No description available.

No description available.

In excitable tissues, voltage-gated calcium channel activity is critical in the linkage of electrical stimuli to physiological responses, and so modulation of calcium channels therefore has significant implications. The exact mechanisms of calcium channel modulation and membrane expression, however, remain elusive. Previous work suggests that the calcium channel β subunit (Cavβ) modulates the expression and biophysical properties of the pore-forming α1 subunit. Previous research has shown that the core domains of Cavβ subunits are highly conserved, although alternative splicing in the highly variable N-terminus and HOOK regions is commonly observed in invertebrates, teleost fish and mammals. Splicing in these regions can produce unique isoforms that differentially modulate the membrane trafficking and gating properties of high voltage-activated calcium channels. With this in mind, two novel isoforms of an invertebrate Cavβ subunit have been identified and cloned from the pond snail Lymnaea stagnalis, which contain a novel N-terminus not previously identified. In addition, one of these novel isoforms excludes an optional, short exon in the HOOK region of LCavβ. Intron sequencing and amino acid alignments of the variable N-terminal and HOOK regions with mammalian and fish homologs have revealed that the genomic structure of Cavβ subuinits is conserved, despite the divergence in sequence and function between genes and splice isoforms. It was determined that the previously characterized LCavβ isoform, as well as the two new isoforms, can act to fine-tune calcium channel activity by modulating the membrane expression, voltage-dependencies of activation and inactivation and gating kinetics of invertebrate homologs of L-type (LCav1) and neuronal (LCav2) calcium channels. It is hoped that broadening our knowledge of simplified invertebrate calcium channels, like those found in Lymnaea, may advance our understanding the workings of our own highly elaborate and dynamic calcium channel complexes, and the nervous system as a whole.

In excitable tissues, voltage-gated calcium channel activity is critical in the linkage of electrical stimuli to physiological responses, and so modulation of calcium channels therefore has significant implications. The exact mechanisms of calcium channel modulation and membrane expression, however, remain elusive. Previous work suggests that the calcium channel β subunit (Cavβ) modulates the expression and biophysical properties of the pore-forming α1 subunit. Previous research has shown that the core domains of Cavβ subunits are highly conserved, although alternative splicing in the highly variable N-terminus and HOOK regions is commonly observed in invertebrates, teleost fish and mammals. Splicing in these regions can produce unique isoforms that differentially modulate the membrane trafficking and gating properties of high voltage-activated calcium channels. With this in mind, two novel isoforms of an invertebrate Cavβ subunit have been identified and cloned from the pond snail Lymnaea stagnalis, which contain a novel N-terminus not previously identified. In addition, one of these novel isoforms excludes an optional, short exon in the HOOK region of LCavβ. Intron sequencing and amino acid alignments of the variable N-terminal and HOOK regions with mammalian and fish homologs have revealed that the genomic structure of Cavβ subuinits is conserved, despite the divergence in sequence and function between genes and splice isoforms. It was determined that the previously characterized LCavβ isoform, as well as the two new isoforms, can act to fine-tune calcium channel activity by modulating the membrane expression, voltage-dependencies of activation and inactivation and gating kinetics of invertebrate homologs of L-type (LCav1) and neuronal (LCav2) calcium channels. It is hoped that broadening our knowledge of simplified invertebrate calcium channels, like those found in Lymnaea, may advance our understanding the workings of our own highly elaborate and dynamic calcium channel complexes, and the nervous system as a whole.

No description available.

The present research is focused on the topic of inflammation-drug interaction. Inflammation complicates many human diseases and conditions ranging from obesity to cancer. Therefore, the study of the effect of inflammation on drug pharmacokinetics and pharmacodynamics is pivotal. First, we tested the hypothesis that controlling inflammation using valsartan can restore the previously reported altered verapamil pharmacokinetics and pharmacodynamics. Such an effect is expected due to the anti-inflammatory properties of angiotensin II inhibition. Inflammation resulted in L-type calcium channel target protein (Cav1.2) downregulation and reduced verapamil potency in pre-adjuvant arthritis rat model. Valsartan treatment reversed the observed downregulation of L-type calcium channels thereby enhancing verapamil potency. This beneficial interaction, once proven in humans, may be of value in cardiac patients with superimposing inflammatory diseases. Second, we investigated whether the response to verapamil is reduced in experimentally induced acute myocardial injury (AMI) in rats. AMI caused a 75% reduction in verapamil potency and Cav1.2 target protein downregulation. If extrapolated to humans, our observations may suggest that L-type calcium channel downregulation can contribute, at least in part, to the poor outcome in myocardial infarction patients treated with calcium channel blockers (CCBs). Third, we studied the effect of obesity on the pharmacological response of CCBs in children with renal disease. Our data indicated that obese children are less responsive to CCBs than non-obese ones. Therefore, obesity should be considered when initiating antihypertensive drug therapy in children. Last, we were interested in finding out if the expression of other target genes is also altered by inflammation. We used real time polymerase chain reaction, after determination of the best housekeeping gene to be used as an internal control. Inflammation resulted in significant alterations of several molecular targets and transporters affecting the pharmacokinetics and pharmacodynamics of drugs. These findings may provide an insight into the effect of inflammation on drug targets and modulators of disease pathogenesis. In conclusion, inflammation is a missed ring in the chain of therapy. The research presented in this thesis will add to the inflammation-drug interaction field important findings that will help understanding the role of inflammation in pharmacotherapy outcomes. / pharmaceutical sciences

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [64]-71).

Voltage-gated calcium channels (CaV) are key regulators of cellular excitability; they translate electrical information into biochemical responses in excitable cells such as nerve and muscle cells. CaV are separated in three families: CaV1, CaV2 and CaV3. CaV1 and CaV2 typically comprise a pore-forming alpha1 with auxiliary β and alpha2delta subunits. The alpha2delta enhances surface expression and modulates the biophysical properties of CaV. It has been implicated in pain and epilepsy, and the target for anti-epileptic and anti-nociceptive gabapentinoid drugs. Despite its clinical significance, the relationship between the structure and function of this subunit remains poorly understood. Fitzgerald and co-workers recently showed that the N-terminal region of alpha2delta-1, termed the R domain (Rd), is both necessary and sufficient to replicate the effects of full-length alpha2delta on CaV2.2 channels. In order to understand the functional role(s) of Rd and the regions downstream of it, the biochemical and cell biological properties of alpha2delta were explored producing a set of alpha2delta-truncated proteins, in which the delta protein was inserted into an inert type-1 transmembrane reporter protein (PIN-G). The construct was then extended towards the N-terminal of the alpha2delta-1 (C- to N- PIN-constructs). Other sets of constructs, lacking the delta protein, were prepared after successive additions of stop codons (TGA) in the alpha2delta (N- to C- PIN-constructs). The MIDAS motif within the VWA domain of alpha2delta-1/-2 has been suggested to be critical for trafficking of alpha2delta to the cell surface. Whilst the present study supports a role for MIDAS in surface expression of alpha2delta, it is the Rd that appears essential. Mutation of MIDAS reduced expression, whereas the removal of Rd completely abolished the presence of alpha2delta at the cell surface. Examination of the electrophysiological effects of N- to C- terminal truncated constructs (PIN-Rd, PIN-Rd-VWA and PIN-alpha2) on CaV2.2/β1b channels revealed that, in contrast to the full functionality of Rd alone, extension to the end of the VWA domain, or the alpha2 region, abolished typical alpha2delta-mediated current enhancement. Nevertheless, both constructs increased rate of voltage-dependent inactivation, indicating that they interact with the channel via Rd. Thus, Rd appears to contain all the machinery required to support the electrophysiological and trafficking effects of alpha2delta. Preliminary work has generated tools that could be used to conduct competition-based assays to identify the extracellular loops of the CaV2.2 alpha1 subunit that interact with the Rd. Such an approach could be applied to other alpha1 subtypes to determine discrete alpha2-Rd interactions, information that is critical for further therapeutic exploitation of alpha2delta. Finally, the data from this thesis and the existing literature have been used to propose a revised model of how alpha2delta interacts with CaV.

Calcium entry through voltage-gated calcium (CaV) channels is important in diverse cellular processes including neurotransmitter release, gene expression and cardiac pacemaker activity. CaV channels auxiliary CaVβ subunits enhance plasma membrane expression and modify the biophysical properties of CaVα1 subunits. Due to their multi-domain structures - including a conserved SH3-GK 'core' and hypervariable N- and C- terminal domains - CaVβs are also considered to be members of the membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins, and may therefore act as molecular scaffolds both within and outside the CaV channel complex. This project studied the roles of CaVβ N- and C-terminal hypervariable domains in contributing to isoform-specific differences in CaVβ functions both in a) CaV channel complex expression and distribution, and b) interactions with non channel proteins. To analyse such contributions a series of molecular tools were developed to assess the distributions of CaVβs (both within and outside the CaV channel complex) and their interactions with novel potential partner proteins. This involved systematically testing fluorophore- and epitope-tagged CaVβs for co-localisation with both fluorophore-tagged CaV2.2 and a range of myc-tagged potential interaction partners (as quantified either by a 'Membrane Localisation Index' developed during this project or Intensity Correlation Analysis). This approach uncovered much detail about relative isoform specificities of CaVβ non-channel complex protein-protein interactions, however one particularly striking interaction was discovered between CaVβ1b/CaVβ4 and the nuclear protein Heterochromatin 1 γ (HP1γ), where nuclear translocation of CaVβ1b or CaVβ4 was induced upon association with HP1γ. Given the similarity of CaVβ1b and CaVβ4 N termini, a series of CaVβ1b N-terminal chimeras were then created, where the N terminus was exchanged with that of CaVβ3 (which did not interact with HP1γ). Subsequent imaging studies using these chimeras then confirmed that the CaVβ1b N terminus is necessary for co-localisation with HP1γ and subsequent HP1γ mediated CaVβ nuclear uptake. Given that an interaction between the CaVβ3 isoform and Pax6(S) - another nuclear protein - have been reported, where the CaVβ3-Pax6(S) interaction also induces nuclear translocation of both proteins, the CaVβ1b/CaVβ4-HP1γ interaction may represent one of a range of as-yet undiscovered CaVβ1b/gene regulatory protein interactions. As interaction with CaVβ3 suppresses the transcriptional activity of Pax6(S), nuclear targeting may be an important means by which CaVβs modulate gene expression - which in the case of HP1γ interactions may occur via de-repression.

No description available.