Altered expression of voltage-dependent calcium channel alpha(1) subunits in temporal lobe epilepsy with Ammon's horn sclerosis.

Voltage-dependent calcium channels, the initial components in the calcium signalling cascade, are increasingly being recognised as relevant factors in the pathology of epilepsy. To further characterise their role in temporal lobe epilepsy associated with Ammon's horn sclerosis, we investigated the immunohistochemical distribution of five different voltage-dependent calcium channel alpha(1) subunits (alpha(1A), alpha(1B), alpha(1C), alpha(1D), alpha(1E)) in 14 hippocampal specimens of patients with Ammon's horn sclerosis in comparison with eight autopsy control cases. In epilepsy specimens an increased immunoreactivity was observed for alpha(1A), alpha(1B), alpha(1D) and alpha(1E) in the neuropil of the dentate gyrus molecular layer. Dentate gyrus granule cells and residual CA3 pyramidal neurones showed enhanced immunoreactivity for alpha(1A), while labelling of these neurones was decreased for alpha(1C). Astrocytes in Ammon's horn sclerosis specimens were strongly immunoreactive for the alpha(1C) subunit contrasting with an absent astrocytic alpha(1C) labelling in controls. Our results suggest that the expression of calcium channels in neurones and glial cells is dynamically regulated in temporal lobe epilepsy, supporting the relevance of calcium signalling pathways for this disease.

Construction of phosphatidylethanolamine-less strain of Saccharomyces cerevisiae. Effect on amino acid transport.

A triple yeast mutant was constructed which lacks BST1, the gene for sphingosine lyase, besides the phosphatidylserine decarboxylases PSD1 and PSD2. In this yeast mutant, which can only be grown in the presence of exogenous ethanolamine, phosphatidylethanolamine can be depleted to very low levels. Under those conditions, respiration as well as glucose and 3-O-methylglucose uptake proceed unaffected. Plasma membrane ATPase is as active in these cells as that of control cells grown in the presence of ethanolamine. Drastically decreased, however, are H+/amino acid symporters. The activities of arginine (Can1p), proline (Put4p) and general amino acid permease (Gap1p) are decreased more than 20-fold. Amino acid transport in yeast is dependent on coupling to the proton motive force. It can be envisaged that phosphatidylethanolamine might play a role in this process or in the early steps of the secretion pathway common for all amino acid permeases or, eventually, it could affect the transport proteins directly at the plasma membrane Transformation of the triple mutant with a CEN plasmid harbouring BST1 wild-type gene totally reversed its phenotype to that observed in the double mutant.

Properties of a reconstituted eukaryotic hexose/proton symporter solubilized by structurally related non-ionic detergents: specific requirement of phosphatidylcholine for permease stability.

Overexpression of the hexose/proton symporter HUP1 from Chlorella kessleri in S. cerevisiae permits a one-step purification via a biotinylation domain. Milligram amounts of the protein are obtained starting from 2 l of yeast culture. The HUP1 protein is used as a model eukaryotic membrane protein of the 'major facilitator superfamily' (MFS) to study specific lipid requirements for activity and stability. Testing two series of detergents revealed that n-nonyl-beta-D-glucoside (NG) and n-octyl-beta-D-glucoside (OG) solubilize the HUP1 protein efficiently. Only the use of NG resulted in long-term stabilization of the HUP1 protein in the absence of external lipids. When affinity purified protein was extracted with organic solvents, a stoichiometric amount of phosphatidyl choline, phosphatidyl ethanolamine and ergosterol in the ratio of close to 2:1 was detected. These lipids were only observed, however, when the protein purification was carried out in the presence of NG; no lipids were copurified with the HUP1 protein in the presence of OG. Of the three lipids copurified, phosphatidyl choline showed a crucial role in ensuring maximal HUP1 permease activity and stability when added back to the OG-protein. The requirement of phosphatidylcholine documents a specific effect of lipids on vectorial transport mediated by a eukaryotic membrane protein of the MFS family.

The C-terminal tetrapeptide HWFW of the Chlorella HUP1 hexose/H(+)-symporter is essential for full activity and an alpha-helical structure of the C-terminus.

C-terminal tails of plant hexose/H(+)-symporters of the major facilitator superfamily contain a highly conserved motif of four amino acids: HWFW. A deletion of these four amino acids in the Chlorella HUP1 protein leads to a decrease in transport activity by a factor of 3-4. The mutated tail is highly sensitive to trypsin; it does not show alpha-helical conformation in contrast to the wild type C-terminal peptide with an alpha-helical content of at least 15%. The production of monoclonal antibody 416B8 recognizing an epitope within the central loop of HUP1 protein has been a prerequisite for the experiments described.

Expression of eukaryotic plasma membrane transporter HUP1 from Chlorella kessleri in Escherichia coli.

To study the effect of sterols on the activity of the eukaryotic plasma membrane transporter, the hexose-proton symporter HUP1 from the unicellular alga Chlorella kessleri was expressed in Escherichia coli, a prokaryotic microorganism containing virtually no sterols. Under certain conditions, the recombinant protein was partially active in this prokaryotic organism. The heterologously produced HUP1p was purified from membrane fractions of E. coli and reconstituted in an in vitro system. The presence of ergosterol during solubilization, purification and reconstitution resulted in an increased activity of the reconstituted protein. Its activity, however, was 5-6 times lower as compared to the activity of HUP1p produced in Saccharomyces cerevisiae membranes and solubilized, purified, and reconstituted under the same conditions as above.

Alteration of substrate affinities and specificities of the Chlorella Hexose/H+ symporters by mutations and construction of chimeras.

The cDNAs HUP1 and HUP2 of Chlorella kessleri code for monosaccharide/H+ symporters that can be functionally expressed in Schizosaccharomyces pombe. By random mutagenesis three HUP1 mutants with an increased Km value for D-glucose were isolated. The 40-fold increase in Km of the first mutant is due to the amino acid exchange N436I in putative transmembrane helix XI. Two substitutions were found in a second (G97C/I303N) and third mutant (G120D/F292L), which show a 270-fold and 50-fold increase in Km for D-glucose, respectively. An investigation of the individual mutations revealed that the substitutions I303N and F292L (both in helix VII) cause the Km shifts seen in the corresponding double mutants. These mutations together with those previously found support the hypothesis that helices V, VII, and XI participate in the transmembrane sugar pathway. Whereas for most mutants obtained so far the Km change for D-glucose is paralleled by a corresponding change for other hexoses tested, the exchange D44E exclusively alters the Km for D-glucose. Moreover the pH profile of this mutant is shifted by more than 2 pH units to alkaline values, indicating that the activity of the transporter may require deprotonation of the corresponding carboxyl group. Chimeric transporters were constructed to study the 100-fold lower affinity for D-galactose of the HUP1 symporter as compared with that of the HUP2 protein. A crucial determinant for the differential D-galactose recognition was shown to be associated with the first external loop. The effect could be pinpointed to a single amino acid change: replacement of Asn-45 of HUP1 with isoleucine, the corresponding amino acid of HUP2, yields a transporter with a 20 times higher affinity for D-galactose. The reverse substitution (I47N) decreases the affinity of HUP2 for D-galactose 20-fold.