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No / Carbon monoxide (CO) is firmly established as an important, physiological signalling molecule as well as a potent toxin. Through its ability to bind metal-containing proteins, it is known to interfere with a number of intracellular signalling pathways, and such actions can account for its physiological and pathological effects. In particular, CO can modulate the intracellular production of reactive oxygen species, NO and cGMP levels, as well as regulate MAPK signalling. In this review, we consider ion channels as more recently discovered effectors of CO signalling. CO is now known to regulate a growing number of different ion channel types, and detailed studies of the underlying mechanisms of action are revealing unexpected findings. For example, there are clear areas of contention surrounding its ability to increase the activity of high conductance, Ca2+-sensitive K+ channels. More recent studies have revealed the ability of CO to inhibit T-type Ca2+ channels and have unveiled a novel signalling pathway underlying tonic regulation of this channel. It is clear that the investigation of ion channels as effectors of CO signalling is in its infancy, and much more work is required to fully understand both the physiological and the toxic actions of this gas. Only then can its emerging use as a therapeutic tool be fully and safely exploited.

No / The importance of H2S as a physiological signaling molecule continues to develop, and ion channels are emerging as a major family of target proteins through which H2S exerts many actions. The purpose of the present study was to investigate its effects on T-type Ca2+ channels. Using patch-clamp electrophysiology, we demonstrate that the H2S donor, NaHS (10 μM−1 mM) selectively inhibits Cav3.2 T-type channels heterologously expressed in HEK293 cells, whereas Cav3.1 and Cav3.3 channels were unaffected. The sensitivity of Cav3.2 channels to H2S required the presence of the redox-sensitive extracellular residue H191, which is also required for tonic binding of Zn2+ to this channel. Chelation of Zn2+ with N,N,N′,N′-tetra-2-picolylethylenediamine prevented channel inhibition by H2S and also reversed H2S inhibition when applied after H2S exposure, suggesting that H2S may act via increasing the affinity of the channel for extracellular Zn2+ binding. Inhibition of native T-type channels in 3 cell lines correlated with expression of Cav3.2 and not Cav3.1 channels. Notably, H2S also inhibited native T-type (primarily Cav3.2) channels in sensory dorsal root ganglion neurons. Our data demonstrate a novel target for H2S regulation, the T-type Ca2+ channel Cav3.2, and suggest that such modulation cannot account for the pronociceptive effects of this gasotransmitter. / This work was supported by the British Heart Foundation, the Medical Research Council, and the Hebei Medical University

The nodes of Ranvier are short, periodical interruptions in the myelin sheath of myelinated axons, at which voltage-gated sodium channels are highly concentrated. The correct targeting of sodium channels to the nodes of Ranvier permits rapid propagation of action potentials in myelinated axons. The nodes of Ranvier contain a unique set of ion channels, cell-adhesion molecules, and cytoplasmic adaptor proteins. Neurofascins are cell adhesion molecules of the immunoglobulin superfamily and previous work has shown they are involved in the assembly of the node of Ranvier. The Neurofascin (Nfasc) gene is subject to extensive alternative splicing. RT-PCR studies have suggested that there were several different Neurofascin (Nfasc) transcripts. Thus far, research on the Neurofascins has concentrated on two isoforms, Nfasc186 and Nfasc155, which are expressed in neurons and glia respectively. A third Neurofascin isoform, Nfasc140, lacking the Mucin domain and two of the fibronectin repeats was originally identified in the laboratory of V. Bennett. However, neither the location nor function of this protein was known. By RT-PCR I successfully cloned the Nfasc140 cDNA and determined its domain composition, which was confirmed by a series of Western blots using domain-specific antibodies. The developmental expression of Nfasc140 revealed that it is the predominate isoform of Neurofascin during the embryonic stage. Using cell-type-specific conditional Neurofascin knock-out mice, I have also found that Nfasc140 is a neuronal isoform, like Nfasc186. I have used transgenic mouse lines to characterize the location and function of Nfasc140. Like Nfasc186, Nfasc140 is targeted to the nodes of Ranvier and axonal initial segment. Also Nfasc140 alone can reconstitute the nodal complex in Neurofascin knock-out mice in CNS and PNS in the absence of Nfasc186 and Nfasc155. It can also partially restore the electrophysiological function of PNS nerves. In order to address the role of the paranodes in sodium channel clustering, I generated a new neuronal-Cre-expressing transgenic line which, when bred with floxed Nfasc mice, generated early neuronal Neurofascin knock-out mice. Using those animals I have shown that after the ablation of all neuronal Neurofascins, when only glial Nfasc155 is presented, sodium channels can still target to the nodes of Ranvier in both PNS and CNS. These conditional knock-out mice have a longer life span than pan-Neurofascin knock-out mice. This indicates the importance of paranodal junctions, in addition to nodal neuronal Neurofascins, in clustering sodium channels at the node.

published_or_final_version / Surgery / Master / Master of Philosophy

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