Biological transport

Neurons are able to maintain membrane potential and synaptic integrity by an
intricate equilibrium of membrane transporter proteins and ion channels. Two
membrane proteins of particular importance in the vertebrate retina are the
excitatory amino acid transporters (EAATs) which are responsible for the reuptake
of glutamate into both glial and neuronal cells and the sodium potassium
chloride cotransporters (NKCCs) that are responsible for the uptake of chloride
ions into the cell. NKCCs are electro-neutral with the uptake of 2 Cl- coupled to
an exchange of a potassium and Na+ ion into the cells. Therefore, there is little
change of cell membrane potential in the action of NKCCs. In this study the
localization and function of EAATs in the distal retina is investigated. Whole cell
patch clamp recordings in lower vertebrate retina have demonstrated that EAAT2
is the main synaptic EAATs in rod photoreceptors and it is localized to the axon terminals. Furthermore, the action of the transporter seems to be modified by
intracellular calcium concentration. There is also evidence that EAAT2 might be
regulated by feedback from the neuron network by glycinergic and GABAergic
mechanisms. The second half of this study investigates expression of NKCCs in
the retina by western blot analysis and quantitative polymerase chain reaction.
There are two forms of NKCCs, NKCC1 and NKCC2. NKCC1 is mostly
expressed in the central nervous system and NKCC2 was thought to only be
expressed in the kidneys. NKCC1 is responsible for the majority of chloride
uptake into neuronal and epithelial cells and NKCC1 is expressed in the distal
retina where photoreceptors synapse on second order horizontal and bipolar
cells. This study found the expression of NKCC1 in the distal retina to be
regulated by temporal light and dark adaptation. Light adaptation increased
phosphorylated NKCC1 expression (the active form of the cotransporter). The
increase in NKCC1 expression during light adaptation was modulated by
dopamine. Specifically, a D1 receptor agonist increased phosphorylated NKCC1
expression. Dopamine is an essential chemical and receptor known for initiating
light adaptation in retina. Finally, an NKCC1 knockout mouse model was
examined and it revealed that both forms of NKCC are expressed in the
vertebrate retina.

Hydroxylysine is produced as a posttranslational modification mainly in collagens, the most abundant protein in mammals. Lysyl hydroxylase (LH) is the enzyme that catalyzes the formation of hydroxylysyl residues in collagen by hydroxylation of -X-Lys-Gly- sequences, for which it requires Fe 2+, 2-oxoglutarate, O2 and ascorbate. In order to study the hydroxylation reaction catalysed by LH, we have synthesized 4 different peptides [for example, GFP*GLP*GAKGE (P*=hydroxyproline) and the corresponding hydroxylated (hydroxylysine-containing) peptide] using Fmoc solid-phase methodology. Peptides have been characterized by HPLC, MALDI-TOF mass spectrometry and CD spectroscopy. A new method for efficient separation of lysine- from hydroxylysine-containing peptides by HPLC has been developed in both organic phase (1-anthroylnitrile as derivatizating reagent) and aqueous phase (dansyl chloride as derivatizating reagent). These reagents have been used to derivatize peptides prior to HPLC analysis. The products (di- and tetra-substituted lysine- and hydroxylysine-containing peptides) have been fully separated by HPLC and their structure confirmed by MALDI-TOF MS analysis. Efficient separation of derivatized peptides will allow for the convenient and rapid measurement of LH activity by HPLC methods.

Taurine is the second most abundant amino acid in the CNS after glutamate and its functions have been found largely related to intracellular calcium ([Ca2+]i) modulation, osmoregulation, membrane stabilization, reproduction and immunity. The action of taurine has also been implicated in neurotransmission and neuromodulation though its specific sites of action are not fully understood. Isolated retinal neurons from the larval tiger salamanders (Ambystoma tigrinum) were used as a model to study the neuromodulatory role of taurine in the CNS and to gain insights into its potential sites of action. A combination of techniques was used, including whole-cell patch clamp recording to study taurine's regulation of voltage-gated potassium (K+) and Ca2+ channels and Fluo-4AM Ca2+-imaging to study taurine's regulation of glutamate-induced [Ca2+] I,. Taurine was shown to suppress of glutamate-induced [Ca2+] l, in a dose dependent manner. This suppression was mostly sensitive to the glycine rece ptor antagonist Strychnine but insensitive to any GABA receptor antagonist. The remaining strychnine-insensitive effect was inhibited with the protein kinase A (PKA) inhibitor, PKI, suggesting that there was an additional metabotropic pathway. Moreover, using the protein kinase C (PKC) inhibitor, GF109203X, there was an enhancement in strychnine-insensitive taurine's regulation. Taurine inhibits voltage-gated Ca2+ channels in the retinal neurons and has a dual effect on voltage-gated K+ channels. Taurine causes an increase in K+ current amplitude which is further enhanced with PKI and blocked with GF109203X, suggesting that it is through a PKC-dependent pathway negatively controlled by PKA-dependent pathway.

Small conductance calcium-activated potassium (SK) channels are found ubiquitously throughout the brain and modulate the encoding of learning and memory. Systemic injection of 1-ethyl-2-benzimidalzolinoe (EBIO), a SK channel activator, impairs the encoding of novel object memory and locomotion but spares fear memory encoding in C57BL/6NHsd mice. The memory impairments discovered were not due to non-cognitive performance confounds such as ataxia, anxiety, attention or analgesia. Further investigation with intra-hippocampal application of EBIO revealed SK channels in dorsal CA1 contribute to the encoding deficits seen systemically, but do not account for the full extent of the impairment. Concentrated activation of dorsal CA1 SK channels do not influence fear memory encoding or locomotor impairments. Taken together, these data indicate SK channels, especially in the dorsal hippocampus, have a modulatory role on novel object memory encoding, but not retrieval; however, pharmacological activation of hippocampal SK channels does not appear to influence fear memory encoding.

Carbohydrate Active Enzyme family 6 (CA6) glycosyltransferases (GTs) are type II transmembrane proteins localized in the Golgi apparatus. CA6 GTs have a GT-A fold, a type of structure that resembles the Rossman fold and catalyze the transfer either galactose (Gal) or N-acetylgalactosamine (GalNAc) from the UDP nucleotide sugar to an non-reducing terminal Gal or GalNAc on an acceptor via an a-1,3 linkage. In this reaction, the anomeric configuration of the sugar moiety of the donor is retained in the product. CA6 GTs includes the histo-blood group A and B GTs, a-galactosyltransferase (a3GT), Forssman glycolipid synthase (FS), isogloboside 3 synthase (iGb3) in mammals. a3GT and its products (a-Gal epitode) are present in most mammals but are absent in humans and old world primates because of inactivating mutations. The absence of a3GT and its products results in the production of anti-a-Gal epitope natural antibodies in these species.