Oscar Moran

Channels in Mitochondrial Membranes: Knowns, Unknowns, and Prospects for the Future

Rapid diffusion of hydrophilic molecules across the outer membrane of mitochondria has been related to the presence of a protein of 29 to 37 kDa, called voltage-dependent anion channel (VDAC), able to generate large aqueous pores when integrated in planar lipid bilayers. Functional properties of VDAC from different origins appear highly conserved in artificial membranes: at low transmembrane potentials, the channel is in a highly conducting state, but a raise of the potential (both positive and negative) reduces drastically the current and changes the ionic selectivity from slightly anionic to cationic. It has thus been suggested that VDAC is not a mere molecular sieve but that it may control mitochondrial physiology by restricting the access of metabolites of different valence in response to voltage and/or by interacting with a soluble protein of the intermembrane space. The latest application of the patch clamp and tip-dip techniques, however, has indicated both a different electric behavior of the outer membrane and that other proteins may play a role in the permeation of molecules. Biochemical studies, use of site-directed mutants, and electron microscopy of two-dimensional crystal arrays of VDAC have contributed to propose a monomeric beta barrel as the structural model of the channel. An important insight into the physiology of the inner membrane of mammalian mitochondria has come from the direct observation of the membrane with the patch clamp. A slightly anionic, voltage-dependent conductance of 107 pS and one of 9.7 pS, K(+)-selective and ATP-sensitive, are the best characterized at the single channel level. Under certain conditions, however, the inner membrane can also show unselective nS peak transitions, possibly arising from a cooperative assembly of multiple substrates.

Binding site of activators of the cystic fibrosis transmembrane conductance regulator in the nucleotide binding domains.

Abstract.The use of substances that could activate the defective chloride channels of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) has been suggested as possible therapy for cystic fibrosis. Using epithelia formed by cells stably transfected with wildtype or mutant (G551D, G1349D) CFTR, we estimated the apparent dissociation constant, KD, of a series of CFTR activators by measuring the increase in the apical membrane current. Modification of apparent KD of CFTR activators by mutations of the nucleotide-binding domains (NBDs) suggests that the binding site might be in these regions. The human NBD structure was predicted by homology with murine NBD1. An NBD1-NBD2 complex was constructed by overlying monomers to a bacterial ABC transporter NBD dimer in the ‘head-to- tail’ conformation. Binding sites for CFTR activators were predicted by molecular docking. Comparison of theoretical binding free energy estimated in the model to free energy estimated from the apparent dissociation constants, KD, resulted in a remarkably good correlation coefficient for one of the putative binding sites, located in the interface between NBD1 and NBD2.

Endogenous expression of the β1A sodium channel subunit in HEK-293 cells

The expression of the sole α‐subunit of muscle or brain sodium channels in frog oocytes mediates currents with a bimodal inactivation with an abnormal slow mode that is strongly depressed only by co‐expression of the β1‐subunit. In contrast, in the expression of the α‐subunit in the human embryonic kidney cell line, HEK‐293, the slow mode is almost absent, suggesting an endogenous expression of the β1‐subunit. We have tested this hypothesis by reverse transcriptase‐polymerase chain reaction. We found an abundant expression of mRNA encoding the β1A splicing of the putative regulatory sodium channel subunit but no mRNA encoding the β1‐subunit in HEK cells. This finding is consistent with the idea that the endogenous β1A‐subunit is sufficient for suppressing the slowly inactivating mode of sodium currents by co‐assembly with α‐subunits, and calls attention to the reliability of effects attributed in HEK cells to α–β1 co‐expression.

Molecular determinants of ammonia and urea conductance in plant aquaporin homologs

Aquaporins and/or aquaglyceroporins regulate the permeability of plant membranes to water and small, uncharged molecules. Using molecular simulations with a plant plasma membrane aquaporin tetramer, the residues in the channel constriction region were identified as the crucial determinants of ammonia and urea conductance. The impact of these residues was experimentally verified using AtPIP2;1 pore mutants. Several, but not all, mutants with a NIP‐like selectivity filter promoted yeast growth on urea or ammonia as sole sources of nitrogen. TIP‐like mutants conducted urea but not NH3, and a residue without direct contact to the pore lumen was critical for conduction in the mutants.

A quantitative description of the activation and inhibition of CFTR by potentiators: Genistein

The CFTR, encoded by the gene mutated in cystic fibrosis (CF) patients, is responsible for cAMP dependent chloride transport in epithelia. Substances that activate CFTR have been suggested as possible CF therapy. Most substances investigated so far exert a dual effect on the CFTR: low concentrations stimulate CFTR, whereas higher concentrations inhibit CFTR. Besides, the CFTR phosphorylation level determines the apparent affinity of the drug. We have studied the properties of genistein, the well known CFTR potentiator, by measuring apical membrane current on epithelia formed by cells stably transfected with CFTR and stimulated with different concentrations of CPTcAMP. We propose a quantitative model to describe the activatory and inhibitory effect of genistein, accounting also for the cAMP dependent activation.

Functional Analysis of Mutations in the Putative Binding Site for Cystic Fibrosis Transmembrane Conductance Regulator Potentiators INTERACTION BETWEEN ACTIVATION AND INHIBITION

An increasing number of compounds able to potentiate the activity of mutants of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have been identified by high throughput screening or by individual search of derivatives of known active compounds. Several lines of evidence suggest that most CFTR potentiators act through the same mechanism, probably by binding to the nucleotide binding domains to promote the activity of the protein and then, with lower affinity, to an inhibitory site. With the aim of identifying the activating binding site, we recently modeled the nucleotide binding domain dimer and predicted a common binding site for potentiators in its interface. To validate this model experimentally, we mutated some of the residues involved in the putative binding site, i.e. Arg553, Ala554, and Val1293. The activity of CFTR potentiators was measured as apical membrane currents on polarized cells stably expressing wild type or mutated proteins. CFTR activity was elicited by application of a membrane-permeable cAMP analogue followed by increasing concentrations of potentiators. We found that all three mutants responded to cAMP, although the affinity of R553Q was higher than that of wild type CFTR. In R553Q and V1293G mutants, the dissociation constant of potentiators for the activating site was increased, whereas the dissociation constant for the inhibitory site was reduced. Our results show that the mutated residues are part of the activating binding site for potentiators, as suggested by the molecular model. In addition, these results suggest that the activating and inhibitory sites are not independent of each other.

Activation and deactivation properties of rat brain K+ channels of the Shaker-related subfamily

We studied the activation properties of members of the Shaker-related subfamily of voltage-gated K+ channels cloned from rat brain and expressed in Xenopus oocytes. We find that Kv1.1, Kv1.4, Kv1.5, and Kv1.6 have similar activation and deactivation kinetics. The K+ currents produced by step depolarisations increase with a sigmoidal time course that can be described by a delay and by the derivative of the current at the inflection point. The delay tends to zero and the logarithmic derivative seems to approach a finite value at large positive voltages, but these asymptotic values are not yet reached at +80 mV. Deactivation of the currents upon stepping to negative membrane potentials below -60 mV is fairly well described by a single exponential. The decrease of the deactivation time constant at increasingly negative voltages tends to become less steep, indicating that this parameter also has a finite limiting value, which is not yet reached, however, at −160 mV The various clones studied have very similar voltage dependencies of activation with half-activation voltages ranging between −50 and −11 mV and maximum steepness yielding an e-fold change for voltage increments between 3.8 and 7.0 mV The shallower activation curve of Kv1.4 is likely to be due to coupling with the fast inactivation process present in this clone.

Use dependence of tetrodotoxin block of sodium channels: a revival of the trapped-ion mechanism.

The use-dependent block of sodium channels by tetrodotoxin (TTX) has been studied in cRNA-injected Xenopus oocytes expressing the alpha-subunit of rat brain IIA channels. The kinetics of stimulus-induced extra block are consistent with an underlying relaxation process involving only three states. Cumulative extra block induced by repetitive stimulations increases with hyperpolarization, with TTX concentration, and with extracellular Ca2+ concentration. We have developed a theoretical model based on the suggestion by Salgado et al. that TTX blocks the extracellular mouth of the ion pore less tightly when the latter has its external side occupied by a cation, and that channel opening favors a tighter binding by allowing the escape of the trapped ion. The model provides an excellent fit of the data, which are consistent with Ca2+ being more efficient than Na+ in weakening TTX binding and with bound Ca2+ stabilizing the closed state of the channel, as suggested by Armstrong and Cota. Reports arguing against the trapped-ion mechanism are critically discussed.

Sodium channel heterologous expression in mammalian cells and the role of the endogenous β1-subunits

Sodium currents in cell lines transfected with the sole alpha-subunit, or constitutively expressing sodium channels, have an inactivation that is always prevalently mono-exponential. Differently, expression of alpha-subunit in Xenopus oocytes exerts slow inactivating currents with biphasic decay, while simultaneous co-transfection of alpha and beta1 restores a mono-exponential (normal) inactivation. A hypothesis for such differences is that an endogenous presence of beta1 or beta1-alternative splicing, beta1A, in cells could account for the normal inactivation. To test this hypothesis and to evaluate the role for the beta1A, we inhibited the expression of beta1/beta1A by antisense oligonucleotides on Nav1.4-transfected human embryonic cell line 293 (HEK) cells. Reduction of beta1/beta1A produces no significant functional effects in Nav1.4-HEK. This result invalidates the hypothesis that the lack of slow-mode in cell lines is simply due to a constitutive expression of beta1/beta1A.

Ionic permeability of the mitochondrial outer membrane

The ionic permeability of the outer mitochondrial membrane (OMM) was studied with the patch clamp technique. Electrical recording of intact mitochondria (hence of the outer membrane (OM)), derived from mouse liver, showed the presence of currents corresponding to low conductances (< 50 pS), as well as of four distinct conductances of 99 pS,152 pS, 220 pS and 307 pS (in 150 mM KCl). The latter were voltage gated, being open preferentially at positive (pipette) potentials. Very similar currents were found by patch clamping liposomes containing the isolated OM derived from rat brain mitochondria. Here a conductance of approximately 530 pS, resembling in its electrical characteristics a conductance already attributed to mitochondrial contact sites (Moran et al. 1990), was also detected. Immunoblot assays of mitochondria and of the isolated OM with antibodies against the outer membrane voltage-dependent anion channel (VDAC) (Colombini 1979), showed the presence of the anion channel in each case. However, the typical electrical behaviour displayed by such a channel in planar bilayers could not be detected under our experimental conditions. From this study, the permeability of the OMM appears different from what has been reported hitherto, yet is more in line with that multifarious and dynamic structure which apparently should belong to it, at least within the framework of mitochondrial biogenesis (Pfanner and Neupert 1990).