Combined IFN-γ and JAK inhibition to treat hemophagocytic lymphohistiocytosis in mice.

BACKGROUND: Familial hemophagocytic lymphohistiocytosis is a life-threatening hyperinflammatory disease caused by genetic defects in the granule-mediated cytotoxic pathway. Success of hematopoietic cell transplantation, the only cure, is correlated with the extent of disease control before transplantation. Unfortunately, disease refractoriness and toxicities to standard chemotherapy-based regimens are fatal in a fraction of patients. Novel targeted immunotherapies, such as IFN-γ blocking antibodies or ruxolitinib, a Janus kinase (JAK) 1/2 inhibitor, are promising but only partially effective at controlling disease. OBJECTIVE: We asked whether combinations of cytokine-targeted therapies, using antibodies or JAK inhibitor, work synergistically to counteract HLH. METHODS: Genetically predisposed mice were infected and treated with distinct combinations of immunotherapies. Disease outcome was monitored and compared to monotherapies. RESULTS: We showed that inhibiting IL-6 or IL-18 signaling in combination with IFN-γ blockade or ruxolitinib did not increase disease control compared to anti-IFN-γ antibodies or ruxolitinib monotherapies. In contrast, clinically relevant doses of ruxolitinib combined with low doses of anti-IFN-γ blocking antibodies corrected cytopenias, prevented overt neutrophilia, limited cytokinemia, and resolved HLH immunopathology and symptomatology. CONCLUSIONS: Our findings demonstrate that IFN-γ blockade and ruxolitinib act synergistically to suppress HLH progression. This supports the use of combined cytokine-targeted therapies as a bridge to hematopoietic cell transplantation in severe familial hemophagocytic lymphohistiocytosis.

Aberrant T-cell exhaustion in severe combined immunodeficiency survivors with poor T-cell reconstitution after transplantation.

BACKGROUND: Severe combined immunodeficiency (SCID) comprises rare inherited disorders of immunity that require definitive treatment through hematopoietic cell transplantation (HCT) or gene therapy for survival. Despite successes of allogeneic HCT, many SCID patients experience incomplete immune reconstitution, persistent T-cell lymphopenia, and poor long-term outcomes. OBJECTIVE: We hypothesized that CD4+ T-cell lymphopenia could be associated with a state of T-cell exhaustion in previously transplanted SCID patients. METHODS: We analyzed markers of exhaustion in blood samples from 61 SCID patients at a median of 10.4 years after HCT. RESULTS: Compared to post-HCT SCID patients with normal CD4+ T-cell counts, those with poor T-cell reconstitution showed lower frequency of naive CD45RA+/CCR7+ T cells, recent thymic emigrants, and TCR excision circles. They also had a restricted TCR repertoire, increased expression of inhibitory receptors (PD-1, 2B4, CD160, BTLA, CTLA-4), and increased activation markers (HLA-DR, perforin) on their total and naive CD8+ T cells, suggesting T-cell exhaustion and aberrant activation, respectively. The exhaustion score of CD8+ T cells was inversely correlated with CD4+ T-cell count, recent thymic emigrants, TCR excision circles, and TCR diversity. Exhaustion scores were higher among recipients of unconditioned HCT, especially when further in time from HCT. Patients with fewer CD4+ T cells showed a transcriptional signature of exhaustion. CONCLUSIONS: Recipients of unconditioned HCT for SCID may develop late post-HCT T-cell exhaustion as a result of diminished production of T-lineage cells. Elevated expression of inhibitory receptors on their T cells may be a biomarker of poor long-term T-cell reconstitution.

Common gamma chain cytokines and CD8 T cells in cancer.

Overcoming exhaustion-associated dysfunctions and generating antigen-specific CD8 T cells with the ability to persist in the host and mediate effective long-term anti-tumor immunity is the final aim of cancer immunotherapy. To achieve this goal, immuno-modulatory properties of the common gamma-chain (γc) family of cytokines, that includes IL-2, IL-7, IL-15 and IL-21, have been used to fine-tune and/or complement current immunotherapeutic protocols. These agents potentiate CD8 T cell expansion and functions particularly in the context of immune checkpoint (IC) blockade, shape their differentiation, improve their persistence in vivo and alternatively, influence distinct aspects of the T cell exhaustion program. Despite these properties, the intrinsic impact of cytokines on CD8 T cell exhaustion has remained largely unexplored impeding optimal therapeutic use of these agents. In this review, we will discuss current knowledge regarding the influence of relevant γc cytokines on CD8 T cell differentiation and function based on clinical data and preclinical studies in murine models of cancer and chronic viral infection. We will restate the place of these agents in current immunotherapeutic regimens such as IC checkpoint blockade and adoptive cell therapy. Finally, we will discuss how γc cytokine signaling pathways regulate T cell immunity during cancer and whether targeting these pathways may sustain an effective and durable T cell response in patients.

A three-way inter-molecular network accounts for the CaVα2δ1-induced functional modulation of the pore-forming CaV1.2 subunit.

L-type CaV1.2 channels are essential for the excitation-contraction coupling in cardiomyocytes and are hetero-oligomers of a pore-forming CaVα1C assembled with CaVß and CaVα2δ1 subunits. A direct interaction between CaVα2δ1 and Asp-181 in the first extracellular loop of CaVα1 reproduces the native properties of the channel. A 3D model of the von Willebrand factor type A (VWA) domain of CaVα2δ1 complexed with the voltage sensor domain of CaVα1C suggests that Ser-261 and Ser-263 residues in the metal ion-dependent adhesion site (MIDAS) motif are determinant in this interaction, but this hypothesis is untested. Here, coimmunoprecipitation assays and patch-clamp experiments of single-substitution variants revealed that CaVα2δ1 Asp-259 and Ser-261 are the two most important residues in regard to protein interactions and modulation of CaV1.2 currents. In contrast, mutating the side chains of CaVα2δ1 Ser-263, Thr-331, and Asp-363 with alanine did not completely prevent channel function. Molecular dynamics simulations indicated that the carboxylate side chain of CaVα2δ1 Asp-259 coordinates the divalent cation that is further stabilized by the oxygen atoms from the hydroxyl side chain of CaVα2δ1 Ser-261 and the carboxylate group of CaVα1C Asp-181. In return, the hydrogen atoms contributed by the side chain of Ser-261 and the main chain of Ser-263 bonded the oxygen atoms of CaV1.2 Asp-181. We propose that CaVα2δ1 Asp-259 promotes Ca2+ binding necessary to produce the conformation of the VWA domain that locks CaVα2δ1 Ser-261 and Ser-263 within atomic distance of CaVα1C Asp-181. This three-way network appears to account for the CaVα2δ1-induced modulation of CaV1.2 currents.

Negatively charged residues in the first extracellular loop of the L-type CaV1.2 channel anchor the interaction with the CaVα2δ1 auxiliary subunit.

Voltage-gated L-type CaV1.2 channels in cardiomyocytes exist as heteromeric complexes. Co-expression of CaVα2δ1 with CaVß/CaVα1 proteins reconstitutes the functional properties of native L-type currents, but the interacting domains at the CaV1.2/CaVα2δ1 interface are unknown. Here, a homology-based model of CaV1.2 identified protein interfaces between the extracellular domain of CaVα2δ1 and the extracellular loops of the CaVα1 protein in repeats I (IS1S2 and IS5S6), II (IIS5S6), and III (IIIS5S6). Insertion of a 9-residue hemagglutinin epitope in IS1S2, but not in IS5S6 or in IIS5S6, prevented the co-immunoprecipitation of CaV1.2 with CaVα2δ1. IS1S2 contains a cluster of three conserved negatively charged residues Glu-179, Asp-180, and Asp-181 that could contribute to non-bonded interactions with CaVα2δ1. Substitutions of CaV1.2 Asp-181 impaired the co-immunoprecipitation of CaVß/CaV1.2 with CaVα2δ1 and the CaVα2δ1-dependent shift in voltage-dependent activation gating. In contrast, single substitutions in CaV1.2 in neighboring positions in the same loop (179, 180, and 182-184) did not significantly alter the functional up-regulation of CaV1.2 whole-cell currents. However, a negatively charged residue at position 180 was necessary to convey the CaVα2δ1-mediated shift in the activation gating. We also found a more modest contribution from the positively charged Arg-1119 in the extracellular pore region in repeat III of CaV1.2. We conclude that CaV1.2 Asp-181 anchors the physical interaction that facilitates the CaVα2δ1-mediated functional modulation of CaV1.2 currents. By stabilizing the first extracellular loop of CaV1.2, CaVα2δ1 may up-regulate currents by promoting conformations of the voltage sensor that are associated with the channel's open state.

Proteolytic cleavage of the hydrophobic domain in the CaVα2δ1 subunit improves assembly and activity of cardiac CaV1.2 channels.

Voltage-gated L-type CaV1.2 channels in cardiomyocytes exist as heteromeric complexes with the pore-forming CaVα1, CaVß, and CaVα2δ1 subunits. The full complement of subunits is required to reconstitute the native-like properties of L-type Ca2+ currents, but the molecular determinants responsible for the formation of the heteromeric complex are still being studied. Enzymatic treatment with phosphatidylinositol-specific phospholipase C, a phospholipase C specific for the cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins, disrupted plasma membrane localization of the cardiac CaVα2δ1 prompting us to investigate deletions of its hydrophobic transmembrane domain. Patch-clamp experiments indicated that the C-terminally cleaved CaVα2δ1 proteins up-regulate CaV1.2 channels. In contrast, deleting the residues before the single hydrophobic segment (CaVα2δ1 Δ1059-1063) impaired current up-regulation. CaVα2δ1 mutants G1060I and G1061I nearly eliminated the cell-surface fluorescence of CaVα2δ1, indicated by two-color flow cytometry assays and confocal imaging, and prevented CaVα2δ1-mediated increase in peak current density and modulation of the voltage-dependent gating of CaV1.2. These impacts were specific to substitutions with isoleucine residues because functional modulation was partially preserved in CaVα2δ1 G1060A and G1061A proteins. Moreover, C-terminal fragments exhibited significantly altered mobility in denatured immunoblots of CaVα2δ1 G1060I and CaVα2δ1 G1061I, suggesting that these mutant proteins were impaired in proteolytic processing. Finally, CaVα2δ1 Δ1059-1063, but not CaVα2δ1 G1060A, failed to co-immunoprecipitate with CaV1.2. Altogether, our data support a model in which small neutral hydrophobic residues facilitate the post-translational cleavage of the CaVα2δ1 subunit at the predicted membrane interface and further suggest that preventing GPI anchoring of CaVα2δ1 averts its cell-surface expression, its interaction with CaVα1, and modulation of CaV1.2 currents.

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity.

Alteration in the L-type current density is one aspect of the electrical remodeling observed in patients suffering from cardiac arrhythmias. Changes in channel function could result from variations in the protein biogenesis, stability, post-translational modification, and/or trafficking in any of the regulatory subunits forming cardiac L-type Ca(2+) channel complexes. CaVα2δ1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type CaV1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant CaVα2δ1 proteins. This change was also observed upon simultaneous mutation of the 16 Asn sites. Nonetheless, the mutation of only 6/16 sites was sufficient to significantly 1) reduce the steady-state cell surface fluorescence of CaVα2δ1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the CaVα2δ1-mediated increase in the peak current density and voltage-dependent gating of CaV1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored CaVα2δ1 function. Single mutation N663Q and double mutations N348Q/N468Q, N348Q/N812Q, and N468Q/N812Q decreased protein stability/synthesis and nearly abolished steady-state cell surface density of CaVα2δ1 as well as the CaVα2δ1-induced up-regulation of L-type currents. These results demonstrate that Asn-663 and to a lesser extent Asn-348, Asn-468, and Asn-812 contribute to protein stability/synthesis of CaVα2δ1, and furthermore that N-glycosylation of CaVα2δ1 is essential to produce functional L-type Ca(2+) channels.

Inherited Ventricular Arrhythmias: The Role of the Multi-Subunit Structure of the L-Type Calcium Channel Complex.

The normal heartbeat is conditioned by transient increases in the intracellular free Ca2+ concentration. Ca2+ influx in cardiomyocytes is regulated by the activity of the heteromeric L-type voltage-activated CaV1.2 channel. A complex network of interactions between the different proteins forming the ion channel supports the kinetics and the activation gating of the Ca2+ influx. Alterations in the biophysical and biochemical properties or in the biogenesis in any of these proteins can lead to serious disturbances in the cardiac rhythm. The multi-subunit nature of the channel complex is better comprehended by examining the high-resolution three-dimensional structure of the closely related CaV1.1 channel. The architectural map identifies precise interaction loci between the different subunits and paves the way for elucidating the mechanistic basis for the regulation of Ca2+ balance in cardiac myocytes under physiological and pathological conditions.

Functional characterization of CaVα2δ mutations associated with sudden cardiac death.

L-type Ca(2+) channels play a critical role in cardiac rhythmicity. These ion channels are oligomeric complexes formed by the pore-forming CaVα1 with the auxiliary CaVß and CaVα2δ subunits. CaVα2δ increases the peak current density and improves the voltage-dependent activation gating of CaV1.2 channels without increasing the surface expression of the CaVα1 subunit. The functional impact of genetic variants of CACNA2D1 (the gene encoding for CaVα2δ), associated with shorter repolarization QT intervals (the time interval between the Q and the T waves on the cardiac electrocardiogram), was investigated after recombinant expression of the full complement of L-type CaV1.2 subunits in human embryonic kidney 293 cells. By performing side-by-side high resolution flow cytometry assays and whole-cell patch clamp recordings, we revealed that the surface density of the CaVα2δ wild-type protein correlates with the peak current density. Furthermore, the cell surface density of CaVα2δ mutants S755T, Q917H, and S956T was not significantly different from the cell surface density of the CaVα2δ wild-type protein expressed under the same conditions. In contrast, the cell surface expression of CaVα2δ D550Y, CaVα2δ S709N, and the double mutant D550Y/Q917H was reduced, respectively, by ≈30-33% for the single mutants and by 60% for the latter. The cell surface density of D550Y/Q917H was more significantly impaired than protein stability, suggesting that surface trafficking of CaVα2δ was disrupted by the double mutation. Co-expression with D550Y/Q917H significantly decreased CaV1.2 currents as compared with results obtained with CaVα2δ wild type. It is concluded that D550Y/Q917H reduced inward Ca(2+) currents through a defect in the cell surface trafficking of CaVα2δ. Altogether, our results provide novel insight in the molecular mechanism underlying the modulation of CaV1.2 currents by CaVα2δ.

Cooperative activation of the T-type CaV3.2 channel: interaction between Domains II and III.

T-type CaV3 channels are important mediators of Ca(2+) entry near the resting membrane potential. Little is known about the molecular mechanisms responsible for channel activation. Homology models based upon the high-resolution structure of bacterial NaV channels predict interaction between the S4-S5 helix of Domain II (IIS4-S5) and the distal S6 pore region of Domain II (IIS6) and Domain III (IIIS6). Functional intra- and inter-domain interactions were investigated with a double mutant cycle analysis. Activation gating and channel kinetics were measured for 47 single mutants and 20 pairs of mutants. Significant coupling energies (ΔΔG(interact) ≥ 1.5 kcal mol(-1)) were measured for 4 specific pairs of mutants introduced between IIS4-S5 and IIS6 and between IIS4-S5 and IIIS6. In agreement with the computer based models, Thr-911 in IIS4-S5 was functionally coupled with Ile-1013 in IIS6 during channel activation. The interaction energy was, however, found to be stronger between Val-907 in IIS4-S5 and Ile-1013 in IIS6. In addition Val-907 was significantly coupled with Asn-1548 in IIIS6 but not with Asn-1853 in IVS6. Altogether, our results demonstrate that the S4-S5 and S6 helices from adjacent domains are energetically coupled during the activation of a low voltage-gated T-type CaV3 channel.

Comparison of Omicron breakthrough infection versus monovalent SARS-CoV-2 intramuscular booster reveals differences in mucosal and systemic humoral immunity.

Our understanding of the quality of cellular and humoral immunity conferred by COVID-19 vaccination alone versus vaccination plus SARS-CoV-2 breakthrough (BT) infection remains incomplete. While the current (2023) SARS-CoV-2 immune landscape of Canadians is complex, in late 2021 most Canadians had either just received a third dose of COVID-19 vaccine, or had received their two-dose primary series and then experienced an Omicron BT. Herein we took advantage of this coincident timing to contrast cellular and humoral immunity conferred by three doses of vaccine versus two doses plus BT. Our results show thatBT infection induces cell-mediated immune responses to variants comparable to an intramuscular vaccine booster dose. In contrast, BT subjects had higher salivary immunoglobulin (Ig)G and IgA levels against the Omicron spike and enhanced reactivity to the ancestral spike for the IgA isotype, which also reacted with SARS-CoV-1. Serumneutralizing antibody levels against the ancestral strain and the variants were also higher after BT infection. Our results support the need for the development of intranasal vaccines that could emulate the enhanced mucosal and humoral immunity induced by Omicron BT without exposing individuals to the risks associated with SARS-CoV-2 infection.

A quartet of leucine residues in the guanylate kinase domain of CaVß determines the plasma membrane density of the CaV2.3 channel.

Ca(V)ß subunits are formed by a Src homology 3 domain and a guanylate kinase-like (GK) domain connected through a variable HOOK domain. Complete deletion of the Src homology 3 domain (75 residues) as well as deletion of the HOOK domain (47 residues) did not alter plasma membrane density of Ca(V)2.3 nor its typical activation gating. In contrast, six-residue deletions in the GK domain disrupted cell surface trafficking and functional expression of Ca(V)2.3. Mutations of residues known to carry nanomolar affinity binding in the GK domain of Ca(V)ß (P175A, P179A, M195A, M196A, K198A, S295A, R302G, R307A, E339G, N340G, and A345G) did not significantly alter cell surface targeting or gating modulation of Ca(V)2.3. Nonetheless, mutations of a quartet of leucine residues (either single or multiple mutants) in the α3, α6, ß10, and α9 regions of the GK domain were found to significantly impair cell surface density of Ca(V)2.3 channels. Furthermore, the normalized protein density of Ca(V)2.3 was nearly abolished with the quadruple Ca(V)ß3 Leu mutant L200G/L303G/L337G/L342G. Altogether, our observations suggest that the four leucine residues in Ca(V)ß3 form a hydrophobic pocket surrounding key residues in the α-interacting domain of Ca(V)2.3. This interaction appears to play an essential role in conferring Ca(V)ß-induced modulation of the protein density of Ca(V)α1 subunits in Ca(V)2 channels.

The REinfection in COVID-19 Estimation of Risk (RECOVER) study: Reinfection and serology dynamics in a cohort of Canadian healthcare workers.

BACKGROUND: Understanding the immune response to natural infection by SARS-CoV-2 is key to pandemic management, especially in the current context of emerging variants. Uncertainty remains regarding the efficacy and duration of natural immunity against reinfection. METHODS: We conducted an observational prospective cohort study in Canadian healthcare workers (HCWs) with a history of PCR-confirmed SARS-CoV-2 infection to (i) measure the average incidence rate of reinfection and (ii) describe the serological immune response to the primary infection. RESULTS: Our cohort comprised 569 HCWs; median duration of individual follow-up was 371 days. We detected six cases of reinfection in absence of vaccination between August 21, 2020, and March 1, 2022, for a reinfection incidence rate of 4.0 per 100 person-years. Median duration of seropositivity was 415 days in symptomatics at primary infection compared with 213 days in asymptomatics (p < 0.0001). Other characteristics associated with prolonged seropositivity for IgG against the spike protein included age over 55 years, obesity, and non-Caucasian ethnicity. CONCLUSIONS: Among unvaccinated healthcare workers, reinfection with SARS-CoV-2 following a primary infection remained rare.

Symptomatology during previous SARS-CoV-2 infection and serostatus before vaccination influence the immunogenicity of BNT162b2 COVID-19 mRNA vaccine.

Public health vaccination recommendations for COVID-19 primary series and boosters in previously infected individuals differ worldwide. As infection with SARS-CoV-2 is often asymptomatic, it remains to be determined if vaccine immunogenicity is comparable in all previously infected subjects. This study presents detailed immunological evidence to clarify the requirements for one- or two-dose primary vaccination series for naturally primed individuals. The main objective was to evaluate the immune response to COVID-19 mRNA vaccination to establish the most appropriate vaccination regimen to induce robust immune responses in individuals with prior SARS-CoV-2 infection. The main outcome measure was a functional immunity score (zero to three) before and after vaccination, based on anti-RBD IgG levels, serum capacity to neutralize live virus and IFN-γ secretion capacity in response to SARS-CoV-2 peptide pools. One point was attributed for each of these three functional assays with response above the positivity threshold. The immunity score was compared based on subjects' symptoms at diagnosis and/or serostatus prior to vaccination. None of the naïve participants (n=14) showed a maximal immunity score of three following one dose of vaccine compared to 84% of the previously infected participants (n=55). All recovered individuals who did not have an immunity score of three were seronegative prior to vaccination, and 67% had not reported symptoms resulting from their initial infection. Following one dose of vaccine, their immune responses were comparable to naïve individuals, with significantly weaker responses than individuals who were symptomatic during infection. These results indicate that the absence of symptoms during initial infection and negative serostatus prior to vaccination predict the strength of immune responses to COVID-19 mRNA vaccine. Altogether, these findings highlight the importance of administering the complete two-dose primary regimen and following boosters of mRNA vaccines to individuals who experienced asymptomatic SARS-CoV-2 infection.

Molecular determinants of the CaVbeta-induced plasma membrane targeting of the CaV1.2 channel.

Ca(V)beta subunits modulate cell surface expression and voltage-dependent gating of high voltage-activated (HVA) Ca(V)1 and Ca(V)2 alpha1 subunits. High affinity Ca(V)beta binding onto the so-called alpha interaction domain of the I-II linker of the Ca(V)alpha1 subunit is required for Ca(V)beta modulation of HVA channel gating. It has been suggested, however, that Ca(V)beta-mediated plasma membrane targeting could be uncoupled from Ca(V)beta-mediated modulation of channel gating. In addition to Ca(V)beta, Ca(V)alpha2delta and calmodulin have been proposed to play important roles in HVA channel targeting. Indeed we show that co-expression of Ca(V)alpha2delta caused a 5-fold stimulation of the whole cell currents measured with Ca(V)1.2 and Ca(V)beta3. To gauge the synergetic role of auxiliary subunits in the steady-state plasma membrane expression of Ca(V)1.2, extracellularly tagged Ca(V)1.2 proteins were quantified using fluorescence-activated cell sorting analysis. Co-expression of Ca(V)1.2 with either Ca(V)alpha2delta, calmodulin wild type, or apocalmodulin (alone or in combination) failed to promote the detection of fluorescently labeled Ca(V)1.2 subunits. In contrast, co-expression with Ca(V)beta3 stimulated plasma membrane expression of Ca(V)1.2 by a 10-fold factor. Mutations within the alpha interaction domain of Ca(V)1.2 or within the nucleotide kinase domain of Ca(V)beta3 disrupted the Ca(V)beta3-induced plasma membrane targeting of Ca(V)1.2. Altogether, these data support a model where high affinity binding of Ca(V)beta to the I-II linker of Ca(V)alpha1 largely accounts for Ca(V)beta-induced plasma membrane targeting of Ca(V)1.2.

Carnitine is associated with fatty acid metabolism in plants.

The finding of acylcarnitines alongside free carnitine in Arabidopsis thaliana and other plant species, using tandem mass spectrometry coupled to liquid chromatography shows a link between carnitine and plant fatty acid metabolism. Moreover the occurrence of both medium- and long-chain acylcarnitines suggests that carnitine is connected to diverse fatty acid metabolic pathways in plant tissues. The carnitine and acylcarnitine contents in plant tissues are respectively a hundred and a thousand times lower than in animal tissues, and acylcarnitines represent less than 2% of the total carnitine pool whereas this percentage reaches 30% in animal tissues. These results suggest that carnitine plays a lesser role in lipid metabolism in plants than it does in animals.

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry.

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.