Biology subjects

Csanady, L.

Publications and source records attributed to Csanady, L..

5 recordsLinked to original sources

bioRxiv2025

Evolution of temperature dependence of TRPM2 channel gating and inactivation in vertebrates

TRPM2 is a Ca2+ permeable cation channel activated by cytosolic Ca2+ and ADP ribose (ADPR). In contrast to invertebrate orthologs, vertebrate TRPM2 channels inactivate due to evolutionary alterations in amino acid sequence around the selectivity filter. Human TRPM2 (hsTRPM2) serves as a deep-brain temperature sensor important for body temperature regulation, and its gating is exquisitely temperature dependent. To address whether TRPM2 is temperature sensitive also in ectotherms that lack body temperature regulation, here we investigated the functional properties of zebrafish (Danio rerio) TRPM2 (drTRPM2) across the temperature range of 15{degrees}C to 37{degrees}C. The rate of ion permeation through the open pore was weakly temperature sensitive (Q10[~]1.3) as expected for a diffusion-limited process. In the presence of saturating concentrations of ligands (ADPR and Ca2+) drTRPM2 open probability showed no temperature dependence. Moreover, for both ADPR and Ca2+, the apparent affinities for channel activation were unaffected by temperature, reporting a standard enthalpy of opening near zero for drTRPM2. Inactivation of drTRPM2 is an order of magnitude slower than that of hsTRPM2. To address whether in both orthologs the same mechanism underlies inactivation, we studied its temperature sensitivity. For both drTRPM2 and hsTRPM2 inactivation rate was modestly temperature dependent (Q10 [~]2.7 and [~]4.4). A triple substitution which converts the post-filter sequence of drTRPM2 into the corresponding human sequence accelerated drTRPM2 inactivation by >20-fold. The data suggest that temperature dependence of hsTRPM2 gating evolved in the course of vertebrate evolution, whereas inactivation was temperature dependent already when it first appeared in early vertebrates.

biophysics↗

bioRxiv2024

Structural determinants of protein kinase A essential for CFTR channel activation

CFTR, the anion channel mutated in cystic fibrosis (CF) patients, is activated by the catalytic subunit of protein kinase A (PKA-C). PKA-C activates CFTR both reversibly, through binding, and irreversibly, through phosphorylation of multiple serines in CFTRs regulatory (R) domain. Here we identify key molecular determinants of the CFTR/PKA-C interaction essential for these processes. By comparing CFTR current activation in the presence of ATP or an ATP analog unsuitable for phosphotransfer, as well as pseudosubstrate peptides of various lengths, we identify two distinct specific regions of the PKA-C surface which interact with CFTR to cause reversible and irreversible CFTR stimulation, respectively. Whereas the "substrate site" mediates CFTR phosphorylation, a distinct hydrophobic patch (the "docking site") is responsible for reversible CFTR activation, achieved by stabilizing the R domain in a "released" conformation permissive to channel gating. Furthermore, by comparing PKA-C variants with different posttranslational modification patterns we find that direct membrane tethering of the kinase through its N-terminal myristoyl group is an unappreciated fundamental requirement for CFTR activation: PKA-C demyristoylation abolishes reversible, and profoundly slows irreversible, CFTR stimulation. For the F508del CFTR mutant, present in [~]90% of CF patients, maximal activation by de-myristoylated PKA-C is reduced by [~]10-fold compared to that by myristoylated PKA-C. Finally, in bacterial genera that contain common CF pathogens we identify virulence factors that demyristoylate PKA-C in vitro, raising the possibility that during recurrent bacterial infections in CF patients PKA-C demyristoylation may contribute to the exacerbation of lung disease. Significance StatementCFTR is an anion channel crucial for salt-water transport across epithelia, and is activated by the catalytic subunit of protein kinase A (PKA-C). Reduced activity of mutant CFTR causes cystic fibrosis and CFTR hyperstimulation by sustained PKA-C activity causes diarrhea. PKA-C activates CFTR reversibly through simple binding, and irreversibly by phosphorylating the channel. We uncover here important structural requirements for these two processes. First, two distinct PKA-C surface areas mediate reversible and irreversible CFTR activation. Second, membrane anchoring of PKA-C through a covalently linked fatty (myristic) acid is required for both effects. Finally, we identify bacterial enzymes that cleave the myristic acid from PKA-C, thereby reducing activation of mutant CFTR channels, present in cystic fibrosis patients, by up to tenfold.

physiology↗

bioRxiv2024

The structures of protein kinase A in complex with CFTR: mechanisms of phosphorylation and reversible activation

Protein kinase A (PKA) is a key regulator of cellular functions by selectively phosphorylating numerous substrates, including ion channels, enzymes, and transcription factors. It has long served as a model system for understanding the eukaryotic kinases. Using cryo-electron microscopy, we present complex structures of the PKA catalytic subunit (PKA-C) bound to a full-length protein substrate, the cystic fibrosis transmembrane conductance regulator (CFTR) - an ion channel vital to human health. CFTR gating requires phosphorylation of its regulatory (R) domain. Unphosphorylated CFTR engages PKA-C at two locations, establishing two "catalytic stations" near to, but not directly involving, the R domain. This configuration, coupled with the conformational flexibility of the R domain, permits transient interactions of the eleven spatially separated phosphorylation sites. Furthermore, we determined two structures of the open-pore CFTR stabilized by PKA-C, providing a molecular basis for understanding reversible activation, whereby PKA-C stimulates CFTR function through simple binding.

biophysics↗

bioRxiv2023

Estimating the true stability of the prehydrolytic outward-facing state in an ABC protein

CFTR, the anion channel mutated in cystic fibrosis patients, is a model ABC protein whose ATP-driven conformational cycle is observable at single-molecule level in patch-clamp recordings. Bursts of CFTR pore openings are coupled to tight dimerization of its two nucleotide binding domains (NBDs) and in wild-type (WT) channels are mostly terminated by ATP hydrolysis. The slow rate of non-hydrolytic closure - which determines how tightly bursts and ATP hydrolysis are coupled - is unknown, as burst durations of catalytic site mutants span a range of [~]200-fold. Here we show that Walker A mutation K1250A, Walker B mutation D1370N, and catalytic glutamate mutations E1371S and E1371Q all completely disrupt ATP hydrolysis. True non-hydrolytic closing rate of WT CFTR approximates that of K1250A and E1371S. That rate is slowed [~]15-fold in E1371Q by a non-native inter-NBD H-bond, and accelerated [~]15-fold in D1370N. These findings uncover unique features of the NBD interface in human CFTR.

biophysics↗

bioRxiv2021

Molecular pathology of a cystic fibrosis mutation is explained by loss of a hydrogen bond

The phosphorylation-activated anion channel CFTR is gated by an ATP hydrolysis cycle at its two cytosolic nucleotide binding domains, and is essential for epithelial salt-water transport. A large number of CFTR mutations cause cystic fibrosis. Since recent breakthrough in targeted pharmacotherapy, CFTR mutants with impaired gating are candidates for stimulation by potentiator drugs. Thus, understanding the molecular pathology of individual mutations has become important. The relatively common R117H mutation affects an extracellular loop, but nevertheless causes a strong gating defect. Here we identify a hydrogen bond between the side chain of arginine 117 and the backbone carbonyl group of glutamate 1124 in the cryo-electronmicroscopic structure of phosphorylated, ATP-bound CFTR. We address the functional relevance of that interaction for CFTR gating using macroscopic and microscopic inside-out patch-clamp recordings. Employing thermodynamic double-mutant cycles, we systematically track gating-state dependent changes in the strength of the R117-E1124 interaction. We find that the H-bond is formed only in the open state, but neither in the short-lived "flickery" nor in the long-lived "interburst" closed state. Loss of this H-bond explains the entire gating phenotype of the R117H mutant, including robustly shortened burst durations and strongly reduced intraburst open probability. The findings may help targeted potentiator design.

biophysics↗