of β2a current activation, such that inhibition occurred only at depolarised potentials (> +5 mV) whereas at more negative potentials the current amplitude was enhanced. A marked change in β2a current kinetics, perceived either as decreased activation or increased inactivation, was also associated with UO126 application. A similar effect of UO126 on β4 current kinetics was also observed. The β2a-specific effects of UO126 on current inhibition and voltage dependence of activation were abolished when α1B was co-expressed with de-palmitoylated β2a(C3,4S), in which amino terminal cysteines 3 and 4 had been mutated to serines. In the absence of β subunit, UO126 had no effect on α1B Ca2+ channel current. Together, these data suggest an absolute requirement for β in MAPK-dependent modulation of these channels. Since β subunits vary both in their temporal expression and localisation within neurones, β subunit-dependent modulation of N-type Ca2+ channels via MAPK could provide an important new mechanism by which to fine-tune neurotransmitter release.