A light-gated potassium channel for sustained neuronal inhibition.

Currently available inhibitory optogenetic tools provide short and transient silencing of neurons, but they cannot provide long-lasting inhibition because of the requirement for high light intensities. Here we present an optimized blue-light-sensitive synthetic potassium channel, BLINK2, which showed good expression in neurons in three species. The channel is activated by illumination with low doses of blue light, and in our experiments it remained active over (tens of) minutes in the dark after the illumination was stopped. This activation caused long periods of inhibition of neuronal firing in ex vivo recordings of mouse neurons and impaired motor neuron response in zebrafish in vivo. As a proof-of-concept application, we demonstrated that in a freely moving rat model of neuropathic pain, the activation of a small number of BLINK2 channels caused a long-lasting (>30 min) reduction in pain sensation.

Engineering a Ca⁺⁺⁺-sensitive (bio)sensor from the pore-module of a potassium channel.

Signals recorded at the cell membrane are meaningful indicators of the physiological vs. pathological state of a cell and will become useful diagnostic elements in nanomedicine. In this project we present a coherent strategy for the design and fabrication of a bio-nano-sensor that monitors changes in intracellular cell calcium concentration and allows an easy read out by converting the calcium signal into an electrical current in the range of microampere that can be easily measured by conventional cell electrophysiology apparatus.

Synergistic difference in the effect of stretching on electromechanical delay components.

This study investigated the synergistic difference in the effect of stretching on electromechanical delay (EMD) and its components, using a simultaneous recording of electromyographic, mechanomyographic, and force signals. Twenty-six healthy men underwent plantar flexors passive stretching. Before and after stretching, the electrochemical and mechanical components of the EMD and the relaxation EMD (R-EMD) were calculated in gastrocnemius medialis (GM), lateralis (GL) and soleus (SOL) during a supramaximal motor point stimulation. Additionally, joint passive stiffness was assessed. At baseline, the mechanical components of EMD and R-EMD were longer in GM and GL than SOL (Cohen's d from 1.78 to 3.67). Stretching decreased joint passive stiffness [-22(8)%, d = -1.96] while overall lengthened the electrochemical and mechanical EMD. The mechanical R-EMD components were affected more in GM [21(2)%] and GL [22(2)%] than SOL [12(1)%], with d ranging from 0.63 to 1.81. Negative correlations between joint passive stiffness with EMD and R-EMD mechanical components were found before and after stretching in all muscles (r from -0.477 to -0.926; P from 0.007 to <0.001). These results suggest that stretching plantar flexors affected GM and GL more than SOL. Future research should calculate EMD and R-EMD to further investigate the mechanical adaptations induced by passive stretching in synergistic muscles.

A virus-encoded potassium ion channel is a structural protein in the chlorovirus Paramecium bursaria chlorella virus 1 virion.

Most chloroviruses encode small K(+) channels, which are functional in electrophysiological assays. The experimental finding that initial steps in viral infection exhibit the same sensitivity to channel inhibitors as the viral K(+) channels has led to the hypothesis that the channels are structural proteins located in the internal membrane of the virus particles. This hypothesis was questioned recently because proteomic studies failed to detect the channel protein in virions of the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1). Here, we used a mAb raised against the functional K(+) channel from chlorovirus MA-1D to search for the viral K(+) channel in the virus particle. The results showed that the antibody was specific and bound to the tetrameric channel on the extracellular side. The antibody reacted in a virus-specific manner with protein extracts from chloroviruses that encoded channels similar to that from MA-1D. There was no cross-reactivity with chloroviruses that encoded more diverse channels or with a chlorovirus that lacked a K(+) channel gene. Together with electron microscopic imaging, which revealed labelling of individual virus particles with the channel antibody, these results establish that the viral particles contain an active K(+) channel, presumably located in the lipid membrane that surrounds the DNA in the mature virions.

Chlorella virus ATCV-1 encodes a functional potassium channel of 82 amino acids.

Chlorella virus PBCV-1 (Paramecium bursaria chlorella virus-1) encodes the smallest protein (94 amino acids, named Kcv) previously known to form a functional K+ channel in heterologous systems. In this paper, we characterize another chlorella virus encoded K+ channel protein (82 amino acids, named ATCV-1 Kcv) that forms a functional channel in Xenopus oocytes and rescues Saccharomyces cerevisiae mutants that lack endogenous K+ uptake systems. Compared with the larger PBCV-1 Kcv, ATCV-1 Kcv lacks a cytoplasmic N-terminus and has a reduced number of charged amino acids in its turret domain. Despite these deficiencies, ATCV-1 Kcv accomplishes all the major features of K+ channels: it assembles into a tetramer, is K+ selective and is inhibited by the canonical K+ channel blockers, barium and caesium. Single channel analyses reveal a stochastic gating behaviour and a voltage-dependent conductance that resembles the macroscopic I/V relationship. One difference between PBCV-1 and ATCV-1 Kcv is that the latter is more permeable to K+ than Rb+. This difference is partially explained by the presence of a tyrosine residue in the selective filter of ATCV-1 Kcv, whereas PBCV-1 Kcv has a phenylalanine. Hence, ATCV-1 Kcv is the smallest protein to form a K+ channel and it will serve as a model for studying structure-function correlations inside the potassium channel pore.

The voltage-sensing domain of a phosphatase gates the pore of a potassium channel.

The modular architecture of voltage-gated K(+) (Kv) channels suggests that they resulted from the fusion of a voltage-sensing domain (VSD) to a pore module. Here, we show that the VSD of Ciona intestinalis phosphatase (Ci-VSP) fused to the viral channel Kcv creates Kv(Synth1), a functional voltage-gated, outwardly rectifying K(+) channel. Kv(Synth1) displays the summed features of its individual components: pore properties of Kcv (selectivity and filter gating) and voltage dependence of Ci-VSP (V(1/2) = +56 mV; z of ~1), including the depolarization-induced mode shift. The degree of outward rectification of the channel is critically dependent on the length of the linker more than on its amino acid composition. This highlights a mechanistic role of the linker in transmitting the movement of the sensor to the pore and shows that electromechanical coupling can occur without coevolution of the two domains.