Taufiq Rahman

An NAADP-gated two-pore channel targeted to the plasma membrane uncouples triggering from amplifying Ca2+ signals

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous messenger proposed to stimulate Ca2+ release from acidic organelles via two-pore channels (TPCs). It has been difficult to resolve this trigger event from its amplification via endoplasmic reticulum Ca2+ stores, fuelling speculation that archetypal intracellular Ca2+ channels are the primary targets of NAADP. Here, we redirect TPC2 from lysosomes to the plasma membrane and show that NAADP evokes Ca2+ influx independent of ryanodine receptors and that it activates a Ca2+-permeable channel whose conductance is reduced by mutation of a residue within a putative pore. We therefore uncouple TPC2 from amplification pathways and prove that it is a pore-forming subunit of an NAADP-gated Ca2+ channel.

Synthetic partial agonists reveal key steps in IP3 receptor activation.

Inositol 1,4,5-trisphosphate receptors (IP3R) are ubiquitous intracellular Ca2+ channels. IP3binding to the IP3-binding core (IBC) near the N-terminal initiates conformational changes that lead to opening of a pore. The mechanisms are unresolved. We synthesized 2-O-modified IP3 analogues that are partial agonists of IP3R. These are like IP3 in their interactions with the IBC, but they are less effective than IP3 in rearranging the relationship between the IBC and N-terminal suppressor domain (SD), and they open the channel at slower rates. IP3R with a mutation in the SD occupying a position similar to the 2-O-substituent of the partial agonists has a reduced open probability that is similar for full and partial agonists. Bulky or charged substituents from either the ligand or SD therefore block obligatory coupling of the IBC and SD. Analysis of ΔG for ligand binding shows that IP3 is recognised by the IBC and conformational changes then propagate entirely via the SD to the pore.

The endo-lysosomal system as an NAADP-sensitive acidic Ca2+ store: Role for the two-pore channels

Accumulating evidence suggests that the endo-lysosomal system provides a substantial store of Ca(2+) that is tapped by the Ca(2+)-mobilizing messenger, NAADP. In this article, we review evidence that NAADP-mediated Ca(2+) release from this acidic Ca(2+) store proceeds through activation of the newly described two-pore channels (TPCs). We discuss recent advances in defining the sub-cellular targeting, topology and biophysics of TPCs. We also discuss physiological roles and the evolution of this ubiquitous ion channel family.

Regulation of Inositol 1,4,5-Trisphosphate Receptors by cAMP Independent of cAMP-dependent Protein Kinase

In HEK cells stably expressing type 1 receptors for parathyroid hormone (PTH), PTH causes a sensitization of inositol 1,4,5-trisphosphate receptors (IP3R) to IP3 that is entirely mediated by cAMP and requires cAMP to pass directly from type 6 adenylyl cyclase (AC6) to IP3R2. Using DT40 cells expressing single subtypes of mammalian IP3R, we demonstrate that high concentrations of cAMP similarly sensitize all IP3R isoforms to IP3 by a mechanism that does not require cAMP-dependent protein kinase (PKA). IP3 binding to IP3R2 is unaffected by cAMP, and sensitization is not mediated by the site through which ATP potentiates responses to IP3. In single channel recordings from excised nuclear patches of cells expressing IP3R2, cAMP alone had no effect, but it increased the open probability of IP3R2 activated by a submaximal concentration of IP3 alone or in combination with a maximally effective concentration of ATP. These results establish that cAMP itself increases the sensitivity of all IP3R subtypes to IP3. For IP3R2, this sensitization results from cAMP binding to a novel site that increases the efficacy of IP3. Using stably expressed short hairpin RNA to reduce expression of the G-protein, Gαs, we demonstrate that attenuation of AC activity by loss of Gαs more substantially reduces sensitization of IP3R by PTH than does comparable direct inhibition of AC. This suggests that Gαs may also specifically associate with each AC·IP3R complex. We conclude that all three subtypes of IP3R are regulated by cAMP independent of PKA. In HEK cells, where IP3R2 selectively associates with AC6, Gαs also associates with the AC·IP3R signaling junction.

IP3 receptors: some lessons from DT40 cells.

Summary:  Inositol‐1,4,5‐trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels that are regulated by IP3 and Ca2+ and are modulated by many additional signals. These properties allow them to initiate and, via Ca2+‐induced Ca2+ release, regeneratively propagate Ca2+ signals evoked by receptors that stimulate formation of IP3. The ubiquitous expression of IP3R highlights their importance, but it also presents problems when attempting to resolve the behavior of defined IP3R. DT40 cells are a pre‐B‐lymphocyte cell line in which high rates of homologous recombination afford unrivalled opportunities to disrupt endogenous genes. DT40‐knockout cells with both alleles of each of the three IP3R genes disrupted provide the only null‐background for analysis of homogenous recombinant IP3R. We review the properties of DT40 cells and consider three areas where they have contributed to understanding IP3R behavior. Patch‐clamp recording from the nuclear envelope and Ca2+ release from intracellular stores loaded with a low‐affinity Ca2+ indicator address the mechanisms leading to activation of IP3R. We show that IP3 causes intracellular IP3R to cluster and re‐tune their responses to IP3 and Ca2+, better equipping them to mediate regenerative Ca2+ signals. Finally, we show that DT40 cells reliably count very few IP3R into the plasma membrane, where they mediate about half the Ca2+ entry evoked by the B‐cell antigen receptor.

Dynamic regulation of IP3 receptor clustering and activity by IP3.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels. Their regulation by both IP3 and Ca2+ allows interactions between IP3Rs to generate a hierarchy of intracellular Ca2+ release events. These can progress from openings of single IP3R, through near-synchronous opening of a few IP3Rs within a cluster to much larger signals that give rise to regenerative Ca2+ waves that can invade the entire cell. We have used patch-clamp recording from excised nuclear membranes of DT40 cells expressing only IP3R3 and shown that low concentrations of IP3 rapidly and reversibly cause IP3Rs to assemble into small clusters. In addition to bringing IP3Rs close enough to allow Ca2+ released by one IP3R to regulate the activity of its neighbors, clustering also retunes the regulation of IP3Rs by IP3 and Ca2+. At resting cytosolic [Ca2+], lone IP3R are more sensitive to IP3 and the mean channel open time (~10ms) is twice as long as for clustered IP3R. When the cytosolic free [Ca2+] is increased to 1µM, to mimic the conditions that might prevail when an IP3R within a cluster opens, clustered IP3R are no longer inhibited and their gating becomes coupled. IP3, by dynamically regulating IP3R clustering, both positions IP3R for optimal interactions between them and it serves to exaggerate the effects of Ca2+ within a cluster. During the course of these studies, we have observed that nuclear IP3R stably express one of two single channel K + conductances (γK ~120 or 200pS). Here we demonstrate that for both states of the IP3R, the effects of IP3 on clustering are indistinguishable. These observations reinforce our conclusion that IP3 dynamically regulates assembly of IP3Rs into clusters that underlie the hierarchical recruitment of elementary Ca2+ release events.

Two-pore channels provide insight into the evolution of voltage-gated Ca2+ and Na+ channels

Molecular modeling, pharmacology, and comparative genomics reveal secrets of ion channel evolution. Exploring Channel Evolution Voltage-gated ion channels are critical regulators of multiple aspects of physiology, including muscle contractility and nerve activity. The animal voltage-gated calcium channels (CaVs) and sodium channels (NaVs) are complex proteins that have four repeated regions that each include six transmembrane domains and a P-loop segment that partially penetrates the membrane and is involved in forming the pore. These four regions assemble into a pseudotetramer to create the ion channel. Another family of voltage-gated channels is the two-pore channel (TPC) family, members of which only have two of the repeated regions and are believed to form dimers that assemble into a pseudotetrameric structure similar to that of CaV and NaV channels. Rahman et al. constructed a model of TPCs using the crystal structure of a bacterial NaV that only has one, instead of four, of the repeated domains. Molecular docking analysis indicated that TPCs could bind pharmacological antagonists or agonists of CaVs or NaVs, and functional studies confirmed that TPC activity was altered. The predicted biophysical properties of the molecular docking experiments; sequence analysis of the predicted ion-selectivity filters of the CaVs, NaVs, and TPCs; and phylogenetic analysis of TPCs from unicellular organisms suggested that the evolution of ion specificity may have predated a gene duplication event that resulted in the evolution of CaVs and NaVs with the four repeated regions structure. Four-domain voltage-gated Ca2+ and Na+ channels (CaV, NaV) underpin nervous system function and likely emerged upon intragenic duplication of a primordial two-domain precursor. To investigate if two-pore channels (TPCs) may represent an intermediate in this evolutionary transition, we performed molecular docking simulations with a homology model of TPC1, which suggested that the pore region could bind antagonists of CaV or NaV. CaV or NaV antagonists blocked NAADP (nicotinic acid adenine dinucleotide phosphate)–evoked Ca2+ signals in sea urchin egg preparations and in intact cells that overexpressed TPC1. By sequence analysis and inspection of the model, we predicted a noncanonical selectivity filter in animal TPCs in which the carbonyl groups of conserved asparagine residues are positioned to coordinate cations. In contrast, a distinct clade of TPCs [TPCR (for TPC-related)] in several unicellular species had ion selectivity filters with acidic residues more akin to CaV. TPCRs were predicted to interact strongly with CaV antagonists. Our data suggest that acquisition of a “blueprint” pharmacological profile and changes in ion selectivity within four-domain voltage-gated ion channels may have predated intragenic duplication of an ancient two-domain ancestor.

The N-terminal region of two-pore channel 1 regulates trafficking and activation by NAADP

TPCs (two-pore channels) are NAADP (nicotinic acid-adenine dinucleotide phosphate)-sensitive Ca2+-permeable ion channels expressed on acidic organelles. In the present study we show that deletion of the N-terminal region redirects TPC1 to the ER (endoplasmic reticulum). The introduction of fluorophores at the N-terminus of TPC1 does not affect its subcellular location, but does reversibly abolish NAADP sensitivity. Our results reveal a dual role for the N-terminus in localization and function of TPC1.

Phytochemicals increase the antibacterial activity of antibiotics by acting on a drug efflux pump.

Drug efflux pumps confer resistance upon bacteria to a wide range of antibiotics from various classes. The expression of efflux pumps are also implicated in virulence and biofilm formation. Moreover, organisms can only acquire resistance in the presence of active drug efflux pumps. Therefore, efflux pump inhibitors (EPIs) are attractive compounds to reverse multidrug resistance and to prevent the development of resistance in clinically relevant bacterial pathogens. We investigated the potential of pure compounds isolated from plants to act as EPIs. In silico screening was used to predict the bioactivity of plant compounds and to compare that with the known EPI, phe‐arg‐β‐naphthylamide (PAβN). Subsequently, promising products have been tested for their ability to inhibit efflux. Plumbagin nordihydroguaretic acid (NDGA) and to a lesser degree shikonin, acted as sensitizers of drug‐resistant bacteria to currently used antibiotics and were able to inhibit the efflux pump‐mediated removal of substrate from cells. We demonstrated the feasibility of in silico screening to identify compounds that potentiate the action of antibiotics against drug‐resistant strains and which might be potentially useful lead compounds for an EPI discovery program.

CaBP1, a neuronal Ca2+ sensor protein, inhibits inositol trisphosphate receptors by clamping intersubunit interactions

Calcium-binding protein 1 (CaBP1) is a neuron-specific member of the calmodulin superfamily that regulates several Ca2+ channels, including inositol 1,4,5-trisphosphate receptors (InsP3Rs). CaBP1 alone does not affect InsP3R activity, but it inhibits InsP3-evoked Ca2+ release by slowing the rate of InsP3R opening. The inhibition is enhanced by Ca2+ binding to both the InsP3R and CaBP1. CaBP1 binds via its C lobe to the cytosolic N-terminal region (NT; residues 1–604) of InsP3R1. NMR paramagnetic relaxation enhancement analysis demonstrates that a cluster of hydrophobic residues (V101, L104, and V162) within the C lobe of CaBP1 that are exposed after Ca2+ binding interact with a complementary cluster of hydrophobic residues (L302, I364, and L393) in the β-domain of the InsP3-binding core. These residues are essential for CaBP1 binding to the NT and for inhibition of InsP3R activity by CaBP1. Docking analyses and paramagnetic relaxation enhancement structural restraints suggest that CaBP1 forms an extended tetrameric turret attached by the tetrameric NT to the cytosolic vestibule of the InsP3R pore. InsP3 activates InsP3Rs by initiating conformational changes that lead to disruption of an intersubunit interaction between a “hot-spot” loop in the suppressor domain (residues 1–223) and the InsP3-binding core β-domain. Targeted cross-linking of residues that contribute to this interface show that InsP3 attenuates cross-linking, whereas CaBP1 promotes it. We conclude that CaBP1 inhibits InsP3R activity by restricting the intersubunit movements that initiate gating.