Glycosylation of TRPM4 and TRPM5 channels: molecular determinants and functional aspects

The transient receptor potential channel, TRPM4, and its closest homolog, TRPM5, are non-selective cation channels that are activated by an increase in intracellular calcium. They are expressed in many cell types, including neurons and myocytes. Although the electrophysiological and pharmacological properties of these two channels have been previously studied, less is known about their regulation, in particular their post-translational modifications. We, and others, have reported that wild-type (WT) TRPM4 channels expressed in HEK293 cells, migrated on SDS-PAGE gel as doublets, similar to other ion channels and membrane proteins. In the present study, we provide evidence that TRPM4 and TRPM5 are each N-linked glycosylated at a unique residue, Asn(992) and Asn(932), respectively. N-linked glycosylated TRPM4 is also found in native cardiac cells. Biochemical experiments using HEK293 cells over-expressing WT TRPM4/5 or N992Q/N932Q mutants demonstrated that the abolishment of N-linked glycosylation did not alter the number of channels at the plasma membrane. In parallel, electrophysiological experiments demonstrated a decrease in the current density of both mutant channels, as compared to their respective controls, either due to the Asn to Gln mutations themselves or abolition of glycosylation. To discriminate between these possibilities, HEK293 cells expressing TRPM4 WT were treated with tunicamycin, an inhibitor of glycosylation. In contrast to N-glycosylation signal abolishment by mutagenesis, tunicamycin treatment led to an increase in the TRPM4-mediated current. Altogether, these results demonstrate that TRPM4 and TRPM5 are both N-linked glycosylated at a unique site and also suggest that TRPM4/5 glycosylation seems not to be involved in channel trafficking, but mainly in their functional regulation

Biochemical, single-channel, whole-cell patch clamp, and pharmacological analyses of endogenous TRPM4 channels in HEK293 cells

Human embryonic kidney cells 293 (HEK293) are widely used as cellular heterologous expression systems to study transfected ion channels. This work characterizes the endogenous expression of TRPM4 channels in HEK293 cells. TRPM4 is an intracellular Ca(2+)-activated non-selective cationic channel expressed in many cell types. Western blot analyses have revealed the endogenous expression of TRPM4. Single channel 22pS conductance with a linear current-voltage relationship was observed using the inside-out patch clamp configuration in the presence of intracellular Ca(2+). The channels were permeable to the monovalent cations Na(+) and K(+), but not to Ca(2+). The open probability was voltage-dependent, being higher at positive potentials. Using the whole-cell patch clamp "ruptured patch" configuration, the amplitude of the intracellular Ca(2+)-activated macroscopic current was dependent on time after patch rupture. Initial transient activation followed by a steady-increase reaching a plateau phase was observed. Biophysical analyses of the macroscopic current showed common properties with those from HEK293 cells stably transfected with human TRPM4b, with the exception of current time course and Ca(2+) sensitivity. The endogenous macroscopic current reached the plateau faster and required 61.9±3.5μM Ca(2+) to be half-maximally activated versus 84.2±1.5μM for the transfected current. The pharmacological properties, however, were similar in both conditions. One hundred μM of flufenamic acid and 9-phenanthrol strongly inhibited the endogenous current. Altogether, the data demonstrate the expression of endogenous TRMP4 channels in HEK293 cells. This observation should be taken into account when using this cell line to study TRPM4 or other types of Ca(2+)-activated channels

TRPM4 channels in the cardiovascular system: physiology, pathophysiology, and pharmacology

The transient receptor potential channel (TRP) family comprises at least 28 genes in the human genome. These channels are widely expressed in many different tissues, including those of the cardiovascular system. The transient receptor potential channel melastatin 4 (TRPM4) is a Ca(2+)-activated non-specific cationic channel, which is impermeable to Ca(2+). TRPM4 is expressed in many cells of the cardiovascular system, such as cardiac cells of the conduction pathway and arterial and venous smooth muscle cells. This review article summarizes the recently described roles of TRPM4 in normal physiology and in various disease states. Genetic variants in the human gene TRPM4 have been linked to several cardiac conduction disorders. TRPM4 has also been proposed to play a crucial role in secondary hemorrhage following spinal cord injuries. Spontaneously hypertensive rats with cardiac hypertrophy were shown to over-express the cardiac TRPM4 channel. Recent studies suggest that TRPM4 plays an important role in cardiovascular physiology and disease, even if most of the molecular and cellular mechanisms have yet to be elucidated. We conclude this review article with a brief overview of the compounds that have been shown to either inhibit or activate TRPM4 under experimental conditions. Based on recent findings, the TRPM4 channel can be proposed as a future target for the pharmacological treatment of cardiovascular disorders, such as hypertension and cardiac arrhythmias

Ubiquitin-specific protease USP2-45 acts as a molecular switch to promote α2δ-1-induced downregulation of Ca v1.2 channels

Availability of voltage-gated calcium channels (Cav) at the plasma membrane is paramount to maintaining the calcium homeostasis of the cell. It is proposed that the ubiquitylation/de-ubiquitylation balance regulates the density of ion channels at the cell surface. Voltage-gated calcium channels Cav1.2 have been found to be ubiquitylated under basal conditions both in vitro and in vivo. In a previous study, we have shown that Cav1.2 channels are ubiquitylated by neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4-1) ubiquitin ligases, but the identity of the counterpart de-ubiquitylating enzyme remained to be elucidated. Regarding sodium and potassium channels, it has been reported that the action of the related isoform Nedd4-2 is counteracted by the ubiquitin-specific protease (USP) 2-45. In this study, we show that USP 2-45 also de-ubiquitylates Cav channels. We co-expressed USPs and Cav1.2 channels together with the accessory subunits β2 and α2δ-1, in tsA-201 and HEK-293 mammalian cell lines. Using whole-cell current recordings and surface biotinylation assays, we show that USP2-45 specifically decreases both the amplitude of Cav currents and the amount of Cav1.2 subunits inserted at the plasma membrane. Importantly, co-expression of the α2δ-1 accessory subunit is necessary to support the effect of USP2-45. We further show that USP2-45 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits. Remarkably, α2δ-1, but not Cav1.2 nor β2, co-precipitated with USP2-45. These results suggest that USP2-45 binding to α2δ-1 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits, in order to regulate the expression of Cav1.2 channels at the plasma membrane

Muscling in on TRP channels in vascular smooth muscle cells and cardiomyocytes

The human TRP protein family comprises a family of 27 cation channels with diverse permeation and gating properties. The common theme is that they are very important regulators of intracellular Ca²⠺ signalling in diverse cell types, either by providing a Ca²⠺ influx pathway, or by depolarising the membrane potential, which on one hand triggers the activation of voltage-gated Ca²⠺ channels, and on the other limits the driving force for Ca²⠺ entry. Here we focus on the role of these TRP channels in vascular smooth muscle and cardiac striated muscle. We give an overview of highlights from the recent literature, and highlight the important and diverse roles of TRP channels in the pathophysiology of the cardiovascular system.status: publishe

The Ca(2+)-activated cation channel TRPM4 is a negative regulator of angiotensin II-induced cardiac hypertrophy

Cardiac muscle adapts to hemodynamic stress by altering myocyte size and function, resulting in cardiac hypertrophy. Alteration in myocyte calcium homeostasis is known to be an initial signal in cardiac hypertrophy signaling. Transient receptor potential melastatin 4 protein (TRPM4) is a calcium-activated non-selective cation channel, which plays a role in regulating calcium influx and calcium-dependent cell functions in many cell types including cardiomyocytes. Selective deletion of TRPM4 from the heart muscle in mice resulted in an increased hypertrophic growth after chronic angiotensin (AngII) treatment, compared to WT mice. The enhanced hypertrophic response was also traceable by the increased expression of hypertrophy-related genes like Rcan1, ANP, and α-Actin. Intracellular calcium measurements on isolated ventricular myocytes showed significantly increased store-operated calcium entry upon AngII treatment in myocytes lacking the TRPM4 channel. Elevated intracellular calcium is a key factor in the development of pathological cardiac hypertrophy, leading to the activation of intracellular signaling pathways. In agreement with this, we observed significantly higher Rcan1 mRNA level, calcineurin enzyme activity and protein level in lysates from TRPM4-deficient mice heart compared to WT after AngII treatment. Collectively, these observations are consistent with a model in which TRPM4 is a regulator of calcium homeostasis in cardiomyocytes after AngII stimulation. TRPM4 contributes to the regulation of driving force for store-operated calcium entry and thereby the activation of the calcineurin-NFAT pathway and the development of pathological hypertrophy.status: publishe

Molecular genetics and functional anomalies in a series of 248 Brugada cases with 11 mutations in the TRPM4 channel.

Brugada syndrome (BrS) is a condition defined by ST-segment alteration in right precordial leads and a risk of sudden death. Because BrS is often associated with right bundle branch block and the TRPM4 gene is involved in conduction blocks, we screened TRPM4 for anomalies in BrS cases. The DNA of 248 BrS cases with no SCN5A mutations were screened for TRPM4 mutations. Among this cohort, 20 patients had 11 TRPM4 mutations. Two mutations were previously associated with cardiac conduction blocks and 9 were new mutations (5 absent from ~14'000 control alleles and 4 statistically more prevalent in this BrS cohort than in control alleles). In addition to Brugada, three patients had a bifascicular block and 2 had a complete right bundle branch block. Functional and biochemical studies of 4 selected mutants revealed that these mutations resulted in either a decreased expression (p.Pro779Arg and p.Lys914X) or an increased expression (p.Thr873Ile and p.Leu1075Pro) of TRPM4 channel. TRPM4 mutations account for about 6% of BrS. Consequences of these mutations are diverse on channel electrophysiological and cellular expression. Because of its effect on the resting membrane potential, reduction or increase of TRPM4 channel function may both reduce the availability of sodium channel and thus lead to BrS

The politics of constraints : electoral promises, pending commitments, public concerns and policy agendas in Denmark, France, Spain and the United Kingdom (1980-2008)

Defence date: 8 January 2015Examining Board: Professor Pepper Culpepper, European University Institute (Supervisor); Professor E. Scott Adler, University of Colorado, Boulder (External Supervisor); Professor Stefano Bartolini, European University Institute; Professor Peter John, University College London.Who sets lawmakers' priorities? The aim of the thesis is to provide a convincing theoretical argument able to identify what are the policy problems that demand lawmakers' attention, but also to test this empirically for France, Denmark, Spain and the United Kingdom between 1980 and 2008. This research shows how accounting for the way in which lawmakers deal with competing policy problems integrate two major accounts of the way in which governments set their priorities: party mandate approaches and public policy approaches. The thesis does so by suggesting that given their double role of representatives and administrators, lawmakers have to deliver policies consistent both with electoral and non-electoral mandates. In this framework, parties’ promises, administrative commitments, and the priorities of the public originate policy problems that compete for lawmakers' attention to enter the policy agenda. Compared to classic party mandate approaches, this research does not conceive parties as being the key actors of the game or the major agenda-setters. Compared to public policy approaches, the study does not dismiss the role of parties. The theory argues that a problem-solving approach is key to account for lawmakers' priorities and for the way in which lawmakers select policy problems that need to be addressed in the policy agenda. In this framework, different policy problems demand lawmakers' attention and problems-solving scholars have illustrated that the types of issues that need to be addressed are different in "nature". Existing accounts of the composition of policy agendas distinguish between problems ranging from "compulsory" to "discretionary" concerns (Walker 1977; Adler and Wilkerson 2012) where the former derive from "periodically recurring demands " and the latter from "chosen problems" (Walker 1977:425). Building on these contributions, the theoretical model of the dissertation discusses the "nature" of different policy problems by identifying some 'ideal types' that originate from the double functions that lawmakers shall perform in contemporary democracies as "representatives" of voters' interests and as "responsible" administrators (Mair 2009). In this sense, the dissertation contends that different policy problems emerge from the electoral promises of the governing parties, from commitments related to the responsibility of being in office, and from the 'external world', and that the balance between them determines the composition of the policy agenda. 13 There are four propositions of this study to existing knowledge in the field of policy agendas. The first is that the content of the policy agenda is stable across countries with different institutional settings. Lawmakers' priorities are no less stable in institutional systems that are more 'open' to accommodating policy problems brought by the electoral promises of the parties. At the same time stability persists even when elections approach, questioning the long-lasting assumption that lawmakers may manipulate policies to their will in order to assure re-election. The second is that policy problems brought by the electoral promises of the governing parties impact lawmakers’ priorities, but this is only half of an old story. The results show that the policy problems originating from the electoral promises of the opposition influence the content of the policy agenda confirming that the agenda-setting power of parties is not limited to those who are in office. The third proposition is a theoretical effort and empirical contribution to conceptualise and measure "policy commitments". Studies of public policy have stressed the importance of inherited commitments in everyday law making (Rose 1994; Adler and Wilkerson 2012) since some decisions take longer than a legislature to be realised. Classic analyses have emphasised the importance of budgetary constraints on policy agendas, but the thesis suggests that there is also another striking case of policy commitments for European polities: EU integration, since decisions on EU affairs and delegation of powers taken from previous governments are hard (if not impossible) to reverse by their successors. In this sense, EU decisions are inherited by all governments, and they add complexity to the problem-solving capacity of Member States because they produce extra policy problems that require lawmakers' attention. For lawmakers respecting legally binding EU decisions, this is a way to avoid "reckless and illegal decision making" (Mair 2009). The results highlight that when reflecting on the divisions of competences between the Union and its Member States (MSs), policy commitments derived from the EU directives are concentrated on a narrow set of policy areas. The results show that in most fields where commitments are higher, the agenda-setting power of parties’ electoral promises is weakened. Finally, this research suggests that policy problems originating from the agenda of the public (as approximated by media coverage) are another explanatory factor of policy priorities, but in a very narrow set of policy areas. Media effects appear to be limited to policy areas with the special characteristics of newsworthiness and sensationalism (Soroka 2002) that contribute to boost their policy appeal. In addition, the findings highlight that the agenda-setting power of the media is mediated by the interaction with the electoral promises of the opposition, probably as a result of a blame avoidance game to discredit incumbents. 14 Chapter 1 introduces the concepts of policy agenda and policy problem before summarising existing accounts of the content of policy agendas. Two theoretical traditions are identified. The first one is the "partisan account" highlighting the importance of partisan preferences for lawmakers' priorities. The second is made up of the "public policy accounts" proposing incrementalist and agenda-setting approaches to representatives' priorities. Chapter 2 sets up the theoretical framework that will be tested in this research. Drawing upon theories of "representative and responsible" government (Mair 2009) the research provides an encompassing model of how different policy problems compete for attention in order to enter the agendas of lawmakers. The thesis highlights that different agenda-setters have to be considered as creating policy problems: the electoral promises of the governing parties, the demands addressed to lawmakers by the EU agenda, and the issues that are important for the public as reported by the media. Starting from existing typologies of problems that must be addressed in the policy agenda (Walker 1977; Adler and Wilkerson 2012), the research roughly distinguishes between discretionary and compulsory policy problems, discussing how the three agenda-setters considered in this study fit into those ideal types, as well as the incentives for lawmakers to prioritise one over the other. Chapter 3 presents the data, models and methods that are used to test the theoretical framework. The dissertation relies on data from the Comparative Agendas Project modelled in the form of time series cross sectional models. Chapter 4 introduces the empirical investigation of the content of the policy agenda. It focuses on stability and change in lawmakers' priorities, to understand the extent to which priorities change (or remain the same) across elections. Chapter 5 moves a step further and will assess the connection between policy problems brought by parties' electoral promises and the content of the policy agenda. Chapter 6 will account for one of the most debated sources of policy problems among public policy scholars: policy commitments. This chapter will test the agenda-setting power of policy commitments deriving from the content of the EU directives on lawmakers' priorities and proposing an "EU acquiescence index" to shed light on the 'overlaps' between EU and domestic policy agendas. Finally, Chapter 7 aims at analysing the connection between lawmakers' priorities and media coverage (in terms of print and, where appropriate, audio media) and each of the two relevant types of policy problems competing for lawmakers' attention identified in the previous chapters. In sum the thesis offers a theory of the composition of policy agendas grounded in a problem-solving understanding of politics, and an empirical assessment of its validity. In this sense the study is about how policy problems originating from the dual role of lawmakers in 15 contemporary democracies (representation and administration) affect everyday policy making. More precisely the thesis considers the impact of different agenda venues (parties, EU commitments, and the media) on the way in which lawmakers deliver policies

Biophysical properties of WT and mutants TRPM4 channel in whole-cell configuration.

<p>(<b>A</b>): Representative current tracings recorded in the whole cell conditions (ramp protocol under the traces) (<b>B</b>): Mean current density for WT and mutants estimated using the maximal current recorded during the ending step of 20 ms at Vm = +100 mV (see A) and reported to cell capacitance. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s.: not significant. Numbers in bars = number of experiments. Error bar: standard error of the mean.</p