Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / "December 1998" Typescript. Vita. Includes bibliographical references (l. 152-155). Also available on the Internet.

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 192-205).

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [58]-66).

Thesis (Ph.D.)--Georgetown University, 2008. / Includes bibliographical references.

Dissertation (Ph.D.)--Auburn University, 2009. / Abstract. Vita. Includes bibliographic references (p.94-115).

Membrane proteins play a vital role in cellular processes. In this thesis, we use KcsA, a prokaryotic potassium channel, as a model to investigate the gating mechanism of ion channels and the effect of anionic lipids on the channel activity using solid-state NMR spectroscopy. KcsA activity is known to be highly dependent on the presence of negatively charged lipids. Multiple crystal structures combined with biochemistry assays suggest that KcsA is co-purified with anionic lipids with phosphatidylglycerol headgroup. Here, we identified this specifically bound, isotopically labeled lipid in the protein 13C-13C correlation spectra. Our results reveal that the lipid cross peaks show stronger intensity when the channel is in the inactivated state compared to the activated state, which indicates a stronger protein-lipid interaction when KcsA is inactivated. In addition, our data shows that including anionic lipids into proteoliposomes leads to a weaker potassium ion affinity at the selectivity filter. Considering ion loss as a model of inactivation, our results suggest anionic lipids promote channel inactivation. However, the surface charge is not the only physical parameter that regulates channel gating or conformational preference. We found that the channel adapted to different conformations when reconstituted into liposome either made of DOPC or DOPE, two zwitterionic lipids. Also, we were able to stabilize the open-conformation of KcsA in 3:1 DOPE/DOPG liposome at pH 4.0 and acquired several multi-dimensional solid-state NMR experiments for site-specific resonance assignments. This is the first time that we obtain wild-type full-length KcsA structural information on the transient state. The structure is not only important for understanding channel gating, but can also serve as a homology model for investigating drug binding with more complicated potassium channels such as human voltage gated channel (hERG).

No description available.

The large conductance calcium dependent potassium (BKCa) channels are expressed in a large variety of cell types including neurons where they modulate excitability and action potential shape. Within the enteric nervous system, stretch-sensitive BKCa channels are expressed on intrinsic primary afferent neurons (IPANs) where they decrease the neurons’ excitability during intestinal contractions. A major determinant of peristalsis is slow excitatory neurotransmission (sEPSPs) within the IPAN to IPAN sensory network, and we wondered whether such transmission might also alter BKCa channel opening. All experiments were performed on longitudinal-muscle myenteric preparations prepared from jejunal segment taken from freshly euthanized adult male Swiss Webster mice. With the myenteric plexus exposed by microdissection, BKCa channel activity was recorded in cell-attached mode via the patch clamp technique. BKCa channel activity was recorded before and after presynaptic electrical stimulation, which was designed to evoke postsynaptic sEPSPs. The morphotype was verified by intracellular injection of a marker dye (neurobiotin). In addition, a blocker and opener were used to identify the effects of BKCa currents on IPAN properties. Analysis of unitary channel recordings revealed increased BKCa open probability (NPo) at fixed trans-patch potentials following sEPSPs. All BKCa channels were independently voltage sensitive with increased NPo during patch depolarisation. Analysis of whole-cell experiments also revealed BKCa channels have a significant effect on the undershoot amplitude of action potentials, and the rate at which IPANs repolarise. This study demonstrates that sEPSPs within the enteric nervous system modulate the function of BKCa channels in IPANs adding to the mechanistic understanding of enteric synaptic transmission and providing a potential target for therapeutic modulation of enteric nervous system excitability. / Thesis / Master of Science (MSc)

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

Ca2+-activated potassium channels (KCa) and canonical transient receptor potential (TRPC) channels are essential to endothelial function. In ischemic heart disease, or in cardiac surgery, coronary endothelium is subjected to ischemia-reperfusion (I-R) / hypoxia-reoxygenation (H-R) injury. Hyperkalemic cardioplegic or organ preservation solutions used in cardiac surgery including heart transplantation also impair endothelial function. The present study was designed to mainly investigate whether endothelial dysfunction occurring in H-R or in hyperkalemic exposure is attributable to alterations of intermediate- and small-conductance KCa (IKCa and SKCa) channels, or TRPC channels, in particular, the TRPC3 channel. / Exposure to 60-min hypoxia followed by reoxygenation inhibited the vasorelaxant response of coronary arteries to IKCa / SKCa activator 1-EBIO. H-R reduced endothelial IKCa and SKCa currents and downregulated IKCa expression in PCECs. 1-EBIO enhanced endothelial K+ current that was blunted by H-R. / Exposure to hyperkalemic solutions decreased Ca2+ influx via TRPC3 in PCECs. The reduced Ca2+ influx in PCECs and the attenuated EDHF-mediated vasorelaxation in porcine coronary arteries, which were caused by hyperkalemic or cardioplegic / organ preservation solutions, were restored by OAG. / In PCECs, hypoxia for 60-min with reoxygenation reduced TRPC3 current and Ca2+ influx via TRPC3, which was accompanied by decreased NO release and endothelium-dependent vasorelaxation of porcine coronary arteries. The compromised endothelial function was restored by OAG. The translocation of TRPC3 to endothelial membrane was inhibited by H-R. / In TRPC3-overexpressing HEK293 cells, followed by reoxygenation, short-time hypoxia (10-min) enhanced, whereas prolonged hypoxia (60-min) reduced the current induced by TRPC3/6/7 activator OAG. / Our results indicate that: (1) Endothelial IKCa, SKCa and TRPC3 play an important role in regulating vascular tone; TRPC3 contributes to NO release from endothelial cells and is also involved in the function of EDHF. (2) H-R (60-30 min) reduces endothelial IKCa and SKCa currents with downregulation ofthe protein expression of IKCa. (3) H-R has dual effect on TRPC3 with short-time hypoxia (lO-min) enhancing whereas prolonged hypoxia (60-min) decreasing the electrophysiological activity of this channel. H-R (60-30 min) inhibits the translocation of TRPC3 to endothelial membrane. Furthermore, H-R inhibits Ca2+ influx via TRPC3 and such inhibition is associated with a decrease of NO production. (4) The activator of IKCa / SKCa or TRPC protects coronary endothelium against H-R injury. In coronary endothelium exposed to hyperkalemic or cardioplegic / organ preservation solutions, TRPC activator also exhibits protective effect. / The above findings are likely to have significant implications in ischemic heart disease and in modem cardiopulmonary surgery. / Whole-cell membrane currents of IKCa, SKCa, or TRPC3 were recorded by patch-clamp in primary cultured porcine coronary endothelial cells (PCECs). TRPC3 current was also studied in human embryonic kidney cells (HEK293 cells) transiently overexpressed with TRPC3 gene. Protein or mRNA expression of these channels was detected by Western blot or RT-PCR. Intracellular Ca2+ concentration was measured by Ca2+ imaging technique. Isometric force study was performed in a wire myograph and endothelial nitric oxide (NO) release was measured electrochemically by using a NO-specific microsensor in porcine coronary small arteries. / Huang, Junhao. / "December 2010." / Adviser: Qin Yang. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 138-165). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.