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 Ca_v1.1 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.Calcium plays critical roles in T-cell differentiation and function, such as activation, proliferation, and cytokine production (1, 2). Cytotoxic CD8⁺ effector T cells (CTLs) primarily use the perforin/granule exocytosis pathway to kill virus-infected and tumor cells (3). The role of calcium in CTL-mediated cytolysis has been studied extensively, mainly by using calcium antagonists (e.g., see ref. 4). The exact requirements for calcium and its molecular mode of entry during CTL function still are largely uncertain (4, 5).The calcium release-activated calcium channel (CRAC) pathway is the most studied plasma membrane store operated calcium (SOC) channel through which calcium enters after T-cell stimulation. Surprisingly, ORAI1 (also known as “CRACM1” or “TMEM142A”)-deficient CTLs indeed show reduced calcium entry but only partial IFN-γ production and normal granzyme-B expression, suggesting that other pore subunits, possibly ORAI2 or ORAI3, or different channels altogether, are involved in this process (6).In addition to CRAC channels (7), we and others (8, 9), found that L-type calcium channel (Ca_v1) subunits α1, α2, β, γ, and δ, which constitute the major route of calcium entry in excitable cells and take up calcium in response to membrane depolarization, also are expressed by T cells (10). Previously, we have shown that CD4⁺ T cells express α1 subunits of the Ca_v1 family and that functional Ca_v β4 and β3 regulatory subunits are necessary for normal TCR-triggered calcium response, nuclear factor of activated T cells (NFAT) nuclear translocation, and cytokine production (8, 11).The AHNAK family of scaffold PDZ proteins consists of 2 giant proteins (700 kDa), AHNAK1 (desmoyokin) and AHNAK2 (12–14). AHNAK1 is involved with calcium signaling through protein–protein interactions (15–18). Furthermore, in cardiomyocytes, AHNAK1 associates with the β-subunit of cardiac Ca_v channels at the plasma membrane and is phosphorylated by protein kinase A (PKA) in response to β-adrenoreceptor stimulation (19).Using AHNAK1-deficient mice (14), we recently described a novel mechanism for the regulation of calcium signaling through Ca_v1.1 α1 subunits mediated by AHNAK1 in peripheral CD4⁺ T cells. AHNAK1 is associated with the regulatory β2 subunit of Ca_v1 channels and is required for normal expression of the Ca_v1.1 α1 subunit and intact calcium influx following TCR cross-linking (11). Here, we demonstrate that CTLs employ AHNAK1 to mediate calcium entry required for cytolytic activity late in primary TCR stimulation through the regulation of Ca_v1.1 channels.