Remodeling the cell surface distribution of membrane proteins during the development of epithelial cell polarity.

The development of polarized epithelial cells from unpolarized precursor cells follows induction of cell-cell contacts and requires resorting of proteins into different membrane domains. We show that in MDCK cells the distributions of two membrane proteins, Dg-1 and E-cadherin, become restricted to the basal-lateral membrane domain within 8 h of cell-cell contact. During this time, however, 60-80% of newly synthesized Dg-1 and E-cadherin is delivered directly to the forming apical membrane and then rapidly removed, while the remainder is delivered to the basal-lateral membrane and has a longer residence time. Direct delivery of greater than 95% of these proteins from the Golgi complex to the basal-lateral membrane occurs greater than 48 h later. In contrast, we show that two apical proteins are efficiently delivered and restricted to the apical cell surface within 2 h after cell-cell contact. These results provide insight into mechanisms involved in the development of epithelial cell surface polarity, and the establishment of protein sorting pathways in polarized cells.

Mechanism for regulating cell surface distribution of Na+,K(+)-ATPase in polarized epithelial cells.

Restriction of sodium, potassium adenosine triphosphatase (Na+,K(+)-ATPase) to either the apical or basal-lateral membrane domain of polarized epithelial cells is fundamental to vectorial ion and solute transport in many tissues and organs. A restricted membrane distribution of Na+,K(+)-ATPase in Madin-Darby canine kidney (MDCK) epithelial cells was found experimentally to be generated by preferential retention of active enzyme in the basal-lateral membrane domain and selective inactivation and loss from the apical membrane domain, rather than by vectorial targeting of newly synthesized protein from the Golgi complex to the basal-lateral membrane domain. These results show how different distributions of the same subunits of Na+,K(+)-ATPase may be generated in normal polarized epithelial and in disease states.

Sodium channel expression and assembly during development of retinal ganglion cells.

Acquisition of functional Na+ channels is a critical event in the development of a neuron because it allows the generation of conducted action potentials. alpha subunit mRNA is first detected in developing rat retina at 1% of its maximum level on embryonic day 15, 4 days after the first ganglion cells are formed. alpha subunit protein is detected in the axons of the ganglion cells at this time, but beta 1 subunits, beta 2 subunits, and high affinity saxitoxin binding sites are not detected until after birth. There is an approximately coordinate increase in alpha subunit mRNA, alpha, beta 1, and beta 2 subunit protein, assembled complexes of alpha, beta 1, and beta 2 subunits, and high affinity saxitoxin binding sites between postnatal days 7 and 21. Expression of alpha subunit genes is an early event in ganglion cell differentiation, and both gene transcription and posttranslational assembly are separate, rate-limiting steps in development of Na+ channels.

Biochemical properties of sodium channels in a wide range of excitable tissues studied with site-directed antibodies.

Antibodies against a peptide (SP19) corresponding to a highly conserved, predicted intracellular region of the sodium channel alpha subunit bind rat brain sodium channels with a similar affinity as the peptide antigen, indicating that the corresponding segment of the alpha subunit is fully accessible in the intact channel structure. These antibodies recognize sodium channel alpha subunits from rat or eel brain, rat skeletal muscle, rat heart, eel electroplax, and locust nervous system. alpha subunits from all these tissues except rat skeletal muscle are substrates for phosphorylation by cAMP-dependent protein kinase. Disulfide linkage of alpha and beta 2 subunits was observed for both the RI and RII subtypes of rat brain sodium channels and for sodium channels from eel brain but not for sodium channels from rat heart, eel electroplax, or locust nerve cord. Treatment with neuraminidase reduced the apparent molecular weight of sodium channel alpha subunits from rat and eel brain and eel electroplax by 22,000-58,000, those from heart by 8000, and those from locust nerve cord by less than 4000. Our results provide the first identification of sodium channel alpha subunits from rat heart and locust brain and nerve cord and show that sodium channel alpha subunits are expressed with different subunit associations and posttranslational modifications in different excitable tissues.

Beta 2 subunits of sodium channels from vertebrate brain. Studies with subunit-specific antibodies.

The sodium channel purified from rat brain is composed of three subunits: alpha (Mr 260,000), beta 1 (Mr 36,000), and beta 2 (Mr 33,000). alpha and beta 2 subunits are linked through disulfide bonds. Procedures are described for preparative isolation of the beta 1 and beta 2 subunits under native conditions. Pure beta 2 subunits obtained by this procedure were used to prepare a specific anti-beta 2 subunit antiserum. Antibodies purified from this serum by antigen affinity chromatography recognize only disulfide-linked alpha beta 2 complexes and beta 2 subunits in immunoblots, and immunoprecipitate 32P-labeled alpha subunits of purified sodium channels having intact disulfide bonds, but not those of sodium channels from which beta 2 subunits have been detached by reduction of disulfide bonds. These antibodies also immunoprecipitate 89% of the high affinity saxitoxin-binding sites from rat brain membranes, indicating that nearly all sodium channels in rat brain have disulfide-linked alpha beta 2 subunits. Approximately 22% of beta 2 subunits in adult rat brain are not disulfide-linked to alpha subunits. Anti-beta 2 subunit antibodies are specific for sodium channels in the central nervous system and will not cross-react with sodium channels in skeletal muscle or sciatic nerve. The brains of a broad range of vertebrate species, including electric eel, are shown to express sodium channels with disulfide-linked alpha beta 2 subunits.

Localization of sodium channels in axon hillocks and initial segments of retinal ganglion cells.

Affinity-purified antibodies against the sodium channel from rat brain were employed to localize sodium channels in the retina by immunocytochemical procedures. In rat retina, intense staining was observed in the ganglion cell axon layer and light staining was detected in fibers of the inner plexiform layer. In frog retina, only the ganglion cell axon layer was stained. Examination at higher magnification revealed that axon hillocks and initial segments of ganglion cells had a high density of immunoreactive sodium channels, whereas the cell bodies were devoid of stain. The sharply defined region of high sodium channel density at the axon hillock is likely to be responsible for the low threshold for action potential initiation in this region of vertebrate central neurons.

Antigenic differences among the voltage-sensitive sodium channels in the peripheral and central nervous systems and skeletal muscle.

Antisera prepared against the voltage-sensitive sodium channel purified from rat brain are able to distinguish antigenic differences between the sodium channels in peripheral nerve tissue, the central nervous system, and in skeletal muscle. These results indicate that, in spite of many well-documented similarities among the sodium channels of all excitable tissues, there are biochemical differences that may prove important in future studies of the regulation and molecular mechanisms of electrical excitability.