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Neurodegenerative disorders involve death of cell bodies, axons, dendrites and synapses, but it is surprisingly difficult to determine the spatiotemporal sequence of events and the causal relationships among these events. Neuronal compartments often crucially depend upon one another for survival, and molecular defects in one compartment can trigger cellular degeneration in distant parts of the neuron. Here, we consider the novel approaches used to understand these biologically complex and technically challenging questions in amyotrophic lateral sclerosis, spinal muscular atrophy, glaucoma, Alzheimer's disease, Parkinson's disease and polyglutamine disorders. We conclude that there is partial understanding of what degenerates first and why, but that controversy remains the rule not the exception. Finally, we highlight strategies for resolving these fundamental issues.

The [Na.sup.+]:HC[O.sup.-.sub.3] cotransporter NBC1 (SLC4A4, variant A, kidney specific) is located exclusively on the basolateral membrane of epithelial cells, implying that this molecule has acquired specific signals for targeting to the basolateral membrane. A motif with the sequence QQPFLS (positions 1010-1015) in the cytoplasmic tail of NBC1 was recently demonstrated to mediate targeting of NBC1 to the basolateral membrane. Here, we demonstrate that mutating the amino acid F (phenylalanine) or L (leucine) at positions 1013 or 1014 to alanine, respectively, resulted in the retargeting of NBC1 to the apical membrane. Furthermore, mutation of the FL motif to FF showed similar properties as the wild-type; however, mutation of the FL motif to LL showed significant intracellular retention of NBC1. Mutating the amino acids Q-Q-P and S (positions 1010-1011-1012 and 1015) to A-A-A and A, respectively, did not affect the membrane targeting of NBC1. Functional studies in oocytes with microelectrode demonstrated that the apically targeted mutants, as well as basolaterally targeted mutants, are all functional. We propose that the FL motif in the COOH-terminal tail of NBC1 is essential for the targeting of NBC1 to the basolateral membrane but is distinct from the membrane-targeting di-leucine motif identified in other membrane proteins. basolateral membrane; apical membrane; acid-base regulation; kidney; transmembrane domain

T cell receptor engagement results in the reorganization of intracellular and membrane proteins at the T cell-antigen presenting cell interface forming the immunological synapse (IS), an event required for [Ca.sup.2+] influx. KCa3.1 channels modulate [Ca.sup.2+] signaling in activated T cells by regulating the membrane potential. Nothing is known regarding KCa3.1 membrane distribution during T cell activation. Herein, we determined whether KCa3.1 translocates to the IS in human T cells using YFP-tagged KCa3.1 channels. These channels showed electrophysiological and pharmacological properties identical to wild-type channels. IS formation was induced by either anti-CD3/CD28 antibody-coated beads for fixed microscopy experiments or Epstein-Barr virus-infected B cells for fixed and live cell microscopy. In fixed microscopy experiments, T cells were also immunolabeled for F-actin or CD3[epsilon], which served as IS formation markers. The distribution of KCa3.1 was determined with confocal and fluorescence microscopy. We found that, upon T cell activation, KCa3.1 channels localize with F-actin and CD3[epsilon] to the IS but remain evenly distributed on the cell membrane when no stimulus is provided. Detailed imaging experiments indicated that KCa3.1 channels are recruited in the IS shortly after antigen presentation and are maintained there for at least 15-30 min. Interestingly, pretreatment of activated T cells with the specific KCa3.1 blocker TRAM-34 blocked [Ca.sup.2+] influx, but channel redistribution to the IS was not prevented. These results indicate that KCa3.1 channels are a part of the signaling complex that forms at the IS upon antigen presentation. T cell activation; ion channels; membrane distribution

The kidney [Na.sup.+]:HC[O.sub.3.sup.-] cotransporter NBC1 is located exclusively on the basolateral membrane of kidney proximal tubule cells and is responsible for the reabsorption of majority of filtered bicarbonate. Two well-described missense mutations in NBC1, R510H and $427L, are associated with renal tubular acidosis (RTA). However, the exact relationship between these mutations and NBC1 dysregulation remains largely unknown. To address this question, cDNAs for wildtype kidney NBCI and its mutants R510H and $427L were generated, fused in frame with N[H.sub.2] terminally tagged GFP, and transiently expressed in Madin-Darby canine kidney cells. In parallel studies, oocytes were injected with the wild-type and mutant NBC1 cRNAs and studied for membrane expression and activity. In monolayer cells grown to polarity, the wild-type GFP-NBC1 was exclusively localized on the basolateral membrane domain. However, GFP-NBC1 mutant R510H was detected predominantly in the cytoplasm. GFP-NBC1 mutant $427L, on the other hand, was detected predominantly on the apical membrane with residual cytoplasmic retention and basolateral membrane labeling. In oocytes injected with the wild-type or mutant GFP-NBC1 cRNAs, Western blot analysis showed that wild-type NBC1 is predominantly localized in the membrane fraction, whereas NBC1-R510H mutant was predominantly expressed in the cytoplasm. NBC1-$427L mutant was mostly expressed in the membrane fraction. Functional analysis of NBC1 activity in oocytes by membrane potential recording demonstrated that compared with wild-type GFP-NBCI, the GFP-NBC1 mutants H510R and $427L exhibited significant reduction in activity. These findings suggest that the permanent isolated proximal RTA in patients with H510R or $427L mutation resulted from a combination of inactivation and mistargeting of kidney NBC1, with H510R mutant predominantly retained in the cytoplasm, whereas $427L mutant is mistargeted to the apical membrane. acid-base balance; sodium hydrogen exchanger type 3; bicarbonate absorption

The outer medullary collecting duct (OMCD) plays an important role in bicarbonate reabsorption and acid-base regulation. An apical V-type [H.sup.+]-ATPase and a basolateral [Cl.sup.-]/HC[O.sup.-sub.3] exchanger, located in intercalated cells of OMCD, mediate the bicarbonate reabsorption. Here we report the identification of a new basolateral [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger in OMCD intercalated cells in rat kidney. Northern hybridizations demonstrated the predominant expression of this transporter, also known as SLC26A7, in the outer medulla, with lower expression levels in the inner medulla. SLC26A7 was recognized as a ~90-kDa band in the outer medulla by immunoblot analysis and was localized on the basolateral membrane of a subset of OMCD cells by immunocytochemical staining. No labeling was detected in the cortex. Double-immunofluorescenee labeling with the aquaporin-2 and SLC26A7 antibodies or anion exchanger-1 and SLC26A7 antibodies identified the SLC26A7-expressing cells as a-intercalated cells. Functional studies in oocytes demonstrated that increasing the osmolality of the media (to simulate the physiological milieu in the medulla) increased the [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger activity mediated via SLC26A7 by about threefold (P < 0.02 vs. normal condition). We propose that SLC26A7 is a basolateral [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger in intercalated cells of the OMCD and may play an important role in bicarbonate reabsorption in medullary collecting duct. anion exchanger; basolateral membrane; aquaporin-2

The purpose of the present studies was to examine the renal distribution and functional properties of [Na.sup.+]-HC[O.sup.-.sub.3] cotransporter type 4 (NBC4), the latest NBC isoform to be identified. Zonal distribution studies in rat kidney by Northern blot hybridization and RT-PCR demonstrated that NBC4 is highly abundant in the outer medulla and cortex but is low in the inner medulla. Nephron segment distribution studies indicated that NBC4 is predominantly expressed in the medullary and cortical thick ascending limb of the loop of Henle. Using specific primers on the basis of the published sequence (GenBank accession no. AF-207661), a full-length NBC4 variant was cloned from human liver and examined. The sequence of this variant (called NBC4e) is shorter by 86 amino acids vs. the published sequence. Xenopus laevis oocytes injected with the full-length NBC4e cRNA were compared with NBC 1-expressing oocytes. Although exposure of NBC1-expressing oocytes to C[O.sub.2]/ HC[O.sup.-.sub.3] resulted in immediate hyperpolarization, the NBC4-expressing oocytes did not show any alteration in membrane potential. NBC activity in oocytes, assayed as the [Na.sup.+]-dependent, HC[O.sup.-.sub.3]-mediated intracellular pH recovery from acidosis, indicated that NBC4 is a DIDS-inhibitable NBC. We propose that NBC4 is expressed in the thick ascending limb of the loop of Henle and mediates cellular HC[O.sup.-.sub.3] uptake in this segment. sodium-bicarbonate cotransporter; NBC4; NBC1; acid-base transporters; thick limb of the loop of Henle

Hypoxic vasorelaxation inhibition by organ culture correlates with loss of Kv channels but not [Ca.sup.2+] channels. Am J Physiol Heart Circ Physiol 283: H247-H253, 2002; 10.1152/ajpheart.00569.2001.--We (Thorne GD, Shimizu S, and Paul RJ. Am J Physiol Cell Physiol 281: C24-C32, 2001) have recently shown that organ culture for 24 h specifically inhibits relaxation to acute hypoxia (95% [N.sub.2]-5% C[O.sub.2]) in the porcine coronary artery. Here we show similar results in the porcine carotid artery and the rat and mouse aorta. In the coronary artery, part of the inability to relax to hypoxia after organ culture is associated with a concomitant loss in ability to reduce intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) during hypoxia (Thorne GD, Shimizu S, and Paul RJ. Am J Physiol Cell Physiol 281: C24-C32, 2001). To elucidate the mechanisms responsible for the loss of relaxation to hypoxia, we investigated changes in [K.sup.+] and [Ca.sup.2+] channel activity and gene expression that play key roles in [[[Ca.sup.2+]].sub.i] regulation in vascular smooth muscle (VSM). Reduced mRNA expression of [O.sub.2]-sensitive [K.sup.+] channels (Kv1.5 and Kv2.1) was shown by reverse transcriptase-polymerase chain reaction in the rat aorta. In contrast, no change in other expressed voltage-gated [K.sup.+] channels (Kv1.2 and Kv1.3) or [Ca.sup.2+] channel subtypes was found. Modified [K.sup.+] channel expression is supported by functional evidence indicating a reduced response to general [K.sup.+] channel activation, by pinacidil, and to specific voltage-dependent [K.sup.+] (Ky) channel blockade by 4-aminopyridine. In conclusion, organ culture decreases expression of specific Kv channels. These changes are consistent with altered mechanisms of VSM contractility that may be involved in [Ca.sup.2+]-dependent pathways of hypoxia-induced vasodilation. hypoxia; coronary; potassium channel; smooth muscle