Rise of palmitoylation: A new trick to tune NCX1 activity.

The cardiac Na+/Ca2+ Exchanger (NCX1) controls transmembrane calcium flux in numerous tissues. The only reversible post-translational modification established to regulate NCX1 is palmitoylation, which alters the ability of the exchanger to inactivate. Palmitoylation creates a binding site for the endogenous XIP domain, a region of the NCX1 intracellular loop established to inactivate NCX1. The binding site created by NCX1 palmitoylation sensitizes the transporter to XIP. Herein we summarize our recent knowledge on NCX1 palmitoylation and its association with cardiac pathologies, and discuss these findings in the light of the recent cryo-EM structures of human NCX1.

Palmitoylation of the pore-forming subunit of Ca(v)1.2 controls channel voltage sensitivity and calcium transients in cardiac myocytes.

Mammalian voltage-activated L-type Ca2+ channels, such as Ca(v)1.2, control transmembrane Ca2+ fluxes in numerous excitable tissues. Here, we report that the pore-forming α1C subunit of Ca(v)1.2 is reversibly palmitoylated in rat, rabbit, and human ventricular myocytes. We map the palmitoylation sites to two regions of the channel: The N terminus and the linker between domains I and II. Whole-cell voltage clamping revealed a rightward shift of the Ca(v)1.2 current-voltage relationship when α1C was not palmitoylated. To examine function, we expressed dihydropyridine-resistant α1C in human induced pluripotent stem cell-derived cardiomyocytes and measured Ca2+ transients in the presence of nifedipine to block the endogenous channels. The transients generated by unpalmitoylatable channels displayed a similar activation time course but significantly reduced amplitude compared to those generated by wild-type channels. We thus conclude that palmitoylation controls the voltage sensitivity of Ca(v)1.2. Given that the identified Ca(v)1.2 palmitoylation sites are also conserved in most Ca(v)1 isoforms, we propose that palmitoylation of the pore-forming α1C subunit provides a means to regulate the voltage sensitivity of voltage-activated Ca2+ channels in excitable cells.

Palmitoylation regulates cellular distribution of and transmembrane Ca flux through TrpM7.

The bifunctional cation channel/kinase TrpM7 is ubiquitously expressed and regulates embryonic development and pathogenesis of several common diseases. The TrpM7 integral membrane ion channel domain regulates transmembrane movement of divalent cations, and its kinase domain controls gene expression via histone phosphorylation. Mechanisms regulating TrpM7 are elusive. It exists in two populations in the cell: at the cell surface where it controls divalent cation fluxes, and in intracellular vesicles where it controls zinc uptake and release. Here we report that TrpM7 is palmitoylated at a cluster of cysteines at the C terminal end of its Trp domain. Palmitoylation controls the exit of TrpM7 from the endoplasmic reticulum and the distribution of TrpM7 between cell surface and intracellular pools. Using the Retention Using Selective Hooks (RUSH) system, we demonstrate that palmitoylated TrpM7 traffics from the Golgi to the surface membrane whereas non-palmitoylated TrpM7 is sequestered in intracellular vesicles. We identify the Golgi-resident enzyme zDHHC17 and surface membrane-resident enzyme zDHHC5 as responsible for palmitoylating TrpM7 and find that TrpM7-mediated transmembrane calcium uptake is significantly reduced when TrpM7 is not palmitoylated. The closely related channel/kinase TrpM6 is also palmitoylated on the C terminal side of its Trp domain. Our findings demonstrate that palmitoylation controls ion channel activity of TrpM7 and that TrpM7 trafficking is dependant on its palmitoylation. We define a new mechanism for post translational modification and regulation of TrpM7 and other Trps.

Insulin-induced palmitoylation regulates the Cardiac Na+/Ca2+ exchanger NCX1.

The cardiac Na+/Ca2+ Exchanger (NCX1) controls Ca2+ extrusion from the cytosol by mediating bidirectional exchange of Na+ for Ca2+, and therefore controls cardiac relaxation. Insulin regulates Ca2+ handling in cardiac tissue through NCX1, however how insulin changes NCX1 activity is poorly understood. Palmitoylation is the only post-translational modification identified to alter NCX1 activity. Here we show that insulin triggers local structural re-arrangements within existing NCX1 dimers by inducing their palmitoylation, thus tunes NCX1 inactivation through a zDHHC5-dependent mechanism in multiple cell types. By activating fatty acid and fatty acyl CoA synthesis insulin promotes palmitoylation of the zDHHC5 active site, which leads to enhanced NCX1 palmitoylation. Our findings represent a new mechanism to regulate the palmitoylation of numerous zDHHC5 substrates.

Phosphodiesterase type 4 anchoring regulates cAMP signaling to Popeye domain-containing proteins.

Cyclic AMP is a ubiquitous second messenger used to transduce intracellular signals from a variety of Gs-coupled receptors. Compartmentalisation of protein intermediates within the cAMP signaling pathway underpins receptor-specific responses. The cAMP effector proteins protein-kinase A and EPAC are found in complexes that also contain phosphodiesterases whose presence ensures a coordinated cellular response to receptor activation events. Popeye domain containing (POPDC) proteins are the most recent class of cAMP effectors to be identified and have crucial roles in cardiac pacemaking and conduction. We report the first observation that POPDC proteins exist in complexes with members of the PDE4 family in cardiac myocytes. We show that POPDC1 preferentially binds the PDE4A sub-family via a specificity motif in the PDE4 UCR1 region and that PDE4s bind to the Popeye domain of POPDC1 in a region known to be susceptible to a mutation that causes human disease. Using a cell-permeable disruptor peptide that displaces the POPDC1-PDE4 complex we show that PDE4 activity localized to POPDC1 modulates cycle length of spontaneous Ca2+ transients firing in intact mouse sinoatrial nodes.

Mechanical Impedance of Rat Glabrous Skin and Its Relation With Skin Morphometry.

The mechanical impedance of intact and epidermis-peeled rat glabrous skin was studied at two sites (digit and sole) and at two frequencies (40 Hz and 250 Hz). The thicknesses of skin layers at the corresponding regions were measured histologically from intact- and peeled-skin samples in every subject. Compared to intact sole skin, digital rat skin has thicker layers and higher mechanical resistance, and it is less stiff. The resistance of the skin significantly decreased after epidermal peeling at both the digit and the sole. Furthermore, peeling caused the reactance to become positive due to inertial effects. As the frequency was increased from 40 to 250 Hz, the resistance and stiffness also increased for the intact skin, while the peeled skin showed less frictional (i.e., resistance) but more inertial (i.e., positive reactance) effects. We estimated the mechanical properties of epidermis and dermis with lumped-element models developed for both intact and peeled conditions. The models predicted that dermis has higher mass, lower stiffness, and lower resistance compared to epidermis, similar to the experimental impedance results obtained in the peeled condition which consisted mostly of dermis. The overall impedance was simulated more successfully at 40 Hz. When both frequencies are considered, the models produced consistent results for resistance in both conditions. The results imply that most of the model parameters should be frequency-dependent and suggest that mechanical properties of epidermis can be related to its thickness. These findings may help in designing artificial skin for neuroprosthetic limbs.

Insights into the molecular basis of the palmitoylation and depalmitoylation of NCX1.

Catalyzed by zDHHC-PAT enzymes and reversed by thioesterases, protein palmitoylation is the only post-translational modification recognized to regulate the sodium/calcium exchanger NCX1. NCX1 palmitoylation occurs at a single site at position 739 in its large regulatory intracellular loop. An amphipathic ɑ-helix between residues 740-756 is a critical for NCX1 palmitoylation. Given the rich background of the structural elements involving in NCX1 palmitoylation, the molecular basis of NCX1 palmitoylation is still relatively poorly understood. Here we found that (1) the identity of palmitoylation machinery of NCX1 controls its spatial organization within the cell, (2) the NCX1 amphipathic ɑ-helix directly interacts with zDHHC-PATs, (3) NCX1 is still palmitoylated when it is arrested in either Golgi or ER, indicating that NCX1 is a substrate for multiple zDHHC-PATs, (4) the thioesterase APT1 but not APT2 as a part of NCX1-depalmitoylation machinery governs subcellular organization of NCX1, (5) APT1 catalyzes NCX1 depalmitoylation in the Golgi but not in the ER. We also report that NCX2 and NCX3 are dually palmitoylated, with important implications for substrate recognition and enzyme catalysis by zDHHC-PATs. Our results could support new molecular or pharmacological strategies targeting the NCX1 palmitoylation and depalmitoylation machinery.

Topical review: Shedding light on molecular and cellular consequences of NCX1 palmitoylation.

Palmitoylation is the post-translational, covalent and reversible conjugation of a 16C saturated fatty acid to cysteine residues of proteins. The sodium calcium exchanger NCX1 is palmitoylated at a single cysteine residue in its large regulatory intracellular loop. Inactivation, mediated by the NCX1 inhibitory region XIP, is drastically impaired in unpalmitoylatable NCX1. The ability of XIP to bind and inactivate NCX1 is largely determined by NCX1 palmitoylation, which induces local conformational changes in the NCX1 intracellular loop to enable XIP to engage its binding site. Consequently, NCX1 palmitoylation regulates intracellular calcium by changing NCX1 sensitivity to inactivation. NCX1 palmitoylation is a dynamic phenomenon which is catalyzed by the palmitoyl acyl transferase zDHHC5 and reversed by the thioesterase APT1, with the switch between palmitoylated and depalmitoylated states, which has profound effects on NCX1 lipid interactions, influenced by NCX1 conformational poise. Herein we review the molecular and cellular consequences of NCX1 palmitoylation and its physiological relevance and highlight the importance of palmitoylation for NCX1 activity. We discuss the cellular control of protein palmitoylation and depalmitoylation, the relationship between lipid microdomains and lipidated and phospholipid binding proteins, and highlight the important unanswered questions in this emerging field.

Dynamic Palmitoylation of the Sodium-Calcium Exchanger Modulates Its Structure, Affinity for Lipid-Ordered Domains, and Inhibition by XIP.

The transmembrane sodium-calcium (Na-Ca) exchanger 1 (NCX1) regulates cytoplasmic Ca levels by facilitating electrogenic exchange of Ca for Na. Palmitoylation, the only reversible post-translational modification known to modulate NCX1 activity, controls NCX1 inactivation. Here, we show that palmitoylation of NCX1 modifies the structural arrangement of the NCX1 dimer and controls its affinity for lipid-ordered membrane domains. NCX1 palmitoylation occurs dynamically at the cell surface under the control of the enzymes zDHHC5 and APT1. We identify the position of the endogenous exchange inhibitory peptide (XIP) binding site within the NCX1 regulatory intracellular loop and demonstrate that palmitoylation controls the ability of XIP to bind this site. We also show that changes in NCX1 palmitoylation change cytosolic Ca. Our results thus demonstrate the broad molecular consequences of NCX1 palmitoylation and highlight a means to manipulate the inactivation of this ubiquitous ion transporter that could ameliorate pathologies linked to Ca overload via NCX1.

Regulation of NCX1 by palmitoylation.

Palmitoylation (S-acylation) is the reversible conjugation of a fatty acid (usually C16 palmitate) to intracellular cysteine residues of proteins via a thioester linkage. Palmitoylation anchors intracellular regions of proteins to membranes because the palmitoylated cysteine is recruited to the lipid bilayer. NCX1 is palmitoylated at a single cysteine in its large regulatory intracellular loop. The presence of an amphipathic α-helix immediately adjacent to the NCX1 palmitoylation site is required for NCX1 palmitoylation. The NCX1 palmitoylation site is conserved through most metazoan phlya. Although palmitoylation does not regulate the normal forward or reverse ion transport modes of NCX1, NCX1 palmitoylation is required for its inactivation: sodium-dependent inactivation and inactivation by PIP2 depletion are significantly impaired for unpalmitoylatable NCX1. Here we review the role of palmitoylation in regulating NCX1 activity, and highlight future questions that must be addressed to fully understand the importance of this regulatory mechanism for sodium and calcium transport in cardiac muscle.

Small-molecule inhibition of STOML3 oligomerization reverses pathological mechanical hypersensitivity.

The skin is equipped with specialized mechanoreceptors that allow the perception of the slightest brush. Indeed, some mechanoreceptors can detect even nanometer-scale movements. Movement is transformed into electrical signals via the gating of mechanically activated ion channels at sensory endings in the skin. The sensitivity of Piezo mechanically gated ion channels is controlled by stomatin-like protein-3 (STOML3), which is required for normal mechanoreceptor function. Here we identify small-molecule inhibitors of STOML3 oligomerization that reversibly reduce the sensitivity of mechanically gated currents in sensory neurons and silence mechanoreceptors in vivo. STOML3 inhibitors in the skin also reversibly attenuate fine touch perception in normal mice. Under pathophysiological conditions following nerve injury or diabetic neuropathy, the slightest touch can produce pain, and here STOML3 inhibitors can reverse mechanical hypersensitivity. Thus, small molecules applied locally to the skin can be used to modulate touch and may represent peripherally available drugs to treat tactile-driven pain following neuropathy.