Identification And Functional Study Of The Interaction Between Cav1.3 Calcium And Snapin

Background and ObjectiveCardiac L-type voltage-gated calcium channel(LVCC) is a calcium selective permeable,hydrophilic channel protein.The influx of Ca²⁺ through LVCC plays an essential role in cardiac excitability and in coupling excitation to contraction.The depolarizing current through L-type Ca²⁺ channels(I_Ca) contributes to the plateau phase of the cardiac action potential as well as to pacemaker activity in nodal cells. This influx of Ca²⁺ triggers not only the release of intracellular stores of Ca²⁺ from the sarcoplasmic reticulum,and the ensuing intracellular Ca²⁺ transient results in activation of the myofilaments,but also impacts on other cellular processes modulated by intracellular Ca²⁺,such as gene expression and excitation-secretion coupling.The LVCC that have been characterized biochemically are complex proteins composed of four or five distinct subunits,which are encoded by multiple genes.Theα1 subunit is the major functional subunit of the calcium channel organized in four homological transmembrane domains(Ⅰ-Ⅳ) with six transmembrane segments (S1-S6) in each,which incorporates the conduction pore,the voltage sensor and gating apparatus,and the known sites of channel regulation by second messengers, drugs,and toxins,and determines the different electrophysiological and pharmacological characters of the LVCC.There are four genes encodingα1 subunit of LVCC calcium channel in mammalian tissue,which are named asα₁S(skeletal muscle),α₁C(in heart and brain),α₁D(mainly in brain and endocrine tissue),andα₁F (retina) according to their tissue source,corresponding with nomenclature Cav1.1, Cav1.2,Cav1.3 and Cav1.4,respectively.Cav1.2(α₁C) has the distribution advantage in cardiac system,while Cav1.3 (α₁D) has a high level of expression in neuron and neuroendocrine cell mainly.It is well established that LVCC encoded by Cav1.2 plays a key role in cardiac physiological and pathophysiological activity and is a therapeutic target for heart disease.However,challenging evidences from Takimoto,Mangoni etc.showed that the existence of Cav1.3 mRNA in rat,mouse and human cardiocyte,particularly with a high level of expression in atrium but not in ventricle.Furthermore,Numkung, Stefan and Zhang etc.reported that Cav1.3^-/- mice displayed obvious sinus bradycardia and atrial ventricular block.Their unique voltage dependence of activation also implies that Cav1.3 LVCC mediate cellular processes that depend on calcium influx at relatively hyperpolarized membrane potentials,which might explain the role of Cav1.3 LVCC in the spontaneous diastolic depolarization for sinoatrial node cells.What's more,the density of I_Ca-L reduced by 79%in the heart of Cav1.3^-/- mice as compared with Cav1.3^+/+ mice.All of these indicated that Cav1.3 might be an important component in the constitution of I_Ca-L cardiac cells.The facts that Cav1.2 calcium channel are expressed mainly in working cardiocyte,whereas Cav1.3 calcium channel are found in conductional tissue(SA nodes and AV nodes etc.) and cardiac atrium might be correspondent with their functional role in the heart respectively. Interestingly,why does cardiac LVCC encoded by two distinct gene expresse in different regions of the heart? What is their signification of the difference in cardiac myocytes? How about their signling pathways involved in functional regulation? In previous study,by using intracellular domain of Cav1.3 as the bait,a protein associated with C terminals of Cav1.3 was screened from human cardiac cDNA library in yeast two hybrid systems.The positive protein was identified as snapin after sequecing.We hypothesize that snapin might be a candidate that is responsible for functional regulation of cardiac Cav1.3 LVCC.This study was designed to determine the interation of Cav1.3 and snapin protein in mammalian cells and examine the functional role of their interaction by means of immuoprecipitation, immunofluorescence assay and electrophysiological recording as well.Methods1.Cell culture and cDNAs transfection:HEK293 cells were grown in DMEM medium supplemented with 10%bovine calf serum,penicillin and streptomycin at 37℃in an air/5%CO₂.Cells were placed on a dish(100mm) with a density of about 2×10⁵ cells/cm² when they become 70%～80%confluence.For transient transfection of Cav1.3 and snapin,10μg of a mix of rat Cav1.3,β2a(or with snapin) cDNAs in a molar ratio 1:1:1 were added using the calcium phosphate method.48～60 hours after transfection,cells were seeded for either patch clamp or immunoprecipitation and immunofluorescence assaies.2.Isolation of atrial myocytes.Single atrial myocytes were isolated from adult C57BL/6J mice as previously described.3.Immunoprecipatation and Western Blot.HEK293 cells co-expressed Cav1.3 channel and snapin,adult C57BL/6J mice,Sprague-Dawley rats and human atrium tissue were prepared for co-immunoprecipitation and Western blot analysis with standard protocol.4.Double immunofluorescence labeling.Single isolated atrium myocytes or HEK293 cells expressed Cav1.3 channel or snapin were fixed with 4%PFA (paraformadehyde),and then blocked with 3%BSA,incubated overnight at 4℃with primary antibodies.Then the samples were treated with FITC-conjugated antibody and TRITC-conjugated antibody respectively.Control experiments were performed by secondary antibody only under the same conditions.5.Recording of I_Ca-L in HEK293 cells.Whole cell I_Ca-L was recorded at room temperature with the Axopatch 200B(Axon Instruments) by patch-clamp techniques.ResultsThe results show that snapin forms a complex with Cav1.3 in all tested mammalian cells,inclulding transfected HEK293 cells and cardiac myocytes isolated from mouse,rat as well as human atrium(See Fig5～8 ).Snapin/Cav1.3 interactions is supported by colocalization at cells surface in cultured HEK293 cells or atrial cells isolated from mouse atria(See Fig3,4).In electrophysiological recordings,the dencity of I_Ca-L recorded from HEK293 cells co-expressed Cav1.3 and snapin was 1.25pA/pF while that from HEK293 cells expressed Cav1.3 alone was 3.5pA/pF,suggeting a suppressed role of snapin on Cav1.3 calcium currents(see Fig.8).The activation and steady-state inactivation of Cav1.3 calcium currents are shown in Fig.8 Fitting the current activation gave a V_1/2,act of-20.1±4.1mV for I_Ca-L recorded from HEK293 cells co-expressed Cav1.3 and snapin whereas gave a V_1/2,act of 22.6±1.8mV for that expressed Cav1.3 alone.Expression of snapin caused a about 2 mV left shift of V_1/2,act in activation process but the difference is not significant.In addition,examining the steady-state inactivation properties of the I_Ca-L showed that there was about 1.4mV difference in inactivation process in the cells expressed Cav1.3 with and without snapin,suggesting an influence on the inactive dynamics of the Cav1.3 channel. However,there are no marktable difference in the shift for either activation or inactivation curve.Summary:The interaction between Cav1.3 calcium channel and snapin exists not only in YTH system but also in mammalian system.Snapin might facilitate the activation and inactivation processes for Cav1.3 calcium channel.