title: Potentiation of high voltage-activated calcium channels by 4-aminopyridine depends on subunit composition.
abstract: 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) Ca(2+) 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 Ca(2+) channels depends on the specific combination of voltage-activated calcium channel α1 (Cavα1) and voltage-activated calcium channel β (Cavβ) subunits. Among the four Cavβ subunits examined, Cavβ3 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, α2δ1, and Cavβ3. 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 Ca(2+) 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 Cavβ3 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 Cavβ3 subunit crucial to the ability of 4-AP to potentiate L-type and N-type currents. Our findings indicate that 4-AP potentiates HVA Ca(2+) channels by enhancing reciprocal Cav1.2-Cavβ3 and Cav2.2-Cavβ3 interactions. The therapeutic effect of 4-AP on neuromuscular function is probably mediated by its actions on Cavβ3-containing L-type Ca(2+) channels.
authors.affiliation: Center for Neuroscience and Pain Research (L.L., D.-P.L, S.-R.C., J.C., H.-L.P.), Department of Anesthesiology and Perioperative Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas; College of Bioscience and Biotechnology (J.C.), Hunan Agricultural University, Changsha, P.R. China; and Department of Integrative Biology and Pharmacology (H.H.), The University of Texas Medical School, Houston, Texas.
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.affiliation: Center for Neuroscience and Pain Research (L.L., D.-P.L, S.-R.C., J.C., H.-L.P.), Department of Anesthesiology and Perioperative Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas; College of Bioscience and Biotechnology (J.C.), Hunan Agricultural University, Changsha, P.R. China; and Department of Integrative Biology and Pharmacology (H.H.), The University of Texas Medical School, Houston, Texas.
authors.forename: De-Pei
authors.initials: DP
authors.lastname: Li
authors.affiliation: Center for Neuroscience and Pain Research (L.L., D.-P.L, S.-R.C., J.C., H.-L.P.), Department of Anesthesiology and Perioperative Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas; College of Bioscience and Biotechnology (J.C.), Hunan Agricultural University, Changsha, P.R. China; and Department of Integrative Biology and Pharmacology (H.H.), The University of Texas Medical School, Houston, Texas.
authors.forename: Shao-Rui
authors.initials: SR
authors.lastname: Chen
authors.affiliation: Center for Neuroscience and Pain Research (L.L., D.-P.L, S.-R.C., J.C., H.-L.P.), Department of Anesthesiology and Perioperative Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas; College of Bioscience and Biotechnology (J.C.), Hunan Agricultural University, Changsha, P.R. China; and Department of Integrative Biology and Pharmacology (H.H.), The University of Texas Medical School, Houston, Texas.
authors.forename: Jinjun
authors.initials: J
authors.lastname: Chen
authors.affiliation: Center for Neuroscience and Pain Research (L.L., D.-P.L, S.-R.C., J.C., H.-L.P.), Department of Anesthesiology and Perioperative Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas; College of Bioscience and Biotechnology (J.C.), Hunan Agricultural University, Changsha, P.R. China; and Department of Integrative Biology and Pharmacology (H.H.), The University of Texas Medical School, Houston, Texas.
authors.forename: Hongzhen
authors.initials: H
authors.lastname: Hu
authors.affiliation: Center for Neuroscience and Pain Research (L.L., D.-P.L, S.-R.C., J.C., H.-L.P.), Department of Anesthesiology and Perioperative Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas; College of Bioscience and Biotechnology (J.C.), Hunan Agricultural University, Changsha, P.R. China; and Department of Integrative Biology and Pharmacology (H.H.), The University of Texas Medical School, Houston, Texas huilinpan@mdanderson.org.
authors.forename: Hui-Lin
authors.initials: HL
authors.lastname: Pan
mesh_terms: D015761:4-Aminopyridine / Q000494:pharmacology*; D000595:Amino Acid Sequence; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000187:drug effects*; D020864:Calcium Channels, N-Type / Q000737:chemistry / Q000187:drug effects*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D009469:Neuromuscular Junction / Q000502:physiology; D021122:Protein Subunits; D009435:Synaptic Transmission / Q000187:drug effects
chemical_list: D020746:Calcium Channels, L-Type; D020864:Calcium Channels, N-Type; D021122:Protein Subunits; D015761:4-Aminopyridine