Potentiation of High Voltage-Activated Calcium Channels by 4-Aminopyridine Depends on Subunit Composition

4-Aminopyridine (4-AP, fampridine) is used clinically to improve neuromuscular function in patients with multiple sclerosis, spinal cord injury, and myasthenia gravis. 4-AP can increase neuromuscular and synaptic transmission by directly stimulating high voltage-activated (HVA) Ca2+ channels independent of its blocking effect on voltage-activated K+ channels. Here we provide new evidence that the potentiating effect of 4-AP on HVA Ca2+ channels depends on the specific combination of voltage-activated calcium channel alpha 1 (Cav alpha(1)) and voltage-activated calcium channel beta (Cav beta) subunits. Among the four Cav beta subunits examined, Cav beta 3 was the most significant subunit involved in the 4-AP-induced potentiation of both L-type and N-type currents. Of particular note, 4-AP at micromolar concentrations selectively potentiated L-type currents reconstituted with Cav1.2, alpha(2)delta 1, and Cav beta 3. In contrast, 4-AP potentiated N-type currents only at much higher concentrations and had little effect on P/Q-type currents. In a phrenic nerve-diaphragm preparation, blocking L-type Ca2+ channels eliminated the potentiating effect of low concentrations of 4-AP on end-plate potentials. Furthermore, 4-AP enhanced the physical interaction of Cav1.2 and Cav2.2 subunits to Cav beta 3 and also increased their trafficking to the plasma membrane. Site-directed mutagenesis identified specific regions in the guanylate kinase, HOOK, and C-terminus domains of the Cav beta 3 subunit crucial to the ability of 4-AP to potentiate L-type and N-type currents. Our findings indicate that 4-AP potentiates HVA Ca2+ channels by enhancing reciprocal Cav1.2-Cav beta 3 and Cav2.2-Cav beta 3 interactions. The therapeutic effect of 4-AP on neuromuscular function is probably mediated by its actions on Cav beta 3-containing L-type Ca2+ channels.