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Ion Selectivity and Competition in Channelrhodopsins
No full textAbstractChannelrhodopsins are light-gated ion channels of green algae. They are widely used for the analysis of neuronal networks using light in the emerging field of optogenetics. Under steady-state light conditions, the two open states, O1 and O2, mediate the photocurrents with different ion conductance and selectivity. To understand the conducting process as well as its optogenetic applications, it is important to study ion binding and transport of this promiscuous cation channel. Here, we present an enzyme kinetic algorithm that allowed us to calculate the ion composition of the initial and steady-state photocurrents for multication media. The approach is based on current-voltage relations determined for the individual ions H+, Na+, Ca2+, and Mg2+. We identify and quantify the widely different competition of the ions in wild-type channelrhodopsin-2 and two high-performing channelrhodopsin variants CatCh+ and C1V1. Both variants show enhanced Ca2+ conductance, but only CatCh+ displays high steady-state Ca2+ currents at neutral pH due to reduced H+ competition and low inactivation. We demonstrate that for optogenetic applications, one should always take into account that the variable equilibria of the two open states depend on light intensity, voltage, and the ionic composition of the medium
Two Open States with Progressive Proton Selectivities in the Branched Channelrhodopsin-2 Photocycle
No full textAbstractChannelrhodopsins are light-gated ion channels that mediate vision in phototactic green algae like Chlamydomonas. In neurosciences, channelrhodopsins are widely used to light-trigger action potentials in transfected cells. All known channelrhodopsins preferentially conduct H+. Previous studies have indicated the existence of an early and a late conducting state within the channelrhodopsin photocycle. Here, we show that for channelrhodopsin-2 expressed in Xenopus oocytes and HEK cells, the two open states have different ion selectivities that cause changes in the channelrhodopsin-2 reversal voltage during a light pulse. An enzyme kinetic algorithm was applied to convert the reversal voltages in various ionic conditions to conductance ratios for H+ and divalent cations (Ca2+ and/or Mg2+), as compared to monovalent cations (Na+ and/or K+). Compared to monovalent cation conductance, the H+ conductance, α, is ∼3 × 106 and the divalent cation conductance, β, is ∼0.01 in the early conducting state. In the stationary mixture of the early and late states, α is larger and β smaller, both by a factor of ∼2. The results suggest that the ionic basis of light perception in Chlamydomonas is relatively nonspecific in the beginning of a light pulse but becomes more selective for protons during longer light exposures
Rectification of the Channelrhodopsin Early Conductance
No full textAbstractWe analyzed the nonlinear current-voltage relationships of the early conducting state of channelrhodopsin-2 expressed in Xenopus oocytes and human embryonic kidney cells with respect to changes of the electrochemical gradients of H+, Na+/K+, and Ca2+/Mg2+. Several models were tested for wild-type ChR2 and mutations at positions E90, E123, H134, and T159. Voltage-gating was excluded as cause for the nonlinearity. However, a general enzyme kinetic model with one predominant binding site yielded good fits throughout. The empty site with an apparent charge number of about −0.3 and strong external cation binding causes some inward rectification of the uniport function. Additional inward rectification is due to asymmetric competition from outside between the transported ion species. Significant improvement of the fits was achieved by introducing an elastic voltage-divider formed by the voltage-sensitive barriers
Electrokinetics of miniature K+ channel: Open-state V sensitivity and inhibition by K+ driving force
No full textKcv, isolated from a Chlorella virus, is the smallest known K+ channel. When Kcv is expressed in Xenopus oocytes and exposed to 50 mM [K+](o), its open-state current-voltage relationship (I-V) has the shape of a "tilted S'' between -200 and + 120 mV. Details of this shape depend on the conditioning voltage (Vc) immediately before an I-V recording. Unexpectedly, the I-V relationships, recorded in different [K+] o, do intersect. These characteristics are numerically described here by. ts of a kinetic model to the experimental data. In this model, the Vc sensitivity of I-V is mainly assigned to an affinity increase of external K+ association at more positive voltages. The general, tilted-S shape as well as the unexpected intersections of the I-V relationships are kinetically described by a decrease of the cord conductance by the electrochemical driving force for K+ in either direction, like in fast V-dependent blocking by competing ions
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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