Functional basis for calmodulation of the TRPV5 calcium channel.

Within the transient receptor potential (TRP) superfamily of ion channels, TRPV5 is a highly Ca2+ -selective channel important for active reabsorption of Ca2+ in the kidney. Its channel activity is controlled by a negative feedback mechanism involving calmodulin (CaM) binding. Combining advanced microscopy techniques and biochemical assays, this study characterized the dynamic lobe-specific CaM regulation. We demonstrate for the first time that functional (full-length) TRPV5 interacts with CaM in the absence of Ca2+ , and this interaction is intensified at increasing Ca2+ concentrations sensed by the CaM C-lobe that achieves channel pore blocking. Channel inactivation occurs without requiring CaM N-lobe calcification. Moreover, we show a Ca2+ -dependent binding stoichiometry at the single channel level. In conclusion, our study proposes a new model for CaM-dependent regulation - calmodulation - of this uniquely Ca2+ -selective TRP channel TRPV5 that involves apoCaM interaction and lobe-specific actions, which may be of significant physiological relevance given its role as gatekeeper of Ca2+ transport in the kidney. KEY POINTS: The renal Ca2+ channel TRPV5 is an important player in maintenance of the body's Ca2+ homeostasis. Activity of TRPV5 is controlled by a negative feedback loop that involves calmodulin (CaM), a protein with two Ca2+ -binding lobes. We investigated the dynamics of the interaction between TRPV5 and CaM with advanced fluorescence microscopy techniques. Our data support a new model for CaM-dependent regulation of TRPV5 channel activity with CaM lobe-specific actions and demonstrates Ca2+ -dependent binding stoichiometries. This study improves our understanding of the mechanism underlying fast channel inactivation, which is physiologically relevant given the gatekeeper function of TRPV5 in Ca2+ reabsorption in the kidney.

Butyrate reduces cellular magnesium absorption independently of metabolic regulation in Caco-2 human colon cells.

Digestion of dietary fibers by gut bacteria has been shown to stimulate intestinal mineral absorption [e.g., calcium (Ca2+) and magnesium (Mg2+)]. Although it has been suggested that local pH and short-chain fatty acid (SCFA) concentrations determine divalent cation absorption, the exact molecular mechanisms are still unknown. Therefore, this study aimed to determine the effects of SCFAs on intestinal Mg2+ absorption. We show that the butyrate concentration in the colon negatively correlates with serum Mg2+ levels in wildtype mice. Moreover, Na-butyrate significantly inhibited Mg2+ uptake in Caco-2 cells, while Ca2+ uptake was unaffected. Although Na-butyrate significantly lowered total ATP production rate, and resulted in increased phosphorylation of AMP-activated protein kinase (AMPK), inhibition of Mg2+ uptake by butyrate preceded these consequences. Importantly, electrophysiological examinations demonstrated that intracellular butyrate directly reduced the activity of the heteromeric Mg2+ channel complex, transient receptor potential melastatin (TRPM)6/7. Blocking cellular butyrate uptake prevented its inhibitory effect on Mg2+ uptake, demonstrating that butyrate acts intracellularly. Our work identified butyrate as novel regulator of intestinal Mg2+ uptake that works independently from metabolic regulation. This finding further highlights the role of microbial fermentation in the regulation of mineral absorption.

Oligomerization and Spatial Distribution of Kvß1.1 and Kvß2.1 Regulatory Subunits.

Members of the regulatory Kvß family modulate the kinetics and traffic of voltage-dependent K+ (Kv) channels. The crystal structure of Kv channels associated with Kvß peptides suggests a α4/ß4 composition. Although Kvß2 and Kvß1 form heteromers, evidence supports that only Kvß2.1 forms tetramers in the absence of α subunits. Therefore, the stoichiometry of the Kvß oligomers fine-tunes the activity of hetero-oligomeric Kv channel complexes. We demonstrate that Kvß subtypes form homo- and heterotetramers with similar affinities. The Kvß1.1/Kvß2.1 heteromer showed an altered spatial distribution in lipid rafts, recapitulating the Kvß1.1 pattern. Because Kvß2 is an active partner of the Kv1.3-TCR complex at the immunological synapse (IS), an association with Kvß1 would alter this location, shaping the immune response. Differential regulation of Kvßs influences the traffic and architecture of the Kvß heterotetramer, modulating Kvß-dependent physiological responses.

S-acylation-dependent membrane microdomain localization of the regulatory Kvß2.1 subunit.

The voltage-dependent potassium (Kv) channel Kvß family was the first identified group of modulators of Kv channels. Kvß regulation of the α-subunits, in addition to their aldoketoreductase activity, has been under extensive study. However, scarce information about their specific α-subunit-independent biology is available. The expression of Kvßs is ubiquitous and, similar to Kv channels, is tightly regulated in leukocytes. Although Kvß subunits exhibit cytosolic distribution, spatial localization, in close contact with plasma membrane Kv channels, is crucial for a proper immune response. Therefore, Kvß2.1 is located near cell surface Kv1.3 channels within the immunological synapse during lymphocyte activation. The objective of this study was to analyze the structural elements that participate in the cellular distribution of Kvßs. It was demonstrated that Kvß peptides, in addition to the cytoplasmic pattern, targeted the cell surface in the absence of Kv channels. Furthermore, Kvß2.1, but not Kvß1.1, targeted lipid raft microdomains in an S-acylation-dependent manner, which was concomitant with peptide localization within the immunological synapse. A pair of C-terminal cysteines (C301/C311) was mostly responsible for the specific palmitoylation of Kvß2.1. Several insults altered Kvß2.1 membrane localization. Therefore, growth factor-dependent proliferation enhanced surface targeting, whereas PKC activation impaired lipid raft expression. However, PSD95 stabilized Kvß2.1 in these domains. This data shed light on the molecular mechanism by which Kvß2.1 clusters into immunological synapses during leukocyte activation.

Calmodulin-dependent KCNE4 dimerization controls membrane targeting.

The voltage-dependent potassium channel Kv1.3 participates in the immune response. Kv1.3 is essential in different cellular functions, such as proliferation, activation and apoptosis. Because aberrant expression of Kv1.3 is linked to autoimmune diseases, fine-tuning its function is crucial for leukocyte physiology. Regulatory KCNE subunits are expressed in the immune system, and KCNE4 specifically tightly regulates Kv1.3. KCNE4 modulates Kv1.3 currents slowing activation, accelerating inactivation and retaining the channel at the endoplasmic reticulum (ER), thereby altering its membrane localization. In addition, KCNE4 genomic variants are associated with immune pathologies. Therefore, an in-depth knowledge of KCNE4 function is extremely relevant for understanding immune system physiology. We demonstrate that KCNE4 dimerizes, which is unique among KCNE regulatory peptide family members. Furthermore, the juxtamembrane tetraleucine carboxyl-terminal domain of KCNE4 is a structural platform in which Kv1.3, Ca2+/calmodulin (CaM) and dimerizing KCNE4 compete for multiple interaction partners. CaM-dependent KCNE4 dimerization controls KCNE4 membrane targeting and modulates its interaction with Kv1.3. KCNE4, which is highly retained at the ER, contains an important ER retention motif near the tetraleucine motif. Upon escaping the ER in a CaM-dependent pattern, KCNE4 follows a COP-II-dependent forward trafficking mechanism. Therefore, CaM, an essential signaling molecule that controls the dimerization and membrane targeting of KCNE4, modulates the KCNE4-dependent regulation of Kv1.3, which in turn fine-tunes leukocyte physiology.

KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology.

The voltage-dependent potassium channel Kv1.3 plays essential roles in the immune system, participating in leukocyte activation, proliferation and apoptosis. The regulatory subunit KCNE4 acts as an ancillary peptide of Kv1.3, modulates K+ currents and controls channel abundance at the cell surface. KCNE4-dependent regulation of the oligomeric complex fine-tunes the physiological role of Kv1.3. Thus, KCNE4 is crucial for Ca2+-dependent Kv1.3-related leukocyte functions. To better understand the role of KCNE4 in the regulation of the immune system, we manipulated its expression in various leukocyte cell lines. Jurkat T lymphocytes exhibit low KCNE4 levels, whereas CY15 dendritic cells, a model of professional antigen-presenting cells, robustly express KCNE4. When the cellular KCNE4 abundance was increased in T cells, the interaction between KCNE4 and Kv1.3 affected important T cell physiological features, such as channel rearrangement in the immunological synapse, cell growth, apoptosis and activation, as indicated by decreased IL-2 production. Conversely, ablation of KCNE4 in dendritic cells augmented proliferation. Furthermore, the LPS-dependent activation of CY15 cells, which induced Kv1.3 but not KCNE4, increased the Kv1.3-KCNE4 ratio and increased the expression of free Kv1.3 without KCNE4 interaction. Our results demonstrate that KCNE4 is a pivotal regulator of the Kv1.3 channelosome, which fine-tunes immune system physiology by modulating Kv1.3-associated leukocyte functions.

Functional Consequences of the Variable Stoichiometry of the Kv1.3-KCNE4 Complex.

The voltage-gated potassium channel Kv1.3 plays a crucial role during the immune response. The channel forms oligomeric complexes by associating with several modulatory subunits. KCNE4, one of the five members of the KCNE family, binds to Kv1.3, altering channel activity and membrane expression. The association of KCNEs with Kv channels is the subject of numerous studies, and the stoichiometry of such associations has led to an ongoing debate. The number of KCNE4 subunits that can interact and modulate Kv1.3 is unknown. KCNE4 transfers important elements to the Kv1.3 channelosome that negatively regulate channel function, thereby fine-tuning leukocyte physiology. The aim of this study was to determine the stoichiometry of the functional Kv1.3-KCNE4 complex. We demonstrate that as many as four KCNE4 subunits can bind to the same Kv1.3 channel, indicating a variable Kv1.3-KCNE4 stoichiometry. While increasing the number of KCNE4 subunits steadily slowed the activation of the channel and decreased the abundance of Kv1.3 at the cell surface, the presence of a single KCNE4 peptide was sufficient for the cooperative enhancement of the inactivating function of the channel. This variable architecture, which depends on KCNE4 availability, differentially affects Kv1.3 function. Therefore, our data indicate that the physiological remodeling of KCNE4 triggers functional consequences for Kv1.3, thus affecting cell physiology.

Metformin regulates TRPM6, a potential explanation for magnesium imbalance in type 2 diabetes patients.

Metformin therapy is associated with lower serum magnesium (Mg2+) levels in type 2 diabetes patients. The TRPM6 channel determines the fine-tuning of Mg2+ (re)absorption in intestine and kidney. Therefore, we aimed to investigate the short- and long-term effects of metformin on TRPM6. Patch clamp recordings and biotinylation assays were performed upon 1 h of incubation with metformin in TRPM6-transfected HEK293 cells. Additionally, 24 h of treatment of mDCT15 kidney and hCaco-2 colon cells with metformin was applied to measure the effects on endogenous TRPM6 expression by quantitative real-time PCR. To assess Mg2+ absorption, 25Mg2+ uptake measurements were performed using inductively coupled plasma mass spectrometry. Short-term effects of metformin significantly increased TRPM6 activity and its cell surface trafficking. In contrast, long-term effects significantly decreased TRPM6 mRNA expression and 25Mg2+ uptake. Metformin lowered TRPM6 mRNA levels independently of insulin- and AMPK-mediated pathways. Moreover, in type 2 diabetes patients, metformin therapy was associated with lower plasma Mg2+ concentrations and fractional excretion of Mg2+. Thereby, short-term metformin treatment increases TRPM6 activity explained by enhanced cell surface expression. Conversely, long-term metformin treatment results in downregulation of TRPM6 gene expression in intestine and kidney cells. This long-term effect translated in an inverse correlation between metformin and plasma Mg2+ concentration in type 2 diabetes patients.

The calmodulin-binding tetraleucine motif of KCNE4 is responsible for association with Kv1.3.

The voltage-dependent potassium (Kv) channel Kv1.3 regulates leukocyte proliferation, activation, and apoptosis, and altered expression of this channel is linked to autoimmune diseases. Thus, the fine-tuning of Kv1.3 function is crucial for the immune system response. The Kv1.3 accessory protein, potassium voltage-gated channel subfamily E (KCNE) subunit 4, acts as a dominant negative regulatory subunit to both enhance inactivation and induce intracellular retention of Kv1.3. Mutations in KCNE4 also cause immune system dysfunction. Although the formation of Kv1.3-KCNE4 complexes has profound consequences for leukocyte physiology, the molecular determinants involved in the Kv1.3-KCNE4 association are unknown. We now show that KCNE4 associates with Kv1.3 via a tetraleucine motif situated within the carboxy-terminal domain of this accessory protein. This motif would function as an interaction platform, in which Kv1.3 and Ca2+/calmodulin compete for the KCNE4 interaction. Finally, we propose a structural model of the Kv1.3-KCNE4 complex. Our experimental data and the in silico structure suggest that the KCNE4 interaction hides a forward-trafficking motif within Kv1.3 in addition to adding a strong endoplasmic reticulum retention signature to the Kv1.3-KCNE4 complex. Thus, the oligomeric composition of the Kv1.3 channelosome fine-tunes the precise balance between anterograde and intracellular retention elements that control the cell surface expression of Kv1.3 and immune system physiology.-Solé, L., Roig, S. R., Sastre, D., Vallejo-Gracia, A., Serrano-Albarrás, A., Ferrer-Montiel, A., Fernández-Ballester, G., Tamkun, M. M., Felipe, A. The calmodulin-binding tetraleucine motif of KCNE4 is responsible for association with Kv1.3.

The C-terminal domain of Kv1.3 regulates functional interactions with the KCNE4 subunit.

The voltage-dependent K+ channel Kv1.3 (also known as KCNA3), which plays crucial roles in leukocytes, physically interacts with KCNE4. This interaction inhibits the K+ currents because the channel is retained within intracellular compartments. Thus, KCNE subunits are regulators of K+ channels in the immune system. Although the canonical interactions of KCNE subunits with Kv7 channels are under intensive investigation, the molecular determinants governing the important Kv1.3- KCNE4 association in the immune system are unknown. Our results suggest that the tertiary structure of the C-terminal domain of Kv1.3 is necessary and sufficient for such an interaction. However, this element is apparently not involved in modulating Kv1.3 gating. Furthermore, the KCNE4-dependent intracellular retention of the channel, which negatively affects the activity of Kv1.3, is mediated by two independent and additive mechanisms. First, KCNE4 masks the YMVIEE signature at the C-terminus of Kv1.3, which is crucial for the surface targeting of the channel. Second, we identify a potent endoplasmic reticulum retention motif in KCNE4 that further limits cell surface expression. Our results define specific molecular determinants that play crucial roles in the physiological function of Kv1.3 in leukocytes.

A non-canonical di-acidic signal at the C-terminus of Kv1.3 determines anterograde trafficking and surface expression.

Impairment of Kv1.3 expression at the cell membrane in leukocytes and sensory neuron contributes to the pathophysiology of autoimmune diseases and sensory syndromes. Molecular mechanisms underlying Kv1.3 channel trafficking to the plasma membrane remain elusive. We report a novel non-canonical di-acidic signal (E483/484) at the C-terminus of Kv1.3 essential for anterograde transport and surface expression. Notably, homologous motifs are conserved in neuronal Kv1 and Shaker channels. Biochemical analysis revealed interactions with the Sec24 subunit of the coat protein complex II. Disruption of this complex retains the channel at the endoplasmic reticulum. A molecular model of the Kv1.3-Sec24a complex suggests salt-bridges between the di-acidic E483/484 motif in Kv1.3 and the di-basic R750/752 sequence in Sec24. These findings identify a previously unrecognized motif of Kv channels essential for their expression on the cell surface. Our results contribute to our understanding of how Kv1 channels target to the cell membrane, and provide new therapeutic strategies for the treatment of pathological conditions.

KCNE gene expression is dependent on the proliferation and mode of activation of leukocytes.

Voltage-dependent K (+) (Kv) channels are tightly regulated during the immune system response. Leukocytes have a limited repertoire of Kv channels, whose physiological role is under intense investigation. A functional Kv channel is an oligomeric complex composed of pore-forming and ancillary subunits. The KCNE gene family is a novel group of modulatory Kv channel elements in leukocytes. Here, we characterized the gene expression of KCNEs (1-5) in leukocytes and investigated their regulation during leukocyte proliferation and mode of activation. Murine bone-marrow-derived macrophages, human Jurkat T-lymphocytes and human Raji B-cells were analyzed. KCNEs (1-5) are expressed in all leukocytes lineages. Most KCNE mRNAs show cell cycle-dependent regulation and are differentially regulated under specific insults. Our results further suggest a new and yet undefined physiological role for KCNE subunits in the immune system. Putative associations of these ancillary proteins with Kv channels would yield a wide variety of biophysically and pharmacologically distinct channels that fine-tune the immunological response.