Abstract ABSTRACT The mobility of lectin receptors and of two types of ion channels was studied in skeletal muscles of the frog Rana temporaria . Lectin receptors were labeled with fluorescent derivatives of succinyl-concanavalin A (Con A) or wheat germ agglutinin (WGA), and their mobility was measured by fluorescence recovery after photobleaching . Of the receptors for WGA, -53% were free to diffuse in the plane of the membrane, with an average diffusion coefficient as found in other preparations (D = 6 .4 X 10-" cm'/s) . Con A receptors were not measurably mobile . The mobility of voltage-dependent Na and K (delayed rectifier) channels was investigated with the loose-patch clamp method, coupled with through-the-pipette photodestruction of channels by ultraviolet (UV) light. Na channels were not measurably mobile (D <_ 10-'2 cm'/s). With K channels, photodestruction was followed by a small but consistent recovery of K current, which suggested that some K channels diffused in the plane of the membrane . Our results with K currents are best ft if 25% of the K channels diffuse with D = 5 X 10-11 cm2/s, with the remainder being immobile. For both Na and K channels, photodestruction by UV was most effective at a wavelength of -289 nm. At this wavelength, the energy density required for an e-fold reduction in the number of functional channels was 0.40 J/cm2 for Na channels and 0 .94 J/cm2 for K channels. Irradiation at this wavelength and dose did not measurably diminish the mobility of WGA receptors; hence, the immobility of Na and most K channels is not due to UV irradiation . It is concluded that mobile and immobile membrane proteins coexist in the sarcolemma of frog skeletal muscle, and that voltage-dependent Na and K channels are singled out for immobilization. INTRODUCTION THE JOURNAL OF GENERAL PHYSIOLOGY -VOLUME 87 -1986 In fully differentiated cells taken from adult animals, the mobility of integral membrane proteins in the plane of the cell membrane varies over a wide range. In most cases, the diffusion coefficient, D, is much lower than the value of 1-4 X 10-9 cm2/s (Frye and Edidin, 1970; Poo and Cone, 1974 ; Liebman and Entine, 1974 ; Tank et al., 1982) expected if integral membrane proteins could diffuse freely in a fluid lipid bilayer (Saffman and Delbruck, 1975) ; this indicates that the mobility of most integral membrane proteins is restricted . An extreme example is the acetylcholine (ACh) receptor at the neuromuscular junction, whose mobility is so low (<10 -' 2 cm2/s, Axelrod et al., 1976b ; possibly <10-" cm2/s, Almers and Stirling, 1984) that it cannot be detected with current methods. In an innervated adult muscle cell, ACh receptors are strictly confined to the small area directly beneath the innervating nerve terminal (Miledi, 1960; Fertuck and Salpeter, 1976) , even though they survive there for weeks (Bevan and Steinbach, 1983) . Immobility is clearly essential if ACh receptors are to remain concentrated at the endplate region throughout the life of the cell . ACh receptors are probably held in place by being anchored to components of the cytoskeleton (Tank et al., 1982) , as is apparently the case with other integral membrane proteins in postsynaptic membranes (Gulley and Reese, 1981 ; Hirokawa and Heuser, 1982) . INTRODUCTION Voltage-dependent ion channels, such as Na and K channels of nerve and muscle, are also kept at discrete locations in the cell membrane of many cells (Almers and Stirling, 1984) . In skeletal muscle, for instance, Na channels show a patchy distribution on the sarcolemma , are found at especially high concentration at the endplate region (Beam et al., 1985; Roberts and Almers, 1985) , and are also prevented from entering the transverse tubular system at the same density as on the sarcolemma (Jaimovitch et al ., 1976 ; Campbell and Hille, 1976 ; Almers et al., 1982) . This uneven distribution suggests that Na channels are restricted in their mobility . To measure the mobility of voltage-dependent ion channels in cell membranes, a method was developed that combined local patch-clamp recording of membrane current with through-thepipette photodestruction of ion channels by ultraviolet (UV) light. This method yielded the result that Na channels in the sarcolemma are essentially immobile, and hence must be anchored in the sarcolemma (Stuhmer and Almers, 1982 ). An advantage of the technique is that it directly assays function . Furthermore, it does not rely on fluorescent labels that might themselves influence mobility and, in any case, may not be readily available for all ion channels . INTRODUCTION In this paper, the mobility of voltage-dependent K channels is investigated . Most K channels were found to be immobile or severely restricted in their mobility, while there was a large population of relatively mobile lectin receptors. Tests of our method are reported, and comparison mobility measurements for membrane proteins other than ion channels are provided. We conclude that mobile and immobile membrane proteins coexist in the sarcolemma of skeletal muscle, and that Na and K channels are among the proteins that are singled out for immobilization . Our results help to explain how a muscle cell maintains the unequal distribution of these two channels over sarcolemma and the transverse tubular system . Preliminary accounts of this work have been given (Weiss et al., 1984x, b, 1985 . METHODS Fluorescence Recovery After Photobleaching (FRAP) The method followed that of Peters et al. (1974) , Edidin et al. (1976) , Jacobsen et al. (1976) , and Axelrod et al. (1976b) . METHODS Preparation . Small bundles of two to three fibers, or single fibers (70-100 um diam) were dissected in Ringer fluid (composition: 115 mM NaCl, 2 .5 mM KCI, 1 .8 mM CaC12, 3 mM Na-phosphate buffer, pH 7 .2) from semitendinosus muscles of Rana temporaria, mounted on plastic coverslips with fine silk threads tied to the tendon ends, and placed in 10-ml petri dishes for labeling with Fuorescein-conjugated derivatives of wheat germ agglutinin (FITC-WGA) or succinyl-concanavalin A (FITC-sConA) . Fluorescent lectins were obtained from US Biochemical Co., Cleveland, OH. The fibers were labeled during a 20-40-min incubation in a Ringer fluid (20°C) containing 25-60,ug/ml FITC-WGA or 1 .7 tug/ml FITC-sConA, washed three times in lectin-free Ringer, taken off the plastic coverslips, and transferred to the experimental chamber ( Fig. 1 B) . The fibers were mounted at 120% slack length and with the central 5 mm resting on a glass pedestal. On either side of the fibers lay glass microspheres (Duke Scientific, Palo Alto, CA), 62 Am ± 3 .2% SD in diameter, which were held in place on the pedestal by vaseline . A glass or quartz coverslip was then placed on top of the fibers and fixed in place by two drops of dental wax (Dentsply Ltd ., Weybridge, Surrey, England). This compressed the fibers slightly and resulted in an optically flat region of sarcolemma just beneath the coverslip . The microspheres acted as spacers and prevented the coverslip from compressing the fiber to a thickness of less than^"60 ,um. The lectin-free experimental medium, usually a Ca-free, ATP-containing solution (relaxing solution, containing 96 mM K, 42 mM Na, 7 .3 mM Mg, 112 mM morpholinopropanesulfonate, 15.8 mM propionate, 5.5 mM ATP, 15.6 mM creatine phosphate, 20 mM EGTA, pH 7 .0) was then substituted for Ringer fluid ; thereafter the fiber was ready for use . METHODS Recording . Fluorescein was excited with 390-490-nm light and its fluorescence was observed at 510-600 nm with a Leitz Dialux microscope (E. Leitz, Inc ., Rockleigh, NJ) fitted for epifluorescence . A schematic of the light path is shown in Fig . 1A . The excitation light was provided by a mercury arc (Osram HBO-100) in a lamp housing fitted with a quartz condenser (both from Oriel Co., Stamford, CT) . The light passed through an electronic shutter (Uniblitz, Vincent Associates, Rochester, NY), a heat-absorbing filter, a neutral density filter, a 400-nm long-pass glass filter (Schott Optical Glass, Inc ., Duryea, PA), and an interference filter into the epifluorescence port of the Leitz microscope. Except for the dichroic mirror, all lenses and filters were removed from the epifluorescence attachment. The filter cube directed the light through the microscope objective (Leitz 50X water immersion objective, 1 .00 NA, a Nikon 100X glycerol immersion objective, 1 .30 NA, or a 74X UV-transmitting reflecting objective, 0.65 NA, by R. &J. Beck, Watford, England) onto the preparation . In most experiments, the excitation beam also passed through a pinhole in the image plane of the objective (FS in Fig. 1 A) to project a 3 .6-7.2-Am-diam circular spot on the surface of the muscle fiber. The arrangement allowed us to excite and hence to sample fluorescence from a small, well-defined area of sarcolemma centered in the field of view. For local photobleaching of the fluorophore, the excitation beam could be temporarily intensified by removing the neutral density filter . METHODS The fluorescence from the muscle fiber was measured by the photomultiplier tube in 958 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 87 -1986 a Nikon UFX camera unit (Nikon, Inc ., Garden City, NY) . The photomultiplier was sensitive only to light emitted from the central area covering I % of the field of view . The camera unit was modified to provide electrical access to the output of the operational amplifier that measured the current from the photomultiplier anode . This output, which was a linear function of light intensity, was amplified, filtered (100 Hz four-pole Bessel), and passed to the A/D converter of a NOVA 3 computer (Data General Co., Westboro, The light paths used in FRAP experiments . Abbreviations are : Hg lamp, mercury arc lamp; PC, primary condenser ; elect . shut ., electronic shutter ; HF, heat filter ; NDF, neutral density filter ; LPF, long-pass (>400 nm) filter ; FS, field stop ; IF, interference filter ; H2-FC, dichroic mirror with long-pass barrier filter ; PL, projection lens; PM, photomultiplier ; obj ., microscope objective ; C, condenser ; HSM, half-silvered mirror ; PD, photodiode ; man . shut ., manual shutter ; FD, field diaphragm ; Q .H. lamp, quartz halogen incandescent lamp. (B) The experimental chamber used in FRAP experiments . Abbreviations are : LC, Lucite chamber; GB, glass bottom ; ped., glass pedestal ; MS, microspheres (62 um diam) ; CS, coverslip ; SW, dental wax; MF, muscle fiber . MA). The excitation light passing through the preparation and the microscope condenser was deflected by a half-silvered mirror onto an HUV 400B photodiode (EG&G, Salem, MA) so that intensity fluctuations of the excitation beam could be measured. The computer alternately sampled the outputs of the photomultiplier (fluorescent light) and the photodiode (excitation light), each at 5 kHz . METHODS During a typical experiment, fluorescence measurements were made once every 30 s with a 10-100-fold-attenuated excitation beam . Immediately after photobleaching, the frequency of measurement was temporarily increased to 10/min . The electronic shutter opened for 80 ms during each fluorescence measurement . 25 ms after the shutter had opened, the computer began to take samples, 250 each of excitation and fluorescent light . After the 50-ms sampling period was complete, the shutter closed. The computer calculated average values for the intensities of excitation and fluorescence emission during the 50-ms interval and calculated the fluorescence intensity (in arbitrary units) as the ratio of fluorescence emission and excitation intensity . Forming this ratio corrected for fluctuations in the excitation light intensity . METHODS In some experiments, fibers were irradiated with UV light . The UV spectral lines from the mercury arc were selected with the appropriate bandpass interference filters and passed to the preparation through a reflecting objective . The interference filters had a 10-nm bandwidth at half-maximal intensity and were purchased from Ditric Optics Co ., Marlboro, MA, or from Melles-Griot Co ., Irvine, CA . FIGURE 2 . The tip of the micropipette used in the experiment shown in Fig . 9 , viewed at^-45°with respect to the shank axis. Calibration bar, 10 gm. Electrical Recording The muscle cell membrane was voltage-clamped with the loose-seal patch clamp ) using circuitry described previously (Stiihmer et al ., 1984) . Briefly, firepolished glass pipettes of 11-30 km tip diameter (Fig. 2) were pressed against sartorius muscle fibers of R. temporaria to electrically isolate a patch of sarcolemma . Suction was not applied to the pipette, and we avoided the region of the motor endplate . The resistance of the seal between pipette and membrane (R.) was always greater than the pipette resistance (RP , typically 0.15-0 .3 M9), and was monitored throughout the experiment . Electrical Recording Step depolarizations of 4 .5 ms duration were applied to the pipette in order to elicit local currents IN . and IK through Na and K channels, respectively . Unless otherwise indicated, step amplitudes were 130 mV for measuring IK and 90 mV for measuring peak IN. . Na and K currents served as an assay for the number of functional Na and K channels in the patch . The holding potential was set to zero, i .e ., equal to the resting potential of the fiber. Current recordings were corrected for capacitative and leakage currents by a 960 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 87 -1986 combination of digital and analog techniques, filtered (10 kHz low-pass, four-pole Bessel), and stored by the computer. Electrical Recording Unless the resting potential ofthe fiber is measured with a microelectrode, the absolute membrane potential is unknown in loose-patch experiments. However, the resting potential could be estimated from the voltage dependence of Na current activation . In order to exclude depolarized (and hence unhealthy) fibers, data were accepted only if the peak Na current during a 50-mV depolarizing step was <10% of that measured during a 90-mV step. Electrical Recording Local destruction of Na and K channels in the patch was achieved by directing UV light through the pipette and onto the patch of membrane beneath the pipette tip (Stiihmer and . The light from the mercury arc lamp, after passing through a UV bandpass interference filter, was focused onto one end ofa 1-mm-diam quartz fiber (Math Associates, Port Washington, NY) by a 15X reflecting objective (R. & J. Beck) ; the other end of the quartz fiber was abutted against the back end of the pipette . Pipettes were coated to within 50 lm of the tip with an opaque epoxy paint to improve their electrical properties and also to minimize leakage of UV light through the glass wall . To measure the light emerging from the pipette, the tip was passed through a pinhole covering a photodiode (HUV 4000, EG&G). Experiments were rejected if, after UV irradiation, the inward current for a 130-mV step exceeded that for a 90-mV step; this was considered an indication of an unacceptably large rim current . Electrical Recording Unless indicated otherwise, all experiments were carried out at room temperature (22°C) . Error bars represent 1 SEM. RESULTS Labeling of the Sarcolemma with FITC-WGA WGA is a 35,000-mol-wt dimeric lectin that binds to N-acetyl-n-glucosamine and N-acetylneuraminic acid residues (Nagata and Burger, 1974) . In a variety of cell culture lines, most WGA receptors have been shown to represent glycosylated integral membrane proteins (see Nicolson, 1974, for review) . Fig . 3 shows photomicrographs of an isolated single fiber stained with FITC-WGA . The plane of focus led through the center of the fiber, and striations are clearly visible in transmitted light (Fig. 3A) . When the fiber was viewed with fluorescence optics, it appeared that fluorescence was restricted to the edge of the fiber, which indicates that the lectin was bound to the fiber surface (Fig. 3B) . RESULTS In Fig . 4 , the microscope was focused onto the top surface of the fiber, providing a grazing view of the sarcolemma . Under bright-field illumination (Fig. 4A) , faint striations from superficial sarcomeres are visible just below the cell membrane . Also visible is the nucleus of a small cell, probably a satellite cell, that was attached to the surface of this fiber . Fig . 4B was recorded with fluorescence optics . Although the fluorescence from the fiber surface appears granular or somewhat patchy, there were no completely dark areas . Evidently FITC-WGA was bound everywhere on the fiber surface . The outline of the small cell whose nucleus is visible in Fig . 4A appeared brightly . This cell was probably almost entirely within the microscope's plane of focus, since its membrane, labeled with fluorescent WGA, can be seen in edge view. RESULTS When the fluorescence of a small spot on the fiber surface was monitored using a photomultiplier and the protocol of repeated measurements described in 96 1 Methods, the fluorescence was found to remain undiminished for at least 3 h (not shown), even if the fiber was perfused with a bathing fluid that contained no FITC-WGA but had an excess (100 leg/ml) of unlabeled WGA . Evidently FITC-WGA remains stably associated with the sarcolemma for hours. The Mobility ofLectin Receptors in the Plane of the Sarcolemma The mobility of WGA receptors can be investigated by FRAP . In this method, the surface of a cell is first labeled with a fluorescent hgand, the fluorophores in a small area of labeled membrane are then bleached, and the fluorescence of 96 2 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 87 -1986 that area is monitored thereafter to test whether it recovered . Fig. 4 C shows the area of membrane in Fig . 4B , photographed almost immediately after an 80-s illumination with a spot of intense 390-490-nm light . Where the light fell, little fluorescence remained, and the outlines of the bleached region are sharply FIGURE 4 . Surface view of an FITC-WGA-stained single muscle fiber . The fiber was lightly compressed between two coverslips to create a flat region of sarcolemma in the plane of the picture, and the microscope was focused onto that surface . Panels A (bright-field illumination) and B-D (fluorescence optics) are all from the same area of the fiber, and were photographed in the sequence shown. B is before, C is 1 .5 min after, and D is 36 .5 min after bleaching the sarcolemma with a 31-Am-diam spot of intense blue light . The background brightness of the last two fluorescence panels was adjusted during printing to compensate for slight bleaching of the entire field, which occurred during photography . Calibration bar, 10 gm. RESULTS defined . The fluorescence in the bleached area had largely recovered 36 .5 min later (Fig . 4D) ; only a faint outline of the bleached spot remained . Following other investigators, who have performed similar experiments on other types of cells, we interpret the recovery of fluorescence as being due to fresh WGA receptors that, carrying along their unbleached fluorophores, migrate into the bleached area by diffusion within the cell membrane . This interpretation is the simplest, since the bath contained no fluorescent material and the material bound elsewhere on the muscle did not dissociate within the times explored here (see above) . Also consistent with this interpretation was the appearance of the small cell, which was situated entirely within the bleached area and lost its fluorescence completely (Fig . 4 C) . The fluorescent outline of this cell did not reappear (Fig . 4D ) . Evidently the recovery of fluorescence observed here depended on continuity with a large reservoir of unbleached membrane. RESULTS To determine the time course of recovery in this type of experiment, the local concentration of fluorophore in a small area of sarcolemma was periodically assayed by flashing images of a small circular spot of exciting light onto the sarcolemma and measuring the resulting fluorescence with a photomultiplier (see Methods) . Fig. 5A shows an experiment on a fiber labeled with FITC-WGA . The ordinate shows the normalized fluorescence from a 7.2-tm-diam spot on the sarcolemma . Immediately before t = 0, fluorescence measurements were suspended while the exciting beam was intensified 10-fold so that, during 15-s continuous illumination, much of the fluorophore in the spot was bleached . Then periodic sampling of fluorescence with dim excitation light was resumed . It can be seen that fluorescence from the spot was diminished strongly by the intense light, but recovered partially during the following 50 min . As before, we attribute the recovery to lateral diffusion of WGA receptors that carry unbleached fluorophore . RESULTS The continuous curves in Fig. 5 are given by RESULTS n=i a which describes radial diffusion into a disk . The expression gives the average concentration of the diffusing substance inside the disk (e.g., Crank, 1970, Eqs . 5.14 and 5.23 ) after this concentration has been uniformly reduced to a fractional value, Fo . a are the roots of fo(x) = 0, fo(x) being the Bessel function of zero order and the first kind. D is the radial diffusion coefficient and a is the radius of the illuminated (and bleached) spot. It is assumed that not all of the fluorophore is free to diffuse, and that the freely diffusible fraction is m . In Fig. 5A , where FO is measured as 0.35, the best least-squares fit is obtained with D = 5 .6 X 10 -" cm2/s and m = 0.50 . The curve is seen to describe the data well . In 14 similar experiments on 11 fibers, average values were D = 6 .4 X 10 -' cm 2/S (±0 .8 X 10-" cm2/s [SEM] ) and m = 0.53 ± 0 .02. The magnitude of the mobile fraction of sarcolemma) WGA receptors may be somewhat larger than the value of m, since any WGA receptors in the basement membrane and adhering connective tissue strands would appear immobile . When FRAP measurements were performed twice on the same spot on a muscle fiber, the diffusion coefficients obtained were identical within experimental error, with the ratio of the first over the second measurement being D,/D2 = 0 .84 ± 0.13 SEM (n = 5). However, the mobile fraction in the second experiment was always increased, because the immobile fraction does not recover its fluorescence . We conclude that the excitation light used to bleach the fluorophore did not significantly affect the mobility of WGA receptors. Fig . 5B illustrates a FRAP experiment on a fiber stained with FITC-sConA. This dimeric lectin (52,000 mol wt) binds preferentially to a-n-mannose and av-glucose residues, and the receptors for this lectin are thought to be distinct from those for WGA (Ozanne and Sambrook, 1971 ; . No significant recovery of fluorescence was observed, which indicates that only a vanishingly small fraction of the sConA receptors are measurably mobile. The finding could be explained if the label bound more . Fluorescence recorded from small spots of sarcolemma on four muscle fibers labeled with FITC-conjugated lectins . Each point is the average of three measurements. At t = 0, fluorescence was diminished by continuous illumination (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) s) with the 10-100-times-intensified excitation beam. Fluorescence is seen to recover when the label was FITC-WGA (A and C) but not when it was FITC-sConA (B and D) . A and B were in Ca-free relaxing solution (see Methods) ; C and D were in Ringer fluid ; both solutions were lectin-free . The spot size was 7 .2 gym; all measurements were made with a 50X Leitz water immersion objective. heavily to the basement membrane than to cell membrane receptors, so that fluorescence from the rigid basement membrane would mask that from mobile receptors in the sarcolemma . This explanation would be consistent with the composition of the major basement membrane proteins. For instance, collagen has a high content of glucose residues, and fibronectin is rich in mannose, but neither protein contains comparable amounts of WGA binding residues such as sialic acid (Kefalides, 1973 ; Carter and Hakomori, 1971 to bind considerable amounts of concanavalin A (Gahmberg and Hakomori, 1975) and the same appears to be true for the whole basement membrane of skeletal muscle (Wakayama et al., 1980) . Most of our FRAP experiments were done in a Ca-free, K-rich solution (see Methods) that depolarized the muscle sufficiently to make it mechanically refractory . This was done because even the slightest movement during the -r1-h-long experiment produced large errors by diminishing the coincidence between the bleached area and that from which fluorescence was assayed. Nevertheless, a few successful experiments were also carried out in Ringer fluid (Fig. 5, C and D) and gave similar results. About half of the WGA receptors, but virtually none of the sConA receptors, were measurably mobile . RESULTS Destruction ofNa and K Channels by UV Irradiation Our method of studying the mobility of ion channels relies on the localized photodestruction of channels by UV light. Fig. 6 , A-C, shows membrane currents recorded through a patch pipette (see Methods) during step depolarizations of 80 (A), 140 (B), and 130 (C) mV. During the 80-mV pulse, a transient inward current (INa) through voltage-activated, tetrodotoxin-sensitive Na channels predominated. During the 130-or 140-mV pulses, INa was smaller and followed by a more slowly developing outward current (IK), which, on the basis of previous work (Adrian et al., 1970 ; Stanfield, 1970) flows through voltage-activated K channels that are blocked by tetraethylammonium (not shown) and are often referred to as delayed rectifier channels . Currents through Na channels cause the upstroke of the action potential, while delayed rectifier channels contribute to repolarization . RESULTS Each of the panels in Fig. 6 shows two traces . The trace with the deflections of larger amplitude was recorded before, and the other trace after a period of through-the-pipette irradiation with UV light (289 nm in Fig. 6 ). The traces show that UV light caused a loss of current through both Na and K channels . When irradiation was continued for long enough, these currents were lost entirely (Fig. 6 C) . RESULTS In Fig. 6 , D and E, IK and peak IN. are plotted against time . Both IN,, and IK begin to decline exponentially as soon as irradiation starts (t = 0). IN,, declined more rapidly than IK ; evidently Na channels are more sensitive than K channels to UV light . Similar results were previously obtained by Oxford and Pooler (1975) on lobster axons, and by Fox and Stampfli (1971) and Fox (1974) for Na channels in myelinated nerve. These authors found that the effects of UV light are irreversible, and that, at least for IN ., the surviving channels remain normal in kinetics and voltage dependence . These results, along with the exponential decline of IM, and I K , have led to the view that Na and K channels are destroyed by UV light in a one-hit, one-photon event. RESULTS The sensitivity to UV can be measured as the rate constant of decline divided by the power density of the light reaching the membrane beneath the pipette. The power of the light leaving the pipette was measured after each experiment by passing the pipette tip through a pinhole covering the photosensitive surface of a UV-sensitive photodiode . To obtain the power density, the value obtained was divided by the area of the pipette tip orifice, measured from photographs as 966 THE JOURNAL OF GENERAL PHYSIOLOGY -VOLUME 87 -1986 in Fig . 2 . As an approximation, this seems justified because light is known to leave the pipette as a well-defined beam of the same diameter as the pipette tip orifice (St6hmer and . Effects of nonuniform illumination of the patch were neglected . Stuhmer and Almers (1982) with respect to the baseline before depolarization ; I K was measured at the end of the pulse as the amplitude of the current jump at the moment of repolarization. Irradiation started at t = 0. The continuous curves are the best least-squares fit of exponential functions that decline to zero; their time constants are 36 (D) and 102 (E) s. Same experiment as in A and B. Fig . 7 shows an action spectrum, plotting the sensitivity to U V light (S) against wavelength. S is the reciprocal of the radiation dose required for an e-fold reduction Of INa (circles) and IK (squares) . The wavelengths chosen were the mercury lines provided by the arc lamp. Both Na and K channels are most sensitive at 280-289 rim, as if UV light attacked one or more aromatic amino acids forming part of the channel protein . At 289 nm, 0.40 J/cm2 was sufficient to reduce the number of functional Na channels e-fold ; for K channels, 0 .94 J/ cm2 was required . In this series of experiments, K channels were nearly three times less sensitive than Na channels. However, the sensitivity ratio was variable ; in a later series (see Fig . 12 ), Na and K channels were much more similar in their sensitivity to UV light. The reasons for this variability are not understood. FIGURE 7 . Action spectrum for the destruction of Na channels (circles) and K channels (squares) by UV light. The solid circles at 280 and 289 nm were obtained as in Fig . 6 , and the left-hand ordinate appliesto them. They represent the reciprocal ofthe radiation dose required for an e-fold reduction ofIN . (circles) and IK (squares) . S is defined by S = 0/H, where H is the UV light intensity at the pipette tip (in watts per square centimeter) and 0 is the initial rate constant for the decline of IN., calculated as the reciprocal of time constants as obtained in Fig . 6 , D and E, on the assumption that current declines exponentially to zero. For the right-hand ordinate, the mean sensitivities S for INa at 280 and 289 nm were assigned the values 1 .0 and 1 .19, respectively . All other open symbols were normalized relative to measurements of the initial rate constant of IN. decline at either 280 or 289 nm, made on the same patch (squares at 280 and 289 nm) or with the same pipette on the same fiber. The value of H was determined for each pipette and at all wavelengths of interest, generally both before and after each series of electrophysiologic measurements. Patch diameters in these experiments were 20-30 jAm . Half-filled symbols represent values obtained with the vaseline-gap technique . They were calculated from the initial rate constant of IN. decline, and are given relative to measurements on the same fiber carried out at 280 nm . RESULTS We performed similar measurements OfIN . also with the vaseline-gap technique Almers et al., 1982) . A reflecting objective was used to focus UV onto the fiber, and the section of the fiber from which we recorded current, i.e., the A-pool, was covered with a small piece of quartz coverslip to exclude the vaseline film that normally forms at the air-water interface . The 96 8 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 87 " 1986 decline Of IN ;, in these experiments was only poorly described by an exponential, presumably because muscle fibers strongly absorb UV, so that irradiation reaches only the half of the fiber surface facing the UV source . If an apparent value of S is calculated from the initial rate constant of decline of IN., the average value is S = 1 .08 f 0.04 cm2/J (n = 4, ± SEM), roughly half that observed with the loose-patch method. However, in calculating rate constants, it would probably be more reasonable to refer the initial rate of decline to half the current observed, since only half the membrane from which we collect current is likely to be reached by UV. If this is done, the value of S doubles, and is in good agreement with the loose-patch measurements . RESULTS From results obtained with the vaseline-gap technique on myelinated nerve by Fox and Stampfli (1971, their Fig. 1 ), one can calculate S = 0.7 cm'/j fork, in that tissue . These authors reported also that K channels of myelinated nerve are not measurably sensitive to UV. In lobster axons, K channels were 10 times less sensitive to UV than Na channels (Oxford and Pooler, 1975) . The lesser sensitivity of K channels to UV seems to be a general finding. Mobility of Na and K Channels Since the patch pipette allows both local recording and local irradiation, one can use it to investigate the mobility of ion channels (Stuhmer and Almers, 1982) . If channels are mobile, current from an irradiated patch should recover as healthy channels diffuse into the patch from the surrounding membrane . Fig. 8 (left) shows signal-averaged records Of IK and INa (A) and Of INa alone (C), all obtained from the same patch. Each panel shows three traces superimposed . Trace 1 was recorded before, trace 2 immediately after, and trace 3^-16 min after the end of the irradiation period . In Fig. 8A , trace 3 is slightly larger than trace 2, which indicates a slight recovery of IK after irradiation. The difference between the two traces is shown at a 10-fold-higher gain in Fig. 8B (noisy trace), with a scaled version of trace I superimposed . The difference trace is seen to have a component that is kinetically similar to the late outward current, i.e., to IK, but no inward component that resembles IN.. The difference between traces 2 and 3 in Fig. 8 C is shown in Fig. 8D . Once again, it has an outward but no inward component. It can be concluded that after irradiation, there is a slight recovery of IK, but none Of INa. When IK and peak IN. from this experiment were plotted against time (Fig. 9 , A and C), IN. did not show recovery after irradiation, which confirms that Na channels are not measurably mobile by this technique (Stuhmer and Almers, 1982) . There was, however, a slight recovery of IK, which indicates either that the effects of UV on K channels are partially reversible, or that a small number of K channels entered the irradiated patch by diffusion within the plane of the membrane. Mobility of Na and K Channels The experiment in Fig. 9B was similar to that of Fig. 9A , but was carried out with a pipette of large tip diameter . Although measurements at late times (not included) showed that some recovery of IK ultimately did take place, it is clear that any such recovery in Fig. 9B was considerably slower than in C. A depend- depolarizations . Panels A and C show three traces each; each trace is the average of six measurements. Trace I is from measurements obtained between 0 and 3.5 min before, trace 2 from measurements between 1 and 4 min after, and trace 3 from measurements taken between 14 and 17 min (A) or 16 and 18 min (C) after irradiation with 289-nm UV light. Panel B shows the difference between traces 3 and 2 of panel A, and panel D shows the differences between traces 3 and 2 ofpanel C. For comparison, a scaled version oftrace 1 in A (labeled 1) is superimposed onto the difference trace in B. In this figure, it was important to reduce noise, most of which comes from the signal that is subtracted from the measured current to correct for stray capacity and leakage under the seal . To eliminate this source ofnoise, the leakage-capacity signal was divided into the following six sections : (1) before depolarization, (2) 0-0.25 and (3) 0 .25-1 .5 ms after depolarization, (4) between 1 .5 ms after depolarization and repolarization, (5) 0-0.25 ms after repolarization, and (6) from 0.25 ms after repolarization until the end of the trace. Sections 1, 3, 4, and 6 were replaced with least-squares fits of polynomials of order 0, 3, 2, and 3, respectively . The resulting smooth curves were used instead of the raw signal for leakage and capacity subtraction . Immediately after a potential step (sections 2 and 5), the current changed too rapidly to be fitted satisfactorily; during that time, the raw signal was used for subtraction. The difference between the raw signal and its replacement was routinely displayed on the oscilloscope and showed no systematic deviation from zero over the entire length of the trace. Fiber KMB3; pipette diameter, 11 gm. ence on pipette tip diameter is not expected if recovery is simply due to reversion from a hypothetical, UV-induced inactive state. Recovery by lateral diffusion, on the other hand, is expected to depend strongly on pipette tip diameter, being slowed fourfold when the diameter is doubled . Hence, our results favor the view that the recovery in Fig . 9A was due to diffusion of fresh K channels into the irradiated patch . The recovery of IK in Figs. 8 and 9 was only slight, but it was observed consistently . In Fig. 10 , five experiments including those in Fig. 9 , A and C, were combined by signal-averaging. To allow for differences in pipette diameter, the time axis in each experiment was normalized to correspond to a I 0-mm tip diameter; this was done by multiplying the abscissa by (10 pm/diam)2. To allow A B loo, . From Fig. 7 , the energy density can be calculated as having been between 0.43 and 0.88 J/cm2, since at t = O, IN, was reduced to 34% (C) and IK to 48% (A) or 39% (C) . A and C are from an experiment where IN. and IK were measured as in Fig. 6 , D and E, during alternating pulses of 90 and 130 mV amplitude, respectively . Each point is the average of eight measurements except immediately before and during the irradiation period, where single measurements are plotted. At the end of the experiment, the pipette was moved to two other locations, spaced 20 Am apart along the fiber axis; K currents recorded there were at 110 and 92%, and Na currents were at 94 and 70% of the mean value recorded before irradiation . Fiber KMB3; pipette diameter, 11 lim; same experiment as in Fig. 8 . B was obtained from another fiber (KM04) with a larger (20 um diam) pipette tip . The horizontal lines indicate the current immediately after irradiation . Mobility of Na and K Channels for the fact that IK was reduced to slightly different degrees in different experiments, plots as in Fig . 9 , A and C, were treated as follows . First, a constant was added to all ordinates to make IK immediately after irradiation equal to half the average current before irradiation . Then the ordinates were normalized with respect to the mean 1K before irradiation . Finally, all five experiments were superimposed, and adjacent groups of 30 points were averaged . Fig . 10 clearly shows that a small fraction of IK recovered after irradiation . Mobility of Na and K Channels For quantitative analysis, we followed previous practice and assumed that UV emerges from the pipette tip as a beam of Gaussian intensity profile, with the intensity decreasing by a factor of e2 within the pipette tip radius a, i .e ., within 5 Am for Fig. 10 . The efficiency of current collection was assumed to vary with the same profile. In that case, recovery of current should be given by (Axelrod et al ., 1976x) : where K is calculated from cm 2/S . The continuous curve provides the better fit. Hence, we conclude that although most K channels are immobile, there is also a small fraction (25%) whose mobility is about equal to that of the WGA receptors in Fig. 5 . Mobility of Na and K Channels The failure of Na and K channels to diffuse readily into an UV-irradiated patch could be explained if the UV irradiation transformed the sarcolemma into a rigid structure into which membrane proteins could not diffuse. (A) Experiment similar to that in Fig. 5A , except that a UV-transmitting reflecting objective was used, and the spot size was 5 um. (B) As in A, except that the measurement was carried out after irradiation with 289-nm light . UV irradiation was carried out through the same light path that was used to excite fluorescence, except that all optical elements between the electronic shutter and the dichroic mirror were removed and replaced by a 289-nm bandpass interference filter . The UV intensity that arrived at the plane of the muscle fiber was measured before and after the experiment with a UV-sensitive photodiode (see Methods) . From it and the irradiation time, we calculated an energy density of 1 .56 J/cm2 for this experi- possibility, and shows two FRAP experiments on an FITC-WGA-labeled fiber. The data in Fig. 11 A were obtained as in Fig. 5A , and plot the recovery of fluorescence in a 5 .0-jm-diam bleached spot against time. In Fig. 11 B, a 5.0-,cmdiam spot on the same fiber was first irradiated with 1 .5 J/cm2 of 289-nm light, and then the ability of WGA receptors to diffuse within that area was measured as in Fig. 11 A . On the basis of Fig. 7 , the energy density of UV deposited on the membrane would have been large enough to diminish the number of functional K channels four-to fivefold, more severely than in the experiments of Figs. 9 and 10 . Even with this large dose of UV irradiation, however, fluorescence recovery indicated a diffusion coefficient for WGA receptors that was essentially identical to that measured on an unirradiated patch. Measurements after irradiation with 280-or 289-nm light at an average dose of 1 .29 J/ cm2 (range, 0.67-1 .56 J/cm2) were made on a total of four fibers . The average post-irradiation diffusion coefficient was 2 .6 X 10-" cm2/s (±0.5 X 10" cm2/s) and the average mobile fraction 66.2 ± 6.2% ; values are given f SEM. These values are nearly identical to others obtained without irradiation on fibers from the same muscles (D = 2.8 X ±0 .5 X 10 -" cm2/s; m = 58 .6 ± 9.4% ; n = 5). We conclude that the apparent lack of Na and K channel mobility is not due to the UV irradiation. Size of the Irradiated Area The analysis of Fig. 10 assumes that the irradiation destroys Na and K channels only in a strictly localized area. While this assumption is supported by the finding that UV light supplied to the back end of the pipette leaves the tip as a sharply defined beam of the same diameter as the pipette tip orifice (Stiihmer and , it seemed desirable to map the amount of photodestruction directly in nearby and overlapping patches by recording current as a function of position . In achieving this, we faced two difficulties . First, the distribution of Na and K channels is known to be nonuniform even before irradiation ). Second, it seemed possible that repeated attempts to make seals on overlapping membrane areas might cause changes in current unrelated to UV irradiation, possibly through membrane deformation. Recordings were therefore made from only two locations on each fiber, and the results from many fibers were averaged. A first series of experiments served to test for changes in current attributable to forming a patch. The pipette was advanced until a seal resistance of 0.4 MU was attained, and Na currents were measured with a 90-mV step (INa,) and K currents with a 130-mV step (IKI). The pipette was then withdrawn and a new patch with a 0 .4-Mg seal was made, either at the same location or at a distance of up to 50 km along the longitudinal axis of the fiber. The currents recorded after the second approach, INa2 and IK2, were divided by INa, and IK,, respectively, and plotted against patch separation . Patch separation is defined as the centerto-center distance between the two patches. Fig. 12 (solid symbols) shows the results of 108 such experiments. For IN., the average ratio is seen to be independent of patch separation and was not significantly different from unity (Fig. 12A) . IK, however, was slightly reduced when the second patch overlapped the 974 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 87 -1986 first. Fig. 12B suggests that IK was diminished locally by -10% when a 0.4-Mg seal was made with a pipette of 14,um tip diameter. Size of the Irradiated Area To map the effects of UV irradiation on IK and INa, we used the same experimental protocol as above, except that after IN ., and IKI had been measured, UV light was applied through the pipette until IN, fell to half its initial value. The pipette was then withdrawn and re-advanced, either at the same or another location . IN . and IK were recorded once again, normalized as before, and plotted against patch separation . Fig. 12 (open symbols) shows the result from 90 experiments of this kind. As expected, currents close to the irradiation site were strongly reduced. However, the effect of UV rapidly diminished with increasing distance and, for Na channels, vanished altogether when the patch separation was >25,um (Fig. 12A) . With K channels, a small (^-7%) reduction of IK was seen even at the largest separations (Fig. 12B) as if, in addition to acting locally, U V also had a more diffuse effect that spread uniformly over distances at least as large as 50 lm. The significance and mechanism of the diffuse UV effect on K channels are not understood . Size of the Irradiated Area The smooth curves in Fig. 12 show, as a function of patch separation, the decrement in current expected if the overlap of two nearby patches is taken into account. As in Fig. 10 , we assumed UV light arriving at the membrane to have a Gaussian intensity profile that is maximal at the center of the patch, decreases e2-fold within one pipette radius, and continues to decline to zero outside the pipette . The amount of channel photodestruction at each point in the membrane was assumed to be proportional to the local UV intensity. For IK, we assumed a spatially uniform 7% reduction in addition to the local effect of UV light. The efficiency of current collection was assumed to have the same Gaussian profile as the UV intensity. The curves are seen to provide a good fit to the data. Hence, these data support the view that the destruction of Na and K channels by UV irradiation is confined almost entirely to the membrane beneath the pipette tip, and can be described by the Gaussian profiles assumed in Fig. 9 . Size of the Irradiated Area Variability of Na and K Current Densities Along the Fiber Fig. 12 shows that if no irradiation was applied, currents did not vary .systematically with position on the fiber. However, individual pairs of measurements on the same fiber showed a substantial variation that increased with distance between the two patches and most probably reflects a patchy distribution of Na and K channels over the sarcolemma . Fig. 13 plots, against patch separation, the average fractional difference in current recorded at the two locations. For Na currents, the difference is calculated by : , FIGURE 12 . (opposite) Spatial profiles ofaverage Na and K channel densities before (solid symbols) and after (open symbols) photodestruction of Na and K channels by UV irradiation through the pipette. Smooth curves are theoretical current profiles generated for a 14-,m pipette, assuming a Gaussian profile of UV irradiation and current measurement across the pipette tip (for further explanation, see text). The data were obtained from 10 sartorius muscles. Each point .is the mean of between 10 and 30 paired patches. To avoid bias, all accessible fibers in a muscle were used, and results were rejected only if the fiber showed evidence of being depolarized or if the current traces were suggestive of large rim currents (see Methods). The tip diameters of pipettes used for UV irradiation were 14 km when patch separations were <17 um, and 15 lAm for some measurements with larger separations. In experiments without irradiation, smaller pipettes were also used, and we routinely retained the first patch for -1 min after measurements were complete, and only then withdrew the pipette in order to approach the second patch. This waiting time was about equal to the usual time of irradiation. Size of the Irradiated Area where the brackets denote absolute values . For K currents, the situation is complicated by the fact that forming a patch in itself reduces IK by~10% . To allow for this systematic variation, one can calculate the difference DIK by Size of the Irradiated Area where r, a function of patch separation, is the average value Of IKI/1K2 as obtained from Fig. 12 B (circles) . Eq. 5 was used for IK instead of Eq. 4 for patch separations of <20 um. Size of the Irradiated
{'paper_id': '7038520', 'title': 'Mobility of voltage-dependent ion channels and lectin receptors in the sarcolemma of frog skeletal muscle', 's2_url': 'https://api.semanticscholar.org/CorpusID:7038520', 'source': 'S2ORC'}