The large conductance calcium-sensitive potassium (BK) channel is widely expressed in many organs and tissues, but its in vivo physiological functions have not been fully defined. Here we report a genetic locus associated with a human syndrome of coexistent generalized epilepsy and paroxysmal dyskinesia on chromosome 10q22 and show that a mutation of the alpha subunit of the BK channel causes this syndrome. The mutant BK channel had a markedly greater macroscopic current. Single-channel recordings showed an increase in open-channel probability due to a three- to fivefold increase in Ca(2+) sensitivity. We propose that enhancement of BK channels in vivo leads to increased excitability by inducing rapid repolarization of action potentials, resulting in generalized epilepsy and paroxysmal dyskinesia by allowing neurons to fire at a faster rate. These results identify a gene that is mutated in generalized epilepsy and paroxysmal dyskinesia and have implications for the pathogenesis of human epilepsy, the neurophysiology of paroxysmal movement disorders and the role of BK channels in neurological disease.

We have examined the expression and function of a previously undescribed human member (SGLT3͞SLC5A4) of the sodium͞glu-cose cotransporter gene family (SLC5) that was first identified by the chromosome 22 genome project. The cDNA was cloned and sequenced, confirming that the gene coded for a 659-residue protein with 70% amino acid identity to the human SGLT1. RT-PCR and Western blotting showed that the gene was transcribed and mRNA was translated in human skeletal muscle and small intestine. Immunofluorescence microscopy indicated that in the small intestine the protein was expressed in cholinergic neurons in the submucosal and myenteric plexuses, but not in enterocytes. In skeletal muscle SGLT3 immunoreactivity colocalized with the nicotinic acetylcholine receptor. Functional studies using the Xenopus laevis oocyte expression system showed that hSGLT3 was incapable of sugar transport, even though SGLT3 was efficiently inserted into the plasma membrane. Electrophysiological assays revealed that glucose caused a specific, phlorizin-sensitive, Na ؉ -dependent depolarization of the membrane potential. Uptake assays under voltage clamp showed that the glucose-induced inward currents were not accompanied by glucose transport. We suggest that SGLT3 is not a Na ؉ ͞glucose cotransporter but instead a glucose sensor in the plasma membrane of cholinergic neurons, skeletal muscle, and other tissues. This points to an unexpected role of glucose and SLC5 proteins in physiology, and highlights the importance of determining the tissue expression and function of new members of gene families.Na͞sugar cotransporter ͉ human SGLT3 ͉ muscle

GABA transporters play an important but poorly understood role in neuronal inhibition. They can reverse, but this is widely thought to occur only under pathological conditions. Here we use a heterologous expression system to show that the reversal potential of GAT-1 under physiologically relevant conditions is near the normal resting potential of neurons and that reversal can occur rapidly enough to release GABA during simulated action potentials. We then use paired recordings from cultured hippocampal neurons and show that GABAergic transmission is not prevented by four methods widely used to block vesicular release. This nonvesicular neurotransmission was potently blocked by GAT-1 antagonists and was enhanced by agents that increase cytosolic [GABA] or [Na(+)] (which would increase GAT-1 reversal). We conclude that GAT-1 regulates tonic inhibition by clamping ambient [GABA] at a level high enough to activate high-affinity GABA(A) receptors and that transporter-mediated GABA release can contribute to phasic inhibition.

The Na ؉ /glucose cotransporter (SGLT1) is highly selective for its natural substrates, D-glucose and D-galactose. We have investigated the structural basis of this sugar selectivity on the human isoform of SGLT1, single site mutants of hSGLT1, and the pig SGLT3 isoform, expressed in Xenopus oocytes using electrophysiological methods and the effects of cysteine-specific reagents. Kinetics of transport of glucose analogues, each modified at one position of the pyranose ring, were determined for each transporter. Correlation of kinetics with amino acid sequences indicates that residue Gln-457 sequentially interacts with O1 of the pyranose in the binding site, and with O5 in the translocation pathway. Furthermore, correlation of the selectivity characteristics of the SGLT isoforms (SGLT1 transports both glucose and galactose, but SGLT2 and SGLT3 transport only glucose) with amino acid sequence differences, suggests that residue 460 (threonine in SGLT1, and serine in SGLT2 and SGLT3) are involved in hydrogen bonding to O4 of the pyranose. In addition, the results show that substrate specificity of binding is not correlated to substrate specificity of transport, suggesting there are at least two steps in the sugar translocation process.The Na ϩ /glucose cotransporters (SGLTs) 1 transform the energy of the Na ϩ electrochemical gradient into mechanical work to drive sugar across the membrane, potentially against its concentration gradient (e.g. Refs. 1-3). Previously we have used the interaction of large aromatic glycosides to probe the vestibule to the sugar-binding site and the translocation pathway. These studies determined essential aglycone-protein interactions of human SGLT1 (hSGLT1) and pig SGLT3 (pS-GLT3) and provided a minimum size for the vestibule and sugar transport "pore" (e.g. Refs. 4 -6). In this study we focus our attention on the importance of sugar-protein hydrogen bonding in substrate specificity of transport. Determination of interactions between the sugar and the protein is an important step in elucidating the structural factors that determine specificity in the sugar-binding site and will allow future studies on characterization of the translocation pathway.Our strategy was to: 1) determine the importance of the interactions at each position of the sugar for transport through hSGLT1; 2) test the proposed interactions in cotransporters mutated in a single residue identified through sequence analysis; and 3) compare the interactions of these sugars in an SGLT isoform with known differences in sugar recognition (pSGLT3).In this work we have taken advantage of advances in gene technology and application of biophysical methods to cotransport to extend classic studies of the selectivity of sugar cotransport to the molecular level. We studied sugar specificity in four clones: hSGLT1, two hSGLT1 mutants, Q457C and Q457E, and pSGLT3. Interactions of sugars with the SGLTs were studied by analyzing the transport of sugars that differ in only one position of the ring. The importance of each position in the gl...

Excitatory amino acid transporters (EAATs) remove glutamate from synapses. They maintain an efficient synaptic transmission and prevent glutamate from reaching neurotoxic levels. Glutamate transporters couple the uptake of one glutamate to the cotransport of three sodium ions and one proton and the countertransport of one potassium ion. The molecular mechanism for this coupled uptake of glutamate and its co-and counter-transported ions is not known. In a crystal structure of the bacterial glutamate transporter homolog, Glt Ph , only two cations are bound to the transporter, and there is no indication of the location of the third sodium site. In experiments using voltage clamp fluorometry and simulations based on molecular dynamics combined with grand canonical Monte Carlo and free energy simulations performed on different isoforms of Glt Ph as well on a homology model of EAAT3, we sought to locate the third sodium-binding site in EAAT3. Both experiments and computer simulations suggest that T370 and N451 (T314 and N401 in Glt Ph ) form part of the third sodium-binding site. Interestingly, the sodium bound at T370 forms part of the binding site for the amino acid substrate, perhaps explaining both the strict coupling of sodium transport to uptake of glutamate and the ion selectivity of the affinity for the transported amino acid in EAATs.excitatory amino acid transporters | fluorescence | the sodium/aspartate symporter from Pyrococcus horikoshii (Glt Ph ) | simulations G lutamate, the main excitatory neurotransmitter in the central nervous system, is removed from the extracellular synaptic space by the glutamate excitatory amino acid transporters EAAT1-5 (1, 2). These transporters thereby maintain an efficient synaptic communication between neurons and prevent extracellular glutamate from reaching neurotoxic levels (1, 2). EAATs are trimeric proteins in which each subunit functions as an independent transporter (3, 4). Each subunit has eight transmembrane domains and two membrane inserted hair-pin (HP) loops (5-7). EAATs use the Na + and K + gradients in driving the uptake of glutamate against a concentration gradient (8). The uptake of one glutamate is coupled to the cotransport of three Na + ions and one H + ion and the countertransport of one K + ion (9). How the thermodynamically coupled transport of glutamate, H + , Na + , and K + ions is accomplished by glutamate transporters is not known. Here we present evidence for a binding site for Na + ions that suggests a mechanism for the coupling of sodium and glutamate transport.At least one extracellular Na + ion appears to bind before glutamate can bind, and at least one extracellular Na + ion appears to bind after glutamate has bound to the transporter (10-12). For example, in the absence of glutamate, a fluorophore attached to a cysteine at position A430 on HP2 in EAAT3 reported voltageand Na + -dependent fluorescence changes, consistent with Na + binding to the glutamate-free transporter and inducing a conformational change in HP2. Li + also supports glutamat...