R. Lea Sanford

Thiazolidinedione insulin sensitizers alter lipid bilayer properties and voltage-dependent sodium channel function: implications for drug discovery.

The thiazolidinediones (TZDs) are used in the treatment of diabetes mellitus type 2. Their canonical effects are mediated by activation of the peroxisome proliferator–activated receptor γ (PPARγ) transcription factor. In addition to effects mediated by gene activation, the TZDs cause acute, transcription-independent changes in various membrane transport processes, including glucose transport, and they alter the function of a diverse group of membrane proteins, including ion channels. The basis for these off-target effects is unknown, but the TZDs are hydrophobic/amphiphilic and adsorb to the bilayer–water interface, which will alter bilayer properties, meaning that the TZDs may alter membrane protein function by bilayer-mediated mechanisms. We therefore explored whether the TZDs alter lipid bilayer properties sufficiently to be sensed by bilayer-spanning proteins, using gramicidin A (gA) channels as probes. The TZDs altered bilayer elastic properties with potencies that did not correlate with their affinity for PPARγ. At concentrations where they altered gA channel function, they also altered the function of voltage-dependent sodium channels, producing a prepulse-dependent current inhibition and hyperpolarizing shift in the steady-state inactivation curve. The shifts in the inactivation curve produced by the TZDs and other amphiphiles can be superimposed by plotting them as a function of the changes in gA channel lifetimes. The TZDs’ partition coefficients into lipid bilayers were measured using isothermal titration calorimetry. The most potent bilayer modifier, troglitazone, alters bilayer properties at clinically relevant free concentrations; the least potent bilayer modifiers, pioglitazone and rosiglitazone, do not. Unlike other TZDs tested, ciglitazone behaves like a hydrophobic anion and alters the gA monomer–dimer equilibrium by more than one mechanism. Our results provide a possible mechanism for some off-target effects of an important group of drugs, and underscore the importance of exploring bilayer effects of candidate drugs early in drug development.

Volatile anesthetics inhibit sodium channels without altering bulk lipid bilayer properties

Volatile anesthetics act directly on neuronal sodium channels, independently of effects on the lipid bilayer.

Clinical concentrations of chemically diverse general anesthetics minimally affect lipid bilayer properties

Significance General anesthetics are essential to modern medicine, yet their molecular mechanisms remain poorly understood. Whether general anesthetics primarily act by altering lipid bilayer properties or by interacting directly with specific membrane proteins is a longstanding controversy. We now show that diverse classes of general anesthetics do not alter bilayer properties at concentrations that induce clinical anesthesia. Although anesthetics can have bilayer-perturbing effects at supratherapeutic (toxic) concentrations, this has little pharmacological relevance. Our findings exclude indirect ion channel effects due to perturbations of lipid bilayer properties, supporting the notion that general anesthetics interact directly with therapeutically relevant membrane protein targets. General anesthetics have revolutionized medicine by facilitating invasive procedures, and have thus become essential drugs. However, detailed understanding of their molecular mechanisms remains elusive. A mechanism proposed over a century ago involving unspecified interactions with the lipid bilayer known as the unitary lipid-based hypothesis of anesthetic action, has been challenged by evidence for direct anesthetic interactions with a range of proteins, including transmembrane ion channels. Anesthetic concentrations in the membrane are high (10–100 mM), however, and there is no experimental evidence ruling out a role for the lipid bilayer in their ion channel effects. A recent hypothesis proposes that anesthetic-induced changes in ion channel function result from changes in bilayer lateral pressure that arise from partitioning of anesthetics into the bilayer. We examined the effects of a broad range of chemically diverse general anesthetics and related nonanesthetics on lipid bilayer properties using an established fluorescence assay that senses drug-induced changes in lipid bilayer properties. None of the compounds tested altered bilayer properties sufficiently to produce meaningful changes in ion channel function at clinically relevant concentrations. Even supra-anesthetic concentrations caused minimal bilayer effects, although much higher (toxic) concentrations of certain anesthetic agents did alter lipid bilayer properties. We conclude that general anesthetics have minimal effects on bilayer properties at clinically relevant concentrations, indicating that anesthetic effects on ion channel function are not bilayer-mediated but rather involve direct protein interactions.

Effect of Statins on the Nanomechanical Properties of Supported Lipid Bilayers.

Many drugs and other xenobiotics may reach systemic concentrations where they interact not only with the proteins that are their therapeutic targets but also modify the physicochemical properties of the cell membrane, which may lead to altered function of many transmembrane proteins beyond the intended targets. These changes in bilayer properties may contribute to nonspecific, promiscuous changes in membrane protein and cell function because membrane proteins are energetically coupled to their host lipid bilayer. It is thus important, for both pharmaceutical and biophysical reasons, to understand the bilayer-modifying effect of amphiphiles (including therapeutic agents). Here we use atomic force microscopy topography imaging and nanomechanical mapping to monitor the effect of statins, a family of hypolipidemic drugs, on synthetic lipid membranes. Our results reveal that statins alter the nanomechanical stability of the bilayers and increase their elastic moduli depending on the lipid bilayer order. Our results also suggest that statins increase bilayer heterogeneity, which may indicate that statins form nanometer-sized aggregates in the membrane. This is further evidence that changes in bilayer nanoscale mechanical properties may be a signature of lipid bilayer-mediated effects of amphiphilic drugs.

Bilayer Modifying Effects of Antipsychotics of Different Generations

Antipsychotics are used to manage the symptoms of schizophrenia (delusions, hallucinations, etc.), to treat bipolar mania, and are commonly used as adjective agents in the treatment of depression. They are known to alter the function of diverse membrane proteins, and for certain antipsychotics (e.g. quintepine), this polypharmacology is thought to be desirable. Because antipsychotics are amphiphiles that modulate the function of different, structurally unrelated membrane proteins, we investigated whether antipsychotics alter lipid bilayer properties at concentrations where they alter membrane protein function. To this end, we used a gramicidin-based fluorescence assay (GBFA) as well as single-channel electrophysiology and we find that antipsychotics like chlorpromazine increase the lifetime and appearance rate of gramicidin (gA) channels, thus shifting the gA monomer dimer equilibrium toward the conducting dimers. We are currently examining other commonly prescribed antipsychotics with the goal of exploring whether there are systematic differences between antipsychotics from the first and second generations. The expectation is that the newer antipsychotics will be less bilayer active and therefore would be expected to have fewer undesired effects. Our other studies have found that an increased alteration of bilayer properties, above a given threshold, may indicate toxicity. Thus, we were keen to learn whether antipsychotics substantially altered bulk bilayer properties and more specifically, we hope to observe whether or not there is a correlation between how well an antipsychotic is tolerated (and thus more commonly prescribed) and its bilayer modifying propensity.