title: Characterization of Cav1.4 complexes (α11.4, β2, and α2δ4) in HEK293T cells and in the retina.
abstract: In photoreceptor synaptic terminals, voltage-gated Cav1.4 channels mediate Ca(2+) signals required for transmission of visual stimuli. Like other high voltage-activated Cav channels, Cav1.4 channels are composed of a main pore-forming Cav1.4 α1 subunit and auxiliary β and α2δ subunits. Of the four distinct classes of β and α2δ, β2 and α2δ4 are thought to co-assemble with Cav1.4 α1 subunits in photoreceptors. However, an understanding of the functional properties of this combination of Cav subunits is lacking. Here, we provide evidence that Cav1.4 α1, β2, and α2δ4 contribute to Cav1.4 channel complexes in the retina and describe their properties in electrophysiological recordings. In addition, we identified a variant of β2, named here β2X13, which, along with β2a, is present in photoreceptor terminals. Cav1.4 α1, β2, and α2δ4 were coimmunoprecipitated from lysates of transfected HEK293 cells and mouse retina and were found to interact in the outer plexiform layer of the retina containing the photoreceptor synaptic terminals, by proximity ligation assays. In whole-cell patch clamp recordings of transfected HEK293T cells, channels (Cav1.4 α1 + β2X13) containing α2δ4 exhibited weaker voltage-dependent activation than those with α2δ1. Moreover, compared with channels (Cav1.4 α1 + α2δ4) with β2a, β2X13-containing channels exhibited greater voltage-dependent inactivation. The latter effect was specific to Cav1.4 because it was not seen for Cav1.2 channels. Our results provide the first detailed functional analysis of the Cav1.4 subunits that form native photoreceptor Cav1.4 channels and indicate potential heterogeneity in these channels conferred by β2a and β2X13 variants.
authors.affiliation: From the Departments of Molecular Physiology and Biophysics, Otolaryngology Head-Neck Surgery, and Neurology, University of Iowa, Iowa City, Iowa 52242.
authors.forename: Amy
authors.initials: A
authors.lastname: Lee
authors.affiliation: From the Departments of Molecular Physiology and Biophysics, Otolaryngology Head-Neck Surgery, and Neurology, University of Iowa, Iowa City, Iowa 52242.
authors.forename: Shiyi
authors.initials: S
authors.lastname: Wang
authors.affiliation: From the Departments of Molecular Physiology and Biophysics, Otolaryngology Head-Neck Surgery, and Neurology, University of Iowa, Iowa City, Iowa 52242.
authors.forename: Brittany
authors.initials: B
authors.lastname: Williams
authors.affiliation: From the Departments of Molecular Physiology and Biophysics, Otolaryngology Head-Neck Surgery, and Neurology, University of Iowa, Iowa City, Iowa 52242.
authors.forename: Jussara
authors.initials: J
authors.lastname: Hagen
authors.affiliation: the Departments of Ophthalmology and Visual Sciences and Biomedical Engineering, University of Iowa, Iowa City, Iowa 52242, and.
authors.forename: Todd E
authors.initials: TE
authors.lastname: Scheetz
authors.affiliation: the Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195 fanfan@u.washington.edu.
authors.forename: Françoise
authors.initials: F
authors.lastname: Haeseleer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015220:Calcium Channels / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D012160:Retina / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D017386:Sequence Homology, Amino Acid
chemical_list: C113330:CACNA1F protein, human; C480684:Cacna1f protein, mouse; D015220:Calcium Channels; D020746:Calcium Channels, L-Type
keywords: Calcium Channel; Calcium-binding Protein; Electrophysiology; Immunohistochemistry; Photoreceptor; Retina