Abstract
over another in performing a specific kind of computation (22) .
Abstract
In conclusion, the enhanced performance of IC-related tasks below the horizontal meridian indicates that physiological studies and theoretical models of scene segmentation should take into account early cortical processing inhomogeneities and functional specialization. In addition, testing other visual tasks (for example, perceptual grouping) for up/down asymmetry may help to establish relations between the different components of visual processing.

. because an (~Ilusory) edge In any orlentailon other than the horizontal would result n a dispar~iy in the left-and r~ght-eye Images for which the only ecologically valid nterpretatlon IS one of there beng an illusory occuder. 8. D. R~ngach and R. Shapley, Perception isuppi.) 22, 51 (1993) . 9. Subjects CT and CH were naive about the purpose of the experiment. A subjects rece~ved extenslve practce in petformng the shape-d~scrmnaton task w~th the st~mulus centered around the f~xation point before the collecton of the data presented. 10. The stmuus subtended a v~sual angle of 2.75" and was centered 3.4" above or below the fxation pont. The stmulus was presented for 83 ms, followed by a blank screen for 83 ms and then by a mask for 250 ms. The stmulus and the mask were whte (12 cd/ m2) on a dark background The subject was restraned with a chn and forehead rest, and the experment room was dark.

11 A sigmod curve ({1 + tanh[b(x -a)]]/2 )was f~t to the data of each psychometrc function, wth the slope (b) and b~as (a) as free parameters, the thresholds were computed from the fitted curves. 12. The differences In thresholds between the upper and lower vsual hemifelds were statist~cally sgn~fcant in the IC condition (Fig. 3A) for all subjects. The d~ffer-ences n the two control conditions were not significant for any of the subjects. 
Use-Dependent Blockers and Exit Rate of the Last Ion from the Multi-Ion Pore of a K+ Channel
Thomas Baukrowitz and Gary Yellen* Quaternary ammonium blockers inhibit many voltage-activated potassium (K-) channels from the intracellular side. When applied to Drosophila Shaker potassium channels expressed in mammalian cells, these rapidly reversible blockers produced use-dependent inhibition through an unusual mechanism-they promoted an intrinsic conformational change known as C-type inactivation, from which recovery is slow. The blockers did so by cutting off potassium ion flow to a site in the pore, which then emptied at a rate of 1 O5 ions per second. This slow rate probably reflected the departure of the last ion from the multi-ion pore: Permeation of ions (at 1 O7 per second) occurs rapidly because of ion-ion repulsion, but the last ion to leave would experience no such repulsion.
Use-Dependent Blockers and Exit Rate of the Last Ion from the Multi-Ion Pore of a K+ Channel
U s e dependence is a valuable property for therapeutic inhibitors of ion channels. The abilitv to block channels during oeriods of -partidularly high activity while leaving resting channels relativelv unaffected makes use-dependent channel inhibitors valuable as anticonvulsant or antiarrhvthmic agents u (1 ). The accepted mechanism of use-dependent inhibition is that an inhibitor binds better when the channel is used and then dissociates slowly (2) . In studying openchannel blockers of voltage-activated Kt channels, we found a different mechanism of use deoendence. Althoueh the blockers by a distinct molecular mechanism (C-type) W e studied the action of classical ~l u athat prod~ices a confornlat~onal change at ternary ammoni~im blockers, n h i c h block the outer mouth of the pore (4, 5) . . The long pulse current shows the onset of nact~vation; the symbols ind~cate the n t a l currents for the second pulse In a seres of two-pulse recovery expermenis, plotted aganst the t m e allowed for recovery between the two pulses. Best f~t s~ngle exponent~als are shown w~t h the estmated time constants. The long-pulse current w~t h blocker consists of a rap~d block phase (complete w~t h n 50 ms) followed by a snge-exponent~al ~nact~vation phase. (D) Currents from rat Kv1.4 channels expressed in HEK 293 cells after oxidaton to eminate N-type inact~vat~on (19) . When blocker is present, the rap~d C-type inactvaton becomes faster. The external souton contains 150 mM NaCl and 10 mM K C . (E) Dependence of C-type ~nactvat~on rate on the fract~onal block by C.,-TEA. S h l currents were measured [as shown In (C), bottom panel] w~t h d~fferent blocker concentrat~ons. The ~nact~vation rate (second relaxation phase) 1s plotted as a funct~on of the fract~onal steady-state block w~thin a sngle pulse. The lhnear dependence 1s consistent with the overall rate being the weghted average of a slow rate for the unblocked channels and a fast rate for the blocked channels.
Use-Dependent Blockers and Exit Rate of the Last Ion from the Multi-Ion Pore of a K+ Channel
-- (6) (7) (8) . After channel opening, C,,-TEA hinds within milliseconds to the channel and blocks it; on repolari:ation, the drug dissociates within tells of millisecollds (8) .
Use-Dependent Blockers and Exit Rate of the Last Ion from the Multi-Ion Pore of a K+ Channel
In spite of the rapid hinding and dissociation of this c o m p o~~n d , it produced (in addition to the transient blockade during each pulse) a use-dependent accum~ilative blockade at moderate stimulation frequency (Fig. 1, A and B) . As (Fig. 1 C ) . LWhen the blocker was present, there was a very rapid decline in current caused by the onset of blockade, which was followed by a n ~nactivation that was faster and more complete than normal, hut recovery o c c~~r r e d at the ~l o r~n a l rate (Fig. I C ) . 4 similar effect o n the onset of inacti\.ation n.as observed for the ~n a~n n l a l i a n Kv 1.4 channel (Fig. 1 D) .
Use-Dependent Blockers and Exit Rate of the Last Ion from the Multi-Ion Pore of a K+ Channel
T h e overall rate of the inactivation phase that f o l l o a d blockade aw a weighted average betareen a rapid rate for the blocked channels and the normal slow rate for the unblocked channels (Fig. 1E) . T o determine the rate of inactivation of blocked channels, we used a high degree of blockade (>90%) and looked at the disappearance of the remaining current. T h e rate of inactivation of the blocked channels was very sensitive to external potassium concentration ([Kf]) in the physiologic range: It nras fast in iero external [K'] and became much slower as [K-] increased, with an apparent affinity (KIr,-) of 2 mh1 (Fig. ?A) . T h e same [K-] depelldence for the rate of C-type inactivation occurs in tn.o other sit~~ations in which the outward tlon. of K' ions is eliminated elther n.1lel-i the inner inc~uth of the pore is blocked by N-type inact~vation or when all internal K' is temporarily removed and inactivaticm is monitored by measuring ~nward current (9, 19) . Our workiqg lhypc~thesis is that the rate of C-type inactlvatic~n is governed by the occupancy of a n ion-binding site near the external mouth of the pore, and that blocking K-efflux starves this control site and speeds up the rate of C-type inactivation (9) , particularly in the absence of estemal K' .
Use-Dependent Blockers and Exit Rate of the Last Ion from the Multi-Ion Pore of a K+ Channel
This hypotl~esis seems to suggest that all blockers prc~ducing a given level of fraction al block s h o~~l d have a n equal effect o n the inactivation rate. However, even for a series of closely related alkyltriethylammonium blockers, there was substantial variation in the effect o n the C-type inactivation rate (Fig. 3, A and R) . W e observed the maximal effect of the blockers with zero external [K+] and normal internal [K+] . T h e compounds showed various degrees of enhancement (from 2-to 10-fold), except for TEA, which had little or n o effect. Some of this variation in the effects of blockers on C-type inactivation had nothing to do ~v i t h blocking K-flow; variation occurred in the complete absence of K+ ions. W e measured the rate of inactivation with zero lKtl o n both sides of the membrane and L .
Use-Dependent Blockers and Exit Rate of the Last Ion from the Multi-Ion Pore of a K+ Channel
the effects of various blockers o n this rate (Fig. 3C ). T h e largest effect was produced by TEA, which slowed inactivation by about fivefold. Two other low-affinity blockers (C,-TEA and QX-3 14) had similar but smaller effects. A small (about 1.5-fold) enhancement of the rate was ~nduced bv C,-TEA and C1,,-TEA, whereaa C,-TEA, C,-TEA, and hretvl~um (Br) had n o effect These alloster~c effects of the blockera In the absence of K+ were amall and In a few cases opposlte to the overall effect of the blocker on lnactlvatmn n h e n lnternal K+ !\as present (compare Fig. 3B ). Two hlockers with n o allosteric effect, C4-TEA and C,-TEA, caused substantial but different degrees of increase in the rate of inactivation. Thus, it appeared that the dominant effect of all hlockers except TEA depended on the presence of internal K+ and that the effect varied among the blockers. W e hypothesized that this variation might arise from the different dwell times of the blockers. Roughly speaking, long-lived .
Use-Dependent Blockers and Exit Rate of the Last Ion from the Multi-Ion Pore of a K+ Channel
hlockers would prevent K+ flow long enough to affect the occupancy of the control site, whereas short-lived hlockers would not. T o test this correlation, we first determined the kinetics of blockade from relaxation or noise analysis and then plotted the effect for each blocker against its dwell time (Fig. 3L3) . T h e inactivation rate for each blocker in the presence of K+ was normalized to the rate for that blocker in the absence of K + , thus isolating the component of the blocker effect that could be attributed to K+ depletion.
Use-Dependent Blockers and Exit Rate of the Last Ion from the Multi-Ion Pore of a K+ Channel
T h e effects of the blockers were well correlated ~v i t h their dwell times. Shortlived hlockers produced less increase in the inactivation rate than long-lived blockers; as the dwell time increased, the inactivation rate became faster and asymptomaticallr approached a maximum rate. T h e half-maximal effect occurred at a blocker dwell time of -150 ps (Fig. 3D) .
Use-Dependent Blockers and Exit Rate of the Last Ion from the Multi-Ion Pore of a K+ Channel
Where does this characteristic dwell time come from? For a blocker with this dwell time, the control site would be occupied by a K+ ion for about half of the total duration of the blocker's residence and ~vould be empty for the other half; after that, the blocker would dissociate, and outward K+ flux would flood the site with fresh K+ ions. Accordingly, the occupancy of the control site would adjust to the blocked state of the channel with a n average time of 150 ps. Although this seems fast, it takes much longer than the average time that a single Kt ion resides in the pore during active permeation, about 0.1 us. Even if we envision a cloud of accumulated Kt ions in the outer entryway of the pore, this cloud should dissipate by diffusion in < 1 k s ( I I ). Thus, the rate-limiting step that determines the occupancy of the con-
Use-Dependent Blockers and Exit Rate of the Last Ion from the Multi-Ion Pore of a K+ Channel
trol site is likely to be the actual rate of release of K+ from the control site. Recause this site is highly selective for Kf over N a f ions ( 1 2 ) , we propose that t h e site controlling t h e rate of C-type inactivation is o n e of t h e Kt binding sites involved in permeation. O n average, any permeatmn slte must unhlnd mna at the throuehnut rate of t h e channel. about 10i , , A s p l . However, K + channels are known t o have multiple K + ions moving in single file (13) with repulsive interactions between them. In theory [and by analogy with multi-ion permeation in C a L + channels ( I 4 ) ] , a channel with only a single K + ion remaining would hold that ion much tighter t h a n a channel with two Kt ions. Thus, this last K+ ion to leave t h e pore would have a dwell time of 150 p s ( t h a t is, a n exit rate of 6700 s p ' ) , and t h e effect of blocker d~v e l l time o n t h e rate of C-type inactivation would arise from a race hetween the dissociation of a blocker molecule and the dissociation of this K L ion (Fig. 3E) (15) . The chromatic dimensions of human color vision have a neural basis in the retina. Ganglion cells, the output neurons of the retina, exhibit spectral opponency; they are excited by some wavelengths and inhibited by others. The hypothesis that the opponent circuitry emerges from selective connections between horizontal cell interneurons and cone photoreceptors sensitive to long, middle, and short wavelengths (L-, M-, and S-cones) was tested by physiologically and anatomically characterizing cone connections of horizontal cell mosaics in macaque monkeys. H1 horizontal cells received input only from L-and M-cones, whereas H2 horizontal cells received a strong input from S-cones and a weaker input from L-and M-cones. All cone inputs were the same sign, and both horizontal cell types lacked opponency. Despite cone type selectivity, the horizontal cell cannot be the locus of an opponent transformation in primates, including humans.
Use-Dependent Blockers and Exit Rate of the Last Ion from the Multi-Ion Pore of a K+ Channel
T h e retina is the site of two f~~ndamental stages in the neural representation of color. '13 whom correspondence should be addressed nearly 40 years, it has been known that these opponent signals are a property of retinal ganglion cells, the output neurons of the retina, yet the interneuronal circuitry that creates the opponent transformation has remained virtually unstudied because of the technical difficulties of making intracellular recordings from the intact retina of primates (4, 5) .
Use-Dependent Blockers and Exit Rate of the Last Ion from the Multi-Ion Pore of a K+ Channel
In nonrnammals with trichromatic vision, horizontal cells--one class of interneuron-display spectral opponency (6) . A long-held view is that an opponent transformation occurs by cone type-selective negative feedback from horizontal cells to cones (7) . Indirect evidence for such opponent feedback in primates is conflicting. Boycott and Wassle reported that two horizontal cell types ( H I and H2) contact all cone types nonselectively and should not, therefore, subserve opponency (8) ; the first SCIENCE VOL 271 2 FEBRU4RY 1996

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