The role of tunneling in enzyme catalysis of C–H activation

AbstractRecent data from studies of enzyme catalyzed hydrogen transfer reactions implicate a new theoretical context in which to understand C–H activation. This is much closer to the Marcus theory of electron transfer, in that environmental factors influence the probability of effective wave function overlap from donor to acceptor atoms. The larger size of hydrogen and the availability of three isotopes (H, D and T) introduce a dimension to the kinetic analysis that is not available for electron transfer. This concerns the role of gating between donor and acceptor atoms, in particular whether the system in question is able to tune distance between reactants to achieve maximal tunneling efficiency. Analysis of enzyme systems is providing increasing evidence of a role for active site residues in optimizing the inter-nuclear distance for nuclear tunneling. The ease with which this optimization can be perturbed, through site-specific mutagenesis or an alteration in reaction conditions, is also readily apparent from an analysis of the changes in the temperature dependence of hydrogen isotope effects

Synaptic Transmission from Horizontal Cells to Cones Is Impaired by Loss of Connexin Hemichannels

In the vertebrate retina, horizontal cells generate the inhibitory surround of bipolar cells, an essential step in contrast enhancement. For the last decades, the mechanism involved in this inhibitory synaptic pathway has been a major controversy in retinal research. One hypothesis suggests that connexin hemichannels mediate this negative feedback signal; another suggests that feedback is mediated by protons. Mutant zebrafish were generated that lack connexin 55.5 hemichannels in horizontal cells. Whole cell voltage clamp recordings were made from isolated horizontal cells and cones in flat mount retinas. Light-induced feedback from horizontal cells to cones was reduced in mutants. A reduction of feedback was also found when horizontal cells were pharmacologically hyperpolarized but was absent when they were pharmacologically depolarized. Hemichannel currents in isolated horizontal cells showed a similar behavior. The hyperpolarization-induced hemichannel current was strongly reduced in the mutants while the depolarization-induced hemichannel current was not. Intracellular recordings were made from horizontal cells. Consistent with impaired feedback in the mutant, spectral opponent responses in horizontal cells were diminished in these animals. A behavioral assay revealed a lower contrast-sensitivity, illustrating the role of the horizontal cell to cone feedback pathway in contrast enhancement. Model simulations showed that the observed modifications of feedback can be accounted for by an ephaptic mechanism. A model for feedback, in which the number of connexin hemichannels is reduced to about 40%, fully predicts the specific asymmetric modification of feedback. To our knowledge, this is the first successful genetic interference in the feedback pathway from horizontal cells to cones. It provides direct evidence for an unconventional role of connexin hemichannels in the inhibitory synapse between horizontal cells and cones. This is an important step in resolving a long-standing debate about the unusual form of (ephaptic) synaptic transmission between horizontal cells and cones in the vertebrate retina

Ephaptic communication in the vertebrate retina

In the vertebrate retina, cones project to the horizontal cells (HCs) and bipolar cells (BCs). The communication between cones and HCs uses both chemical and ephaptic mechanisms. Cones release glutamate in a Ca2+-dependent manner, while HCs feed back to cones via an ephaptic mechanism. Hyperpolarization of HCs leads to an increased current through connexin hemichannels located on the tips of HC dendrites invaginating the cone synaptic terminals. Due to the high resistance of the extracellular synaptic space, this current makes the synaptic cleft slightly negative. The result is that the Ca2+-channels in the cone presynaptic membrane experience a slightly depolarized membrane potential and therefore more glutamate is released. This ephaptic mechanism forms a very fast and noise free negative feedback pathway. These characteristics are crucial, since the retina has to perform well in demanding conditions such as low light levels. In this mini-review we will discuss the critical components of such an ephaptic mechanism. Furthermore, we will address the question whether such communication appears in other systems as well and indicate some fundamental features to look for when attempting to identify an ephaptic mechanis

Ephaptic communication in the vertebrate retina

In the vertebrate retina, cones project to the horizontal cells (HCs) and bipolar cells (BCs). The communication between cones and horizontal cells uses both chemical and ephaptic mechanisms. Photoreceptors release glutamate in a Ca2+-dependent manner, while HCs feed back to cones via an ephaptic mechanism. Hyperpolarization of HCs leads to an increased current through connexin hemichannels located on the tips of HC dendrites invaginating the cone synaptic terminals. This current makes the extracellular synaptic space slightly negative. The result is that the Ca2+-channels in the cone pre-synaptic membrane experience a slightly depolarized membrane potential and therefore more glutamate is released. This ephaptic mechanism forms a very fast and noise free negative feedback pathway. These characteristics are crucial, since the retina has to perform well in demanding conditions such as low light levels and detecting fast events. In this mini-review we will discuss the critical components of such an ephaptic mechanism. Furthermore, we will address the question whether such communication appears in other systems as well and indicate some fundamental features to look for when attempting to identify an ephaptic mechanism

Action spectra of individual cells and averages per cone-type.

<p>In the top four panels the relative sensitivity is plotted against the stimulus wavelength of individual cones grouped according to cone-type as indicated in the graph. The bottom panel displays the average spectral sensitivity for the different cone-types.</p

Comparison of zebrafish cone spectral sensitivity data.

<p>λ_max values (in nm) of zebrafish A1-based photopigments and cone-types from literature. Parameters are presented as mean ± <i>SD</i>. <i>^a</i> Chinen et al. (2003); <i>^b</i> Nawrocki et al. (1985); <i>^c</i> Robinson et al. (1993); <i>^d</i> Cameron (2002); <i>^e</i> Govardovskii et al. (2000); <i>^f</i> Allison et al. (2004).</p

Parameters of photopigment template fits.

<p>Mean parameter values for the various cone types. Parameters are presented as mean ± <i>SD.</i></p

Properties of zebrafish cones.

<p>Average properties of the various zebrafish cones types. V_rest, resting membrane potential; R_max, maximum response amplitude relative to V_rest; n, coefficient of fit Hill-relation; S_abs absolute sensitivity (see Methods section for details). Parameters are presented as mean ± <i>SD.</i></p

Fits of pigment template to experimental data.

<p>This figure displays the fits (solid lines) of the photopigment template <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068540#pone.0068540-Govardovskii1" target="_blank">[15]</a> to the average experimental data per cone-type with the peak wavelength of the A1-based photopigment, the ratio between A1- and A2-based photopigment and the presence of the β-wave relative to the original photopigment template as free parameters. For comparison the spectral sensitivity functions of corresponding A1- (dashed lines) and A2-based (dotted lines) photopigments are also plotted. These were constructed according to the generic photopigment template in combination with their peak absorbance wavelength as measured <i>in vitro </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068540#pone.0068540-Chinen1" target="_blank">[7]</a>. The UV-cone action spectra deviates considerable from the UV template, presumably due to the small number of data points (top row, left). The action spectrum of the SWS cones was fully overlapped the SWS-2 adsorption spectrum (top row, right). For the MWS cones, both RH2–2 and RH2–3 templates were covering the MWS action spectrum, while RH2–1 and RH2–4 templates could not describe the action spectrum properly. Finally both LWS-1 and LWS-2 templates covered the LWS action spectrum.</p

Specific connectivity between photoreceptors and horizontal cells in the zebrafish retina

The functional and morphological connectivity between various horizontal cell (HC) types (H1, H2, H3 and H4), and photoreceptors was studied in zebrafish retina. Since HCs are strongly coupled by gap-junctions and feedback from HCs to photoreceptors strongly depends on connexin (Cx) hemichannels we characterized the various HC Cxs (Cx52.6, Cx52.7, Cx52.9 and Cx55.5) in Xenopus oocytes. All Cxs formed hemichannels which were conducting at physiological membrane potentials. The Cx hemichannels differed in kinetic properties and voltage dependence, allowing for specific tuning of the coupling of HCs and the feedback signal from HCs to cones. The morphological connectivity between the HC layers and the cones was determined next. We used zebrafish expressing GFP under the control of Cx promoters. We found that all HCs showed Cx55.5 promoter activity. Cx52.7 promoter activity was exclusively present in H4 cells while Cx52.9 promoter activity occurred only in H1 cells. Cx52.6 promoter activity was present in H4 cells and in the ventral quadrant of the retina also in H1 cells. Finally we determined the spectral sensitivities of the HC layers. Three response types were found. Monophasic responses were generated by HCs that contacted all cones (H1 cells), biphasic responses were generated by HCs that contacted M-, S- and UV-cones (H2 cells), and triphasic responses were generated by HCs that contacted either S- and UV-cones (H3 cells) or rods and UV-cones (H4 cells). Electronmicroscopy confirms that H4 cells innervate cones. This indicates that rod driven HCs process spectral information during photopic and luminance information during scotopic conditions