Paul J. Bauer

Subunit Stoichiometry of the CNG Channel of Rod Photoreceptors

Cyclic nucleotide-gated (CNG) channels play a central role in the conversion of sensory stimuli into electrical signals. CNG channels form heterooligomeric complexes built of A and B subunits. Here, we study the subunit stoichiometry of the native rod CNG channel by chemical crosslinking. The apparent molecular weight (M(w)) of each crosslink product was determined by SDS-PAGE, and its composition was analyzed by Western blotting using antibodies specific for the A1 or B1 subunit. The number of crosslink products and their M(w) as well as the immunological identification of A1 and B1 subunits in the crosslink products led us to conclude that the native rod CNG channel is a tetramer composed of three A1 and one B1 subunit. This is an example of violation of symmetry in tetrameric channels.

Mutual inhibition of the dimerized Na/Ca-K exchanger in rod photoreceptors.

In the dark, rod photoreceptors sustain a continuous influx of Na and Ca ions through the cGMP-gated channels of the rod outer segments (ROS). Whereas Na ions are extruded in the inner segment by the Na-pump, Ca ions are extruded already in the ROS by Na/Ca-K exchange. Our previous findings indicate that in the ROS plasma membrane, exchanger and channel form a complex of two exchangers associated per channel. Here, we report evidence of a novel regulatory mechanism of the dimerized exchanger, based on the following findings: (1), thiol-specific cross-linking with dimaleimides resulted in an increase of the Na/Ca-K exchange activity which correlated with the size of the cross-linking reagent, i.e., with increasing separation of the monomers in a dimerized exchanger; (2), partial proteolysis of the exchanger also increased the exchange rate by about a factor of two; (3), disintegration of the channel-exchanger complex by solubilization of the ROS membranes and preparation of proteoliposomes resulted in a twofold enhancement of the exchange rate; however (4), partial proteolysis of proteoliposomes, in which the exchanger molecules exist as monomers, did not result in any enhancement of the exchange rate. These findings suggest an inhibitory protein domain at the contact site of the dimerized exchanger. The physiological implication of this inference will be discussed in terms of a potential allosteric regulation of the exchanger in the channel-exchanger complex.

Direct evidence of Na+/Ca2+ exchange in squid rhabdomeric membranes

Na+/Ca2+exchange has been investigated in squid ( Loligo pealei) rhabdomeric membranes. Ca2+-containing vesicles have been prepared from purified rhabdomeric membranes by extrusion through polycarbonate filters of 1-μm pore size. After removal of external Ca2+, up to 90% of the entrapped Ca2+ could be specifically released by the addition of Na+; this finding indicates that most of the vesicles contained Na+/Ca2+exchanger. The Na+-induced Ca2+ efflux had a half-maximum value ( K 1/2) of ∼44 mM and a Hill coefficient of ∼1.7. The maximal Na+-induced Ca2+ efflux was ∼0.6 nmol Ca2+ ⋅ s-1 ⋅ mg protein-1. Similar Na+-induced Ca2+ effluxes were measured if K+ was replaced with Li+ or Cs+. Vesicles loaded with Ca2+ by Na+/Ca2+exchange also released this Ca2+by Na+/Ca2+exchange, suggesting that Na+/Ca2+exchange operated in both forward and reverse modes. Limited proteolysis by trypsin resulted in a rate of Ca2+ efflux enhanced by approximately fivefold when efflux was activated with 95 mM NaCl. For vesicles subjected to limited proteolysis by trypsin, Na+/Ca2+exchange was characterized by a K 1/2 of ∼25 mM and a Hill coefficient of 1.6. For these vesicles, the maximal Na+-induced Ca2+ efflux was about twice as great as in control vesicles. We conclude that Na+/Ca2+exchange proteins localized in rhabdomeric membranes mediate Ca2+ extrusion in squid photoreceptors.

Identification of stereoisomers based on dielectric studies: dipole moments of chloroalkenes and chlorocumulenes

The use of dielec. measurements to assign mol. configurations was examd. with model compds. of the chloroalkane, chloroalkene and chlorocumulene type. A dielec. microcell is described which allowed measurements of 0.35 mL samples under exclusion of air. Both benzene and cyclohexane were used as solvents. Different theories of dielec. const. are compared to evaluate dipole moments. Dipole moments are analyzed in terms of bond moments. The results can be summarized as follows: (i) dielec. measurements are feasible up to 1 M solns. without substantial deviation from linearity; (ii) in general, the dipole moments found in both solvents are similar; the arom. solvent, however, causes a substantial decrease of the values obtained for chloroalkenes with two bulky substituents at the double bonds; (iii) the (E) isomers of chloroalkenes exhibit significantly higher dipole moments than the corresponding (Z) isomers; (iv) isomers of nonchlorinated alkenes of cumulenes with dipole moments below 0.6 D cannot be discriminated dielec.; (v) on the other hand, if the dipole moments of the compds. exceed 1 D, configurations can be assigned unambiguously on the basis of dielec. data even in cases where other approaches fail. [on SciFinder (R)]

Na+−Ca2+,K+ exchange in bovine retinal rod outer segments: quantitative characterization of normal and reversed mode

Ca2+ homeostasis of bovine retinal rod outer segments is maintained through Na(+)-Ca2+,K+ exchangers and cGMP-gated channels in the plasma membrane. It has recently been demonstrated that both proteins are associated. This novel finding allowed us to investigate quantitatively normal and reversed mode Na(+)-Ca2+,K+ exchange in rod outer segment membrane vesicles and reconstituted proteoliposomes both containing exchangers in rightside-out and inside-out orientations. Addition of Na+ activated both normal and reversed mode exchange; if, however, initially Ca2+ from vesicles containing inside-out oriented exchangers has been released by activation of the associated channels, only normal mode exchange was observed upon addition of Na+. Using this approach, the fractions of vesicles containing rightside-out and inside-out oriented exchangers were about similar in these vesicle preparations. Normal and reversed mode exchange had similar Na+ concentrations of about 70 mM for half maximal activation (in the presence of 115 mM K+) and cooperativity parameters, nHill, of about 2.0. Furthermore, both modes were electrogenic, and showed only little Na(+)-Ca2+,K+ exchange in the absence of K+. The two modes of exchange differed, however, in the maximal exchange rate, the normal mode being about twice as fast as the reversed mode.