Gap junctions are a class of membrane proteins that facilitate cell-to-cell communication by forming channels that directly couple the cytoplasm of neighboring cells. The channels are composed of monomers called connexins. Humans express 21 connexin isoforms in a cell-type specific fashion, and each isoform has distinct mechanisms of permeation and regulation. Co-assembly of multiple isoforms into a single intercellular channel can change channel properties, such as conductance and selectivity to substrates (e.g., ions, metabolites and signaling molecules). However, the mechanistic basis for this functional diversity has remained poorly understood. This lack of mechanistic insight has been due in large part to the lack of high-resolution (atomic-level) structural knowledge on this class of proteins. Prior to this work, the only high-resolution information available on gap junction structure came from a single connexin isoform, connexin-26 (cx26). CryoEM has recently transformed from a low-resolution technique into one capable of rivaling the atomic-level resolutions achieved by x-ray crystallography -- but without the necessity for crystal formation, which has hindered progress towards understanding many classes of proteins (ie, membrane proteins, intrinsically disordered cell signaling complexes and other structurally dynamic systems). For my thesis research, I applied novel methods in single particle electron cryo-microscopy (CryoEM) to study a class of membrane proteins called gap junctions isolated from native lens tissue, as well as two signaling complexes not amenable to other structural techniques. I determined the structure of the lens gap junction, which contains connexin-46 (cx46) and connexin-50 (cx50), to a resolution of 3.4 Å and generated atomic models for both connexin isoforms. Structural analysis paired with molecular dynamics gave insight into energetic features of these channels that determine their isoform-specific conductance and selectivity to electrically charged ions. The cx46/50 gating domain was found to be stabilized by hydrophobic anchors, and also seems to adopt a more stable open state than found in cx26. Genetic mutations associated with congenital cataract formation were found to map to hot-spots of conserved structural and functional importance, rationalizing their disease-causing effects. As part of collaborative efforts, I used methods in single particle EM to characterize two separate signaling complexes that had proven difficult to study with x-ray crystallography and NMR spectroscopy. One system, Ca2+/Calmodulin Kinase II (CaMKII), is a signaling complex in the brain involved in memory formation. Characterization of the CaMKII complex by single particle EM revealed an extended state, which was also shown to be prevalent in cells -- giving more depth to our understanding of how this signaling molecule functions. The second collaboration characterized the multimeric binding sites of the hub protein LC8, which interacts with the disordered region of a transcription factor (ASCIZ). This provided support for a novel model of transcription regulation, wherein LC8 fine-tunes its own transcription levels through multi-valent binding to the disordered region of its own regulatory transcription factor.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 109-118).

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

published_or_final_version / Surgery / Master / Master of Philosophy

How behaviors are generated by neural circuits is one of the central questions in neurobiology. Under standard culture conditions, Caenorhabditis elegans travel by propagating sinusoidal waves, moving primarily forward, punctuated by brief runs of backing. How these behaviors are generated and altered is not well understood. Using a combination of behavioral analyses and neuronal imaging, I reveal that an activity imbalance between cholinergic A- and B-motoneurons is the key determinant of directional locomotion. Furthermore, heterotypic gap junctions that couple command interneurons and motoneurons of the backward motor circuit, mediated by innexins UNC-7 in AVA and UNC-9 in A-motoneurons, respectively, establish the B>A activity pattern required for forward movement. Loss of this coupling results in both the hyperactivation of AVA backward interneurons revealing the unregulated, endogenous activity of A-motoneurons. With equal A-motoneuron activity levels as B-motoneurons, innexin mutant animals exhibit irregular body bending (kinking) instead of executing forward motion, as well as increased backing. Through a genetic screen, I identified two stomatin-like proteins as regulators of innexin UNC-9 activity that affect C. elegans’ directional movement. The loss of function of stomatin-like unc-1 leads to the same kinker phenotype as unc-7 or unc-9 mutants. Like UNC-9, UNC-1 functions primarily in the A-motoneurons to allow forward motion, suggesting that UNC-1 is required for effective UNC-7-UNC-9 coupling between AVA and A-motoneurons. Dominant mutations in UNC-1, and another stomatin-like protein STO-6, exhibit genetic interactions with these innexin mutants. These mutations partially restore the forward movement of unc-7 mutants, in an UNC-9-dependent manner, indicating that they regulate UNC-9 channel activity in motoneurons to re-establish the B>A-motoneuron activity pattern in the absence of heterotypic gap junctions between interneurons and motoneurons. These studies describe a role of gap junctions as regulators of circuit dynamics by establishing an imbalanced motoneuron activity pattern that favors forward motion, which can be modulated by upper layer inputs. This study also identifies stomatin-like regulators of innexin hemichannel and gap junction function. Future work will focus on understanding mechanisms through which these stomatins regulate the activity of specific innexin channels in C. elegans motoneurons, as well as their contribution to the dynamic output of the C. elegans motor circuit.

Vasomotion is the spontaneous oscillation of vascular tone, occurring due to synchronization of internal calcium fluctuations between multiple vascular smooth muscle cells by gap junction and electrical communication. Although altered vasomotion has been observed in a variety of pathological situations, characterization of these alterations has been lacking. Using a novel method of spectral quantification, and two experimental models known to have altered vascular structure, the present thesis was designed to evaluate whether vasomotion characteristics could be correlated with altered vascular structure. Rats with perinatal iron deficiency (PID) have previously been shown to possess altered vascular structure. When phenylephrine-mediated contractile and acetylcholine-mediated dilatory responses were investigated in PID animals, they both displayed blunted relaxation as compared to control vessels. When vasomotion characteristics were quantified, vessels taken from PID animals exhibited a decreased power in the very low frequency window (VLF <0.2 Hz). Changing vessel oxygenation to 10% O2 from 95% O2 did not result in significant alterations of vasomotion characteristics. The primary frequency of oscillation was investigated with a peak finder, and found to be significantly different compared to control in both the aorta and renal arteries obtained from PID animals. To investigate the effect of antihypertensive treatment (enalapril and hydrochlorothiazide) on gap junction communication, spontaneously hypertensive rats (SHR) were subject to a 2-week intensive angiotensin converting enzyme inhibitor treatment. This treatment resulted in significant vascular structural regression. All vessels (aorta, renal, mesenteric) from treated animals had greater proportions of power in the VLF window, with both the mesenteric and renal vessels exhibiting a primary peak of oscillation around 0.2 Hz; whereas the aorta had a primary peak at 0.12 Hz. Investigating altered gap junction communication with the gap junction blocker 18-α glycyrrhetinic acid, revealed that vascular bed location was the determining factor of vasomotion response. Immunoblotting did not indicate differences in connexin 43, a major gap junction protein in the vascular smooth muscle. These studies suggest that vasomotion characteristics can be used as a method of vascular phenotype investigation; vasomotion characteristics were significantly different in vessels taken from PID and hypertensive animals as compared to control and antihypertensive-treated animals, respectively. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2008-09-26 11:39:44.043

How behaviors are generated by neural circuits is one of the central questions in neurobiology. Under standard culture conditions, Caenorhabditis elegans travel by propagating sinusoidal waves, moving primarily forward, punctuated by brief runs of backing. How these behaviors are generated and altered is not well understood. Using a combination of behavioral analyses and neuronal imaging, I reveal that an activity imbalance between cholinergic A- and B-motoneurons is the key determinant of directional locomotion. Furthermore, heterotypic gap junctions that couple command interneurons and motoneurons of the backward motor circuit, mediated by innexins UNC-7 in AVA and UNC-9 in A-motoneurons, respectively, establish the B>A activity pattern required for forward movement. Loss of this coupling results in both the hyperactivation of AVA backward interneurons revealing the unregulated, endogenous activity of A-motoneurons. With equal A-motoneuron activity levels as B-motoneurons, innexin mutant animals exhibit irregular body bending (kinking) instead of executing forward motion, as well as increased backing. Through a genetic screen, I identified two stomatin-like proteins as regulators of innexin UNC-9 activity that affect C. elegans’ directional movement. The loss of function of stomatin-like unc-1 leads to the same kinker phenotype as unc-7 or unc-9 mutants. Like UNC-9, UNC-1 functions primarily in the A-motoneurons to allow forward motion, suggesting that UNC-1 is required for effective UNC-7-UNC-9 coupling between AVA and A-motoneurons. Dominant mutations in UNC-1, and another stomatin-like protein STO-6, exhibit genetic interactions with these innexin mutants. These mutations partially restore the forward movement of unc-7 mutants, in an UNC-9-dependent manner, indicating that they regulate UNC-9 channel activity in motoneurons to re-establish the B>A-motoneuron activity pattern in the absence of heterotypic gap junctions between interneurons and motoneurons. These studies describe a role of gap junctions as regulators of circuit dynamics by establishing an imbalanced motoneuron activity pattern that favors forward motion, which can be modulated by upper layer inputs. This study also identifies stomatin-like regulators of innexin hemichannel and gap junction function. Future work will focus on understanding mechanisms through which these stomatins regulate the activity of specific innexin channels in C. elegans motoneurons, as well as their contribution to the dynamic output of the C. elegans motor circuit.

Plyšinių jungčių kanalai yra sudaryti iš koneksinų baltymų, kurie yra randami įvairiuose audiniuose ir dėka jų ląstelės gali keistis metabolitais bei elektriniais signalais. Plyšinių jungčių kanalai pasižymi dideliu jautrumu viduląsteliniam pH. Šiame darbe tyrėme įvairių inhibitorių poveikį homotipinių plyšinių jungčių, sudarytų iš Cx26, Cx30.2, Cx36, Cx40, Cx43, Cx45, Cx46, Cx47 ir Cx50 laidumui bei šio poveikio priklausomybę nuo viduląstelinio pH.. Plyšinių jungčių blokatoriais buvo pasirinktos kelias skirtingas farmakologines grupes atstovaujančios medžiagos: ilgagrandžiai alkoholiai (heksanolis, oktanolis, nonanolis), izofluranas, meflokvinas, flufenaminė rūgštis, karbenoksolonas ir arachidono rūgštis. Pademonstravome, kad pHi pakėlimas panaudojant NH4Cl iki ~8 didino plyšinių jungčių laidumą tarp ląstelių, turinčių Cx30.2 ar Cx45, tačiau neturėjo jokios įtakos ląstelėms su Cx26, Cx40, Cx46, Cx47 ir Cx50 ir mažino laidumą tarp ląstelių su Cx36 ar Cx43. pHi padidinimas žymiai susilpnino ilgagrandžių alkoholių, izoflurano ir meflokvino poveikį Cx45 plyšinių jungčių laidumui ir neturėjo įtakos flufenaminės rūgšties, karbenoksolono ir arachidono rūgšties poveikiui. Taip pat parodėme, kad nuo pHi priklauso oktanolio poveikio Cx45 plyšinių jungčių laidumui galingumas, t.y., pusinė blokavimo koncentracija IC50 buvo 0.1, 0.25 ir 2.68 mM atitinkamai prie pHi 6.9, 7.2 ir 8.1. Manoma, kad pH poveikis plyšinių jungčiu laidumui pasireiškia protonams jungiantis su koneksino baltymo... [toliau žr. visą tekstą] / Gap junction channels formed from connexin proteins provide a direct pathway for electrical and metabolic cell-cell communication exhibiting high sensitivity to intracellular pH. We examined pHi-dependent modulation of junctional conductance of GJs formed of Cx26, Cx30.2, Cx36, Cx40, Cx43, Cx45, Cx46, Cx47 and Cx50 by reagents representing several distinct groups of uncouplers, such as long carbon chain alkanols, arachidonic acid, carbenoxolone, isoflurane, flufenamic acid and mefloquine. We demonstrate that alkalization by NH4Cl to pH≈8 increased coupling in cells expressing Cx30.2 and Cx45, yet did not affect coupling of Cx26, Cx40, Cx46, Cx47 and Cx50 and decreased it in Cx43 and Cx36 gap junctions. Unexpectedly, cells expressing Cx45, but not other connexins, exhibited full coupling recovery after alkalization with NH4Cl under the continuous presence of alkanols, isoflurane and mefloquine. There was no coupling recovery by alkalization in the presence of arachidonic acid, carbenoxolone and flufenamic acid. In cells expressing Cx45, IC50 for octanol was 0.1, 0.25 and 2.68 mM at pHis of 6.9, 7.2 and 8.1, respectively. Histidine modification of Cx45 protein by N-bromosuccinimide proportionally reduced the coupling-promoting effect of NH4Cl as well as uncoupling effect of octanol. This suggests that long chain alkanols and some other uncouplers may act through the formation of hydrogen bonds with the as-of-yet unidentified histidine/s of the Cx45 gap junction channel protein... [to full text]

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 76-93). / Microfiche. / vii, 93 leaves, bound ill. (some col.) 29 cm

Thesis (M.S.)--Missouri State University, 2008. / "August 2008." Includes bibliographical references (leaves 45-51). Also available online.