Didi Matza

Critical role for the β regulatory subunits of Cav channels in T lymphocyte function

Calcium ion is a universal signaling intermediate, which is known to control various biological processes. In excitable cells, voltage-gated calcium channels (Cav) are the major route of calcium entry and regulate multiple functions such as contraction, neurotransmitter release, and gene transcription. Here we show that T lymphocytes, which are nonexcitable cells, express both regulatory β and pore-forming Cav1 α1 subunits of Cav channels, and we provide genetic evidence for a critical role of the Cav β3 and Cav β4 regulatory subunits in T lymphocyte function. Cav β-deficient T lymphocytes fail to acquire normal functions, and they display impairment in the T cell receptor-mediated calcium response, nuclear factor of activated T cells activation, and cytokine production. In addition, unlike in excitable cells, our data suggest a minimal physiological role for depolarization in Cav channel opening in T cells. T cell receptor stimulation induces only a small depolarization of T cells, and artificial depolarization of T cells using KCl does not lead to calcium entry. These observations suggest that the Cav channels expressed by T cells have adopted novel regulation/gating mechanisms.

Invariant chain, a chain of command

Abstract Invariant chain (Ii) is a type II integral membrane protein, which acts as a chaperone for MHC class II protein expression and facilitates antigen presentation. Recently, an additional role for Ii in the differentiation of immature to mature B cells has been described. These studies showed that Ii acts as a signaling molecule; its cytosolic domain induces B-cell maturation by activation of transcription mediated by the p65 member of the NF-κB family, a process that requires the B-cell-enriched coactivator, TAF II 105. NF-κB activation is mediated by the cytosolic region of Ii, which is liberated from the membrane. The process of intramembrane cleavage followed by nuclear translocation and transcriptional activation is reminiscent of regulated intramembrane cleavage (RIP). In this Review we suggest that the behavior of Ii shows remarkable similarities to the function of proteins processed by RIP and propose that the roles of Ii as a chaperone and as a signaling molecule are intertwined.

Invariant Chain-Induced B Cell Differentiation Requires Intramembrane Proteolytic Release of the Cytosolic Domain

Immature B cells differentiate in the spleen into mature B cells, a process that is essential for their participation in the immune response. Previously, we showed that the MHC class II chaperone, invariant chain (Ii), controls this differentiation to the mature stage. Ii cytosolic domain-induced B cell maturation involves activation of transcription mediated by the NF-kappaB p65/RelA homodimer and requires the B cell enriched coactivator, TAF(II)105. In this study we show that the cytosolic region of Ii is cleaved within the plane of the membrane to generate a cytosolic fragment, which is essential for NF-kappaB activation and B cell differentiation. Our results suggest that Ii functions as a membrane-bound inactive inducer of NF-kappaB transcription that is activated by intramembrane proteolytic cleavage.

Invariant chain induces B cell maturation in a process that is independent of its chaperonic activity

Early stages of B cell development take place in the bone marrow, resulting in formation of immature B cells, which migrate to the spleen for their final differentiation into mature cells. This final maturation step is essential for B cells to become responsive to antigens and to participate in the immune response. Previously, we showed that the MHC class II chaperone, invariant chain (Ii), controls the differentiation of B cells from the immature to the mature stage. In this study, by generating transgenic mice expressing truncated Ii lacking its luminal domain, we could dissect the chaperonin activity of Ii from its role in B cell maturation. We demonstrate in vivo that Ii N-terminal domain is directly involved in the maturation of B cells and is sufficient to promote B cell differentiation.

A Scaffold Protein, AHNAK1, Is Required for Calcium Signaling during T Cell Activation

Engagement of the T cell antigen receptor (TCR) during antigen presentation initiates a coordinated action of a large number of signaling proteins and ion channels. AHNAK1 is a scaffold protein, highly expressed by CD4+ T cells, and is a critical component for calcium signaling. We showed that AHNAK1-deficient mice were highly susceptible to Leishmania major infection. AHNAK1-deficient CD4+ T cells responded poorly to TCR stimulation in vitro with low proliferation and low Interleukin-2 production. Furthermore, AHNAK1 deficiency resulted in a reduced calcium influx upon TCR crosslinking and subsequent poor activation of the transcription factor NFAT. AHNAK1 was required for plasma membrane expression of L-type calcium channels alpha 1S (Cav1.1), probably through its interaction with the beta regulatory subunit. Thus, AHNAK1 plays an essential role in T cell Ca2+ signaling through Cav1 channels, triggered via TCR activation; therefore, AHNAK1 is a potential target for therapeutic intervention.

Roles of Cav channels and AHNAK1 in T cells: the beauty and the beast

Summary:  T lymphocytes require Ca2+ entry though the plasma membrane for their activation and function. Recently, several routes for Ca2+ entry through the T‐cell plasma membrane after activation have been described. These include calcium release‐activated channels (CRAC), transient receptor potential (TRP) channels, and inositol‐1,4,5‐trisphosphate receptors (IP3Rs). Herein we review the emergence of a fourth new route for Ca2+ entry, composed of Cav channels (also known as L‐type voltage‐gated calcium channels) and the scaffold protein AHNAK1 (AHNAK/desmoyokin). Both helper (CD4+) and killer (CD8+) T cells express high levels of Cav1 α1 subunits (α1S, α1C, α1D, and α1F) and AHNAK1 after their differentiation and require these molecules for Ca2+ entry during an immune response. In this article, we describe the observations and open questions that ultimately suggest the involvement of multiple consecutive routes for Ca2+ entry into lymphocytes, one of which may be mediated by Cav channels and AHNAK1.

Requirement for AHNAK1-mediated calcium signaling during T lymphocyte cytolysis

Cytolytic CD8+ T cells (CTLs) kill virally infected cells, tumor cells, or other potentially autoreactive T cells in a calcium-dependent manner. To date, the molecular mechanism that leads to calcium intake during CTL differentiation and function has remained unresolved. We demonstrate that desmoyokin (AHNAK1) is expressed in mature CTLs, but not in naive CD8+ T cells, and is critical for calcium entry required for their proper function during immune response. We show that mature AHNAK1-deficient CTLs exhibit reduced Cav1.1 α1 subunit expression (also referred to as L-type calcium channels or α1S pore-forming subunits), which recently were suggested to play a role in calcium entry into CD4+ T cells. AHNAK1-deficient CTLs show marked reduction in granzyme-B production, cytolytic activity, and IFN-γ secretion after T cell receptor stimulation. Our results demonstrate an AHNAK1-dependent mechanism controlling calcium entry during CTL effector function.

Myocardial Mitochondrial Injury Induced by Pulmonary Exposure to Particulate Matter in Rats

Exposure to air pollution has been associated with acute myocardial ischemia, impaired myocardrial function, and ST-segment depression. Particulate matter (PM)–associated metals, especially vanadium and nickel, have been implicated in observed cardiovascular impairments. We aimed to assess the effect of single intratracheal pulmonary exposure to vanadium-rich respirable oil combustion PM (HP-10) on the intrinsic myocardial ischemic tolerance and mitochondrial integrity in rats. The authors subjected isolated heart tissue slices derived from saline or PM-exposed rats to low glucose low oxygen induced ischemia followed by oxygenated condition with glucose supplementation. Mitochondrial structural integrity was determined by TEM (transmission electron microscopy) and functionality by the 3-(4, 5 dimethylthiazol-2yl)-2, 5 diphenyltetrazolium bromide (MTT) assay. Rats exposed to PM exhibited no apparent inhibition of mitochondrial dehydrogenase activity in oxygenated conditions at 24 or 48 hr post–PM exposure. However, in conditions of simulated ischemia/reoxygenation, these heart slices showed a delayed but consistent and significant decrease in dehydrogenase activity compared to controls at 48 hr after exposure to PM. Electron microscopy revealed significant myocardial mitochondrial injury upon exposure to PM characterized by mitochondrial swelling and fusion. The authors conclude that exposure to soluble vanadium-rich PM induces mitochondrial functional impairment and structural abnormality, which compromises mitochondrial respiration and results in decreased tolerance to ischemia/reoxygenation in rats.

T Cell Receptor Mediated Calcium Entry Requires Alternatively Spliced Cav1.1 Channels

The process of calcium entry in T cells is a multichannel and multi-step process. We have studied the requirement for L-type calcium channels (Cav1.1) α1S subunits during calcium entry after TCR stimulation. High expression levels of Cav1.1 channels were detected in activated T cells. Sequencing and cloning of Cav1.1 channel cDNA from T cells revealed that a single splice variant is expressed. This variant lacks exon 29, which encodes the linker region adjacent to the voltage sensor, but contains five new N-terminal exons that substitute for exons 1 and 2, which are found in the Cav1.1 muscle counterpart. Overexpression studies using cloned T cell Cav1.1 in 293HEK cells (that lack TCR) suggest that the gating of these channels was altered. Knockdown of Cav1.1 channels in T cells abrogated calcium entry after TCR stimulation, suggesting that Cav1.1 channels are controlled by TCR signaling.