Arturo Picones

Permeability and interaction of Ca2+ with cGMP-gated ion channels differ in retinal rod and cone photoreceptors.

We studied the ionic permeability of cGMP-dependent currents in membrane patches detached from the outer segment of retinal cone and rod photoreceptors. Reversal potentials measured in membranes exposed to symmetric Na+ but with varying cytoplasmic Ca2+ concentrations reveal that the permeability ratio, PCa/PNa, is higher in the cGMP-gated channels of cones (7.6 +/- 0.8) than in those of rods (3.1 +/- 1.0). Ca2+ blocks both channels in a voltage-dependent manner. At any Ca2+ concentration, the channel block is maximal near the ionic reversal potential. The maximal block is essentially identical in channels of cones and rods with respect to its extent and voltage and Ca2+ dependence. The Ca2+ block is relieved by voltage, but the features of this relief differ markedly between rods and cones. Whereas the Boltzmann distribution function describes the relief of block by hyperpolarizing voltages, any given voltage is more effective in relieving the Ca2+ block in cones than in rods. Similarly, depolarizing voltages more effectively relieve Ca2+ block in cones than in rods. Our results suggest that channels contain two binding sites for Ca2+, one of which is similar in the two receptor types. The second site either interacts more strongly with Ca2+ than the first one or it is located differently in the membrane, so as to be less sensitive to membrane voltage. The channels in rods and cones differ in the features of this second site. The difference in Ca2+ permeability between the channels is likely to result in light-dependent changes in cytoplasmic Ca2+ concentration that are larger and faster in cones than in rods. The functional differences between channels, therefore, may be critically important in explaining the differences in the phototransduction signal of the two photoreceptor types.

Spontaneous, ligand‐independent activity of the cGMP‐gated ion channels in cone photoreceptors of fish.

1. We studied the electrical conductance of membrane patches detached from the outer segment of single cone photoreceptors isolated from striped bass retina. 2. Only a single class of ion channels exists in the plasma membrane of the cone outer segments; they are gated by cytoplasmic cGMP and select cations over anions, but distinguish poorly among cations. In the absence of added cGMP and of divalent cations, however, membrane patches detached from the outer segments exhibit a small conductance that ideally selects cations over anions, but distinguishes poorly between Na+ and Li+. 3. The cGMP‐independent conductance does not arise from the effect of residual cGMP that may remain associated with the detached membrane, because treatment of the patch with cGMP‐specific phosphodiesterase does not affect this conductance. 4. The cGMP‐independent conductance is pharmacologically indistinguishable from that activated by cGMP. Ca2+ and L‐cis‐diltiazem block both conductances at comparable concentrations and with similar quantitative characteristics. 5. We analysed the noise of Ca(2+)‐ or L‐cis‐diltiazem‐dependent macroscopic currents both in the presence and in the absence of cGMP. In the presence of cGMP, the power density spectrum of the noise is well fitted by the sum of two Lorentzian components. The same function with similar corner frequencies fits the noise of the cGMP‐independent currents. However, the total power in the current fluctuations is smaller in the absence of cGMP than in its presence; also, the ratio of the zero frequency asymptotes of the low over the high frequency components, S1(0)/Sh(0), is larger in the absence of cGMP than in its presence.(ABSTRACT TRUNCATED AT 250 WORDS)

Unitary conductance variation in Kir2.1 and in cardiac inward rectifier potassium channels.

Kir2.1 (IRK1) is the complementary DNA for a component of a cardiac inwardly rectifying potassium channel. When Kir2.1 is expressed in Xenopus oocytes or human embryonic kidney (HEK) cells (150 mM external KCl), the unitary conductances form a broad distribution, ranging from 2 to 33 pS. Channels with a similarly broad distribution of unitary conductance amplitudes are also observed in recordings from adult mouse cardiac myocytes under similar experimental conditions. In all three cell types channels with conductances smaller, and occasionally larger, than the ~30 pS ones are found in the same patches as the ~30 pS openings, or in patches by themselves. The unitary conductances in patches with a single active channel are stable for the durations of the recordings. Channels of all amplitudes share several biophysical characteristics, including inward rectification, voltage sensitivity of open probability, sensitivity of open probability to external divalent cations, shape of the open channel i-V relation, and Cs(+) block. The only biophysical difference found between large and small conductance channels is that the rate constant for Cs(+) block is reduced for the small-amplitude channels. The unblocking rate constant is similar for channels of different unitary conductances. Apparently there is significant channel-to-channel variation at a site in the outer pore or in the selectivity filter, leading to variability in the rate at which K(+) or Cs(+) enters the channel.

Analysis of fluctuations in the cGMP-dependent currents of cone photoreceptor outer segments.

We measured cGMP-dependent currents, under voltage clamp, in membrane patches detached from the outer segment of single-cone photoreceptors isolated from the retina of striped bass. We analyzed the variance of the current about its mean and the spectral density distribution of the current fluctuations. From the analysis of variance, we determined that the cGMP-gated channels increase their probability of opening with increasing cGMP up to a maximum value of 0.87 +/- 0.03. The dependence on cGMP of the probability of opening is well described by a Hill equation with Km = 60.2 +/- 3.7 microM and n = 2.33 +/- 0.32 at -50 mV. At the same voltage, the spectral density distribution is well fit by the sum of two Lorentzians with corner frequencies at 26 +/- 18 and 318 +/- 58 Hz. The single-channel conductance calculated from the current noise by two different methods suggests that the most frequently occupied conductance state has an amplitude of about 18 pS.

A developmental time line in a retinal slice from rainbow trout.

The retina in teleost fish continues to grow throughout much of the life of the animal, in part by the continuing differentiation of new tissue at the retinal margin, an area termed the peripheral growth zone (PGZ) (Lyall, Q J Micros Sci, 1957:98:101-110). We have developed a retinal slice preparation--including the PGZ--from juvenile rainbow trout (Onchorynchus mykiss), a species in which retinal growth is rapid and the PGZ is correspondingly pronounced. The PGZ slice preparation contains a time line of retinal development, with cells at different stages of maturation present side by side. We present evidence that the birth sequence of the various retinal cell types in the PGZ recapitulates the sequence during embryonic development. We also report data on the rate of growth of the PGZ in juvenile trout in vivo. Finally, we have used the PGZ slice preparation to make whole-cell voltage clamp recordings from individual retinal GCs at both early and late stages of maturation. We report that the amplitude of delayed rectifier and A-type potassium currents increases during GC maturation.

Voltage-dependence of Ion Permeation in Cyclic GMP–gated Ion Channels Is Optimized for Cell Function in Rod and Cone Photoreceptors

The kinetics of the photocurrent in both rod and cone retinal photoreceptors are independent of membrane voltage over the physiological range (−30 to −65 mV). This is surprising since the photocurrent time course is regulated by the influx of Ca2+ through cGMP-gated ion channels (CNG) and the force driving this flux changes with membrane voltage. To understand this paradigm, we measured Pf, the fraction of the cyclic nucleotide–gated current specifically carried by Ca2+ in intact, isolated photoreceptors. To measure Pf we activated CNG channels by suddenly increasing free 8-Br-cGMP in the cytoplasm of rods or cones loaded with a caged ester of the cyclic nucleotide. Simultaneous with the uncaging flash, we measured the cyclic nucleotide–dependent changes in membrane current and fluorescence of the Ca2+ binding dye, Fura-2, also loaded into the cells. We determined Pf under physiological solutions at various holding membrane voltages between −65 and −25 mV. Pf is larger in cones than in rods, but in both photoreceptor types its value is independent of membrane voltage over the range tested. This biophysical feature of the CNG channels offers a functional advantage since it insures that the kinetics of the phototransduction current are controlled by light, and not by membrane voltage. To explain our observation, we developed a rate theory model of ion permeation through CNG channels that assumes the existence of two ion binding sites within the permeation pore. To assign values to the kinetic rates in the model, we measured experimental I-V curves in membrane patches of rods and cones over the voltage range −90 to 90 mV in the presence of simple biionic solutions at different concentrations. We optimized the fit between simulated and experimental data. Model simulations describe well experimental photocurrents measured under physiological solutions in intact cones and are consistent with the voltage-independence of Pf, a feature that is optimized for the function of the channel in photoreceptors.

Developmental maturation of passive electrical properties in retinal ganglion cells of rainbow trout

We investigated the electrotonic and anatomical features of the dendritic arbor in developing retinal ganglion cells (RGCs). Cell anatomy was studied by filling individual cells with fluorescent, membrane‐bound dyes and using computer‐assisted image reconstruction. Electrotonic properties were characterized through an analysis of charging membrane currents measured with tight‐seal electrodes in the whole‐cell mode. We studied developing RGCs in the peripheral growth zone (PGZ) of a fish retina. The PGZ presents a developmental time‐line ranging from pluripotent, proliferating cells at the extreme edge, to mature, fully developed retina more centrally. In the PGZ, RGCs mature through three histologically distinct zones (in developmental sequence): bulge, transition and mature zones. In the most peripheral three‐quarters of the bulge zone, cells have rounded somas, lack dendritic extensions and some are coupled so that membrane‐bound dyes traverse from one cell to its immediate neighbours. In the more central quarter of the bulge, cells’ dendrites are few, short and of limited branching. In the transition zone dendritic arbors becomes progressively more expansive and branched and we present a morphometric analysis of these changes. Regardless of the size and branching pattern of the developing RGC dendritic arbor, the ratio of the diameters of parent and progeny dendrites at any branching nodes is well described by Ralls 3/2 power law. Given this anatomical feature, the RGC passive electrical properties are well described by an equivalent electrical circuit consisting of an isopotential cell body in parallel with a single equivalent cylinder of finite length. We measured the values of the electrical parameters that define this equivalent circuit in bulge, transition and mature RGCs. As RGCs develop the electrical properties of their dendritic arbor change in an orderly and tightly regulated manner, not randomly. Electrically, dendritic arbors develop along either of two distinct modes, but only these modes: isoelectrotonic and isometric. In isoelectrotonic growth, electrotonic properties are constant regardless of the absolute dimensions of the dendritic arbor or its branching geometry. These cells maintain unvarying relative synaptic efficacy independently of the size or pattern of their dendritic arbor. In isometric growth, in contrast, electronic properties change, but the ratio of the changing electrotonic length to electrotonic diameter is constant. In these cells relative synaptic efficacy decreases linearly as dendrites extend.