Thomas Zimmer

Proliferation of intracellular membrane structures upon homologous overproduction of cytochrome P-450 in Candida maltosa

In an alkane-assimilating yeast, Candida maltosa, a cultivation on alkane causes both induction of endoplasmic reticulum (ER)-resident membrane proteins, such as cytochrome P-450, and proliferation of ER. In this study, individual genes for alkane-inducible forms of cytochrome P-450 (P-450alk) were homologously overexpressed in C. maltosa using a galactose-inducible expression system developed in this yeast. Immunoelectron microscopy revealed that, upon the overexpression, a dramatic proliferation of ER occurred, in which overproduced P-450alk protein accumulated. The proliferated membranes were mainly tubular forms and stacks of paired membranes were also observed after prolonged expression. The tubular forms were morphologically very similar to the proliferated ER in alkane-induced C. maltosa cells. The observed proliferation of ER membranes by homologous overproduction of P-450alk, here depicted, will provide a unique opportunity for investigating the mechanisms by which cells regulate ER biogenesis, in comparison with the intrinsic form of ER proliferation.

Modulation of Nav1.5 Channel Function by an Alternatively Spliced Sequence in the DII/DIII Linker Region

In the present study, we identified a novel splice variant of the human cardiac Na+ channel Nav1.5 (Nav1.5d), in which a 40-amino acid sequence of the DII/DIII intracellular linker is missing due to a partial deletion of exon 17. Expression of Nav1.5d occurred in embryonic and adult hearts of either sex, indicating that the respective alternative splicing is neither age-dependent nor gender-specific. In contrast, Nav1.5d was not detected in the mouse heart, indicating that alternative splicing of Nav1.5 is species-dependent. In HEK293 cells, splice variant Nav1.5d generated voltage-dependent Na+ currents that were markedly reduced compared with wild-type Nav1.5. Experiments with mexiletine and 8-bromo-cyclic AMP suggested that the trafficking of Nav1.5d channels was not impaired. However, single-channel recordings showed that the whole-cell current reduction was largely due to a significantly reduced open probability. Additionally, steady-state activation and inactivation were shifted to depolarized potentials by 15.9 and 5.1 mV, respectively. Systematic mutagenesis analysis of the spliced region provided evidence that a short amphiphilic region in the DII/DIII linker resembling an S4 voltage sensor of voltage-gated ion channels is an important determinant of Nav1.5 channel gating. Moreover, the present study identified novel short sequence motifs within this amphiphilic region that specifically affect the voltage dependence of steady-state activation and inactivation and current amplitude of human Nav1.5.

Prolonged irradiation of enhanced cyan fluorescent protein or Cerulean can invalidate Förster resonance energy transfer measurements

Since its discovery, green fluorescent protein (GFP) and its variants have proven to be a good and convenient fluorescent label for proteins: GFP and other visible fluorescent proteins (VFPs) can be fused selectively to the protein of interest by simple cloning techniques and develop fluorescence without additional cofactors. Among the steadily growing collection of VFPs, several pairs can be chosen that can serve as donor and acceptor fluorophores in Forster resonance energy transfer (FRET) experiments. Among them, the cyan fluorescent proteins (ECFP/Cerulean) and the enhanced yellow fluorescent protein (EYFP) are most commonly used. We show that ECFP and Cerulean have some disadvantages despite their common use: Upon irradiation with light intensities that are commonly used for intensity- and lifetime-based FRET measurements, both the fluorescence intensity and the fluorescence lifetime of ECFP and Cerulean decrease. This can hamper both intensity- and lifetime-based FRET measurements and emphasizes the need for control measurements to exclude these artifacts.

Role of the S4-S5 linker in CNG channel activation.

Cyclic nucleotide-gated (CNG) channels mediate sensory signal transduction in retinal and olfactory cells. The channels are activated by the binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus that is located at the intracellular side. The molecular events translating the ligand binding to the pore opening are still unknown. We investigated the role of the S4-S5 linker in the activation process by quantifying its interaction with other intracellular regions. To this end, we constructed chimeric channels in which the N-terminus, the S4-S5 linker, the C-linker, and the CNBD of the retinal CNGA1 subunit were systematically replaced by the respective regions of the olfactory CNGA2 subunit. Macroscopic concentration-response relations were analyzed, yielding the apparent affinity to cGMP and the Hill coefficient. The degree of functional coupling of intracellular regions in the activation gating was determined by thermodynamic double-mutant cycle analysis. We observed that all four intracellular regions, including the relatively short S4-S5 linker, are involved in controlling the apparent affinity of the channel to cGMP and, moreover, in determining the degree of cooperativity between the subunits, as derived from the Hill coefficient. The interaction energies reveal an interaction of the S4-S5 linker with both the N-terminus and the C-linker, but no interaction with the CNBD.

Corrigendum: Deciphering the function of the CNGB1b subunit in olfactory CNG channels

This corrects the article DOI: 10.1038/srep29378.

Corrigendum: Quantifying the cooperative subunit action in a multimeric membrane receptor

This corrects the article DOI: 10.1038/srep20974.

Differential Contribution of the Olfactory CNG Channel Subunits to the Activation Process

In olfactory receptor neurons, the cyclic nucleotide-gated (CNG) channels play an important role in converting sensory stimuli into electrical signals. The CNG channels are heterotetrameric proteins composed of three homologue subunits, 2xCNGA2, CNGA4 and CNGB1b. Each subunit has an intracellular cyclic nucleotide binding domain. This suggests that the binding of cGMP or cAMP to each subunit is involved in channel opening. The CNGA4 and CNGB1b subunits produce functional channels only if expressed together with CNGA2. Their contribution to the activation process, in particular their ability to bind cyclic nucleotides, has not been elucidated so far. Herein, heterotetrameric CNGA2A4B1b channels were expressed in Xenopus oocytes and studied in excised patches by monitoring ligand binding and gating under both steady-state and non-steady state conditions. Ligand binding was measured by confocal patch-clamp fluorometry using a fluorescent cGMP analogue (fcGMP). Similar to the homotetrameric CNGA2 channels, we observed in CNGA2A4B1b channels a crossover of the normalized steady-state binding and steady-state activation. Also, the concentration-binding relationships were similar in homo- and heterotetrameric channels. By expressing TFP-labelled CNGA4 and CNGB1b subunits alone we show that these subunits do reach the plasma membrane also in the absence of CNGA2 subunits. In order to study the binding to the CNGA4 and CNGB1b subunits we coexpressed either of them with a mutated CNGA2 subunit (T539M) that has an increased sensitivity to cAMP and is not able to bind fcGMP. Under these conditions, we observed significant binding of fcGMP to the CNGA4, but not to the CNGB1b subunit. The binding to the CNGA4 subunit is rapid (∼30 ms) and opens the channels. Our results suggest that CNGA2A4B1b channels are activated by ligand binding to CNGA2 and CNGA4 subunits, but not to the CNGB1b subunit.

Interaction Energies between Intracellular Regions in CNG Channel Activation

Cyclic nucleotide-gated (CNG) channels mediate sensory signal transduction in retinal and olfactory cells. The channels are activated by the binding of cyclic nucleotides to an intracellular cyclic nucleotide-binding domain (CNBD). The molecular events translating the binding to the pore opening are still unknown. We investigated the role of intracellular channel regions on the activation process by constructing chimeric channels in which the N-terminus, the S4-S5 linker, the C-terminus, and the CNBD of the retinal CNGA1 subunit were systematically replaced by respective regions of the olfactory CNGA2 subunit. Macroscopic concentration-response relations were analyzed, yielding the apparent affinity to cGMP and the Hill coefficient. The degree of functional coupling of intracellular regions in the activation gating was determined by an interaction energy according to the principles of thermodynamic double-mutant cycle analysis. We show that all four intracellular regions, including the short S4-S5 linker, are involved in controlling the apparent affinity of the channel to cGMP and, moreover, in determining the degree of cooperativity between the subunits as determined from the Hill coefficients. The interaction energies are specific for pairs of regions. The interaction energy of the S4-S5 region with both the N-terminus and the C-linker was significantly different from zero at all possible combinations of intracellular regions. In contrast, at all possible combinations of intracellular regions no significant interaction energy was observed for the S4-S5 linker and the CNBD. It is concluded that in CNG channels the S4-S5 linker cooperates with both N-terminus and C-linker in the process of translating ligand binding to the pore opening.