Aging of the human crystalline lens and anterior segment.

Changes in the unaccommodated human crystalline lens were characterized as a function of subject age for 100 normal emmetropes over the age range 18-70 yr by Scheimpflug slit-lamp photography. With increasing age, the lens becomes thicker sagittally, but since the distance from the cornea to the posterior lens surface remains unchanged, this indicates that the center of lens mass moves anteriorly and the anterior chamber becomes shallower. Sagittal nuclear thickness is independent of age, but both anterior and posterior cortical thicknesses increase with age, shifting the location of the nucleus and the central sulcus in the anterior direction. The amount of light scattered by the lens at high angles, as represented by normalized and integrated lens densities from the digitized images, increases with increasing age in an exponential fashion. Similar relationships to age are observed for the major anterior zone of discontinuity (maximum density) and the central sulcus (minimum density). The relationships of these results to accommodation and presbyopia are discussed.

Structure of a putative sodium channel from the sea anemone Aiptasia pallida.

A cDNA encoding a full length putative sodium channel has been cloned from the sea anemone Aiptasia pallida. The deduced protein, named AiNa1, has a predicted molecular weight of 205,000 Da. It shows high structural similarity to other sodium channels from both invertebrates and vertebrates, and its structure is consistent with the four domain, six transmembrane segment motif of all known voltage-gated sodium channels. In the region purported to constitute the tetrodotoxin (TTX) receptor of sodium channels, AiNa1 differs from the TTX-sensitive motif, suggesting that currents carried by this channel would be insensitive to TTX. The presence of a conventional sodium channel protein in anemones indicates, for the first time, that neurons in sea anemones are likely to be capable of producing fast, overshooting action potentials.

Gating of cx46 gap junction hemichannels by calcium and voltage.

Connexin 46 (cx46), when expressed in Xenopus oocytes, not only forms typical gap junction channels between paired cells but also forms open gap junction hemichannels in the plasma membrane of single cells. The gap junction hemichannels share properties with complete gap junction channels in terms of permeability and gating. Here we characterize the gate that closes hemichannels in response to increased calcium concentration with whole-cell and single-channel records. The channels close within a narrow range of extracellular calcium concentrations (1-2 mM) which includes the calcium concentration prevailing in the primary site of cx46 expression, the lens. The effect of calcium on the channels is determined by voltage. A cysteine mutant of cx46, cx46L35C, was used to determine the localization of the gate. Experimental evidence suggests that position 35 is pore lining. The localization protocol tests the accessibility of position 35 for thiol reagents applied extra- or intracellularly to the channel closed by calcium. Channel closure by calcium excluded the thiol reagent from the outside but not from the inside. Consequently, the gate results in a regional closure of the pore and it is located extracellular to the position 35 of cx46. The present data also suggest that the cx46 gap junction hemichannel may exert a physiological function in the lens. Considering the association of calcium with cataract formation, it is feasible that misregulation of cx46 gap junction hemichannels could be a cause for cataract.

Localization of a voltage gate in connexin46 gap junction hemichannels.

Cysteine replacement mutagenesis has identified positions in the first transmembrane domain of connexins as contributors to the pore lining of gap junction hemichannels (Zhou et al. 1997. Biophys. J. 72:1946-1953). Oocytes expressing a mutant cx46 with a cysteine in position 35 exhibited a membrane conductance sensitive to the thiol reagent maleimidobutyryl biocytin (MBB). MBB irreversibly reduced the single-channel conductance by 80%. This reactive cysteine was used to probe the localization of a voltage gate that closes cx46 gap junction hemichannels at negative potentials. MBB was applied to the closed channel either from outside (whole cell) or from inside (excised membrane patches). After washout of the thiol reagent the channels were tested at potentials at which the channels open. After extracellular application of MBB to intact oocytes, the membrane conductance was unaffected. In contrast, channels treated with intracellular MBB were blocked. Thus the cysteine in position 35 of cx46 is accessible from inside but not from the outside while the channel is closed. These results suggest that the voltage gate, which may be identical to the "loop gate" (Trexler et al. 1996. Proc. Natl. Acad. Sci. USA. 93:5836-5841), is located extracellular to the 35 position. The voltage gate results in regional closure of the pore rather than closure along the entire pore length.

A chimeric connexin forming gap junction hemichannels.

Connexins are the subunits of gap junction channels which connect neighboring cells. With the exception of lens connexins, they usually do not form open hemichannels in the cell membrane of single cells. Here we describe a chimeric connexin consisting of cx32 where the first extracellular loop sequence is replaced by the corresponding cx43 sequence. This chimera, cx32E143, forms conventional gap junction channels in the paired oocyte assay. In addition cx32E143 induces a membrane conductance in single oocytes. This membrane conductance is voltage dependent and is similarly sensitive to CO2 as are gap junction channels formed by the chimera or by wild-type cx32. These data suggest that cx32E143 forms patent hemichannels in the plasma membrane of single oocytes. This conclusion is further supported by the observation that oocytes expressing cx32E143 take up from the bath medium tracer molecules known to pass through gap junction channels.

Identification of a pore lining segment in gap junction hemichannels.

The ability of certain connexins to form open hemichannels has been exploited to study the pore structure of gap junction (hemi)channels. Cysteine scanning mutagenesis was applied to cx46 and to a chimeric connexin, cx32E(1)43, which both form patent hemichannels when expressed in Xenopus oocytes. The thiol reagent maleimido-butyryl-biocytin was used to probe 12 cysteine replacement mutants in the first transmembrane segment and two in the amino-terminal segment. Maleimido-butyryl-biocytin was found to inhibit channel activity with cysteines in two equivalent positions in both connexins: I33C and M34C in cx32E(1)43 and I34C and L35C in cx46. These two positions in the first transmembrane segment are thus accessible from the extracellular space and consequently appear to contribute to the pore lining. The data also suggest that the pore structure is complex and may involve more than one transmembrane segment.