title: Two distinct voltage-sensing domains control voltage sensitivity and kinetics of current activation in CaV1.1 calcium channels.
abstract: Alternative splicing of the skeletal muscle CaV1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant CaV1.1a causes an ∼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3-S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (CaV1.1 null) myotubes reconstituted with CaV1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3-S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3-S4 linker is sufficient to confer CaV1.1a-like voltage dependence to the I VSD and that the IS3-S4 linker plus IS4 is sufficient to transfer CaV1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3-S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, CaV1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms within the IV and I VSDs, respectively.
authors.affiliation: Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Innsbruck, A-6020 Innsbruck, Austria.
authors.forename: Petronel
authors.initials: P
authors.lastname: Tuluc
authors.affiliation: Department of Physiology and Medical Physics, Medical University Innsbruck, A-6020 Innsbruck, Austria.
authors.forename: Bruno
authors.initials: B
authors.lastname: Benedetti
authors.affiliation: Department of Physiology and Medical Physics, Medical University Innsbruck, A-6020 Innsbruck, Austria.
authors.forename: Pierre
authors.initials: P
authors.lastname: Coste de Bagneaux
authors.affiliation: Department of Medical Genetics, Molecular and Clinical Pharmacology, Medical University Innsbruck, A-6020 Innsbruck, Austria.
authors.forename: Manfred
authors.initials: M
authors.lastname: Grabner
authors.affiliation: Department of Physiology and Medical Physics, Medical University Innsbruck, A-6020 Innsbruck, Austria bernhard.e.flucher@i-med.ac.at.
authors.forename: Bernhard E
authors.initials: BE
authors.lastname: Flucher
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D015640:Ion Channel Gating; D008564:Membrane Potentials; D051379:Mice; D032446:Myoblasts / Q000378:metabolism / Q000502:physiology; D000072417:Protein Domains; D011994:Recombinant Proteins
chemical_list: D020746:Calcium Channels, L-Type; D011994:Recombinant Proteins