Very little is known about actions of proctolin and FMRFamide-related peptides on ionic currents in insects. This is surprising because proctolin was the earliest identified insect neuropeptide (Starratt and Brown 1975) and FMRFamide-related peptides are ubiquitous in insects, quite a few having been sequenced from locusts (e.g., Lange et al. 1994
; Robb et al. 1989
). It therefore seemed of interest to investigate effects of proctolin and FMRFamide-peptides on ionic conductances in locust jumping muscle [the results from a parallel study on the effects of octopamine will be presented in the companion paper (Walther and Zittlau 1998
)]. Previous studies have suggested an effect of proctolin and FMRFamide-peptides on the membrane resting potential or conductance. The peptides caused slight depolarizations, accompanied by decreases in conductance (May et al. 1979
; Walther and Schiebe 1987
; Walther et al. 1991
). Similarly, proctolin slightly decreased the resting conductance in cockroach muscles (e.g., Adams and O'Shea 1983
, legend of their Fig. 4), and it was hypothesized that this might be due to reduction of a K+ conductance (Hertel and Penzlin 1986
).

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| FIG. 4.
Activation of the K+ conductance, GK,H, at 2 different K+ concentrations in 1 fiber. Tail currents, measured at 55 mV after activating prepulses to the voltages indicated (cf. METHODS and Fig. 3), were converted to conductances according to G(V)= IK/(V EK). EK was obtained from the reversal of K+currents on activation and deactivation as demonstrated by Fig. 2 as well as from the reversal potential of the Ba2+-sensitive resting K+current. EK was 80 mV at 7.5 mM [K+]o and 63 mV at 15 mM [K+]o. Data points are means of the figures obtained by 2 succesive runs at both K+ concentrations. Curves indicate fits of the data to Eq. 1 divided by ( 55 mV EK). Inset, the Boltzmann function G = Gmax × (1/{1 exp[(V V0.5)/S]}) calculated by means of the fit parameters obtained for both K+ concentrations; these were for 7.5 K+ (and for 15 K+): Gmax = 0.76 (1.48) µS; V0.5 = 91.6 ( 88.9) mV; and S = 18.4 (19.6) mV. Circles mark V0.5, i.e., the voltage of half-maximal activation, which was practically unaffected by [K+]o in contrast to the maximal conductance, which was greatly enhanced.
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Because at the outset of our studies native FMRFamide-peptides of locusts were not yet identified and because in our earlier investigations the synthetic analogue YGGFMRFamide (Tyr-Gly-Gly-Phe-Met-Arg-Phe amide) acted strongly in this locust preparation [~50 times more strongly than FMRFamide (Walther et al. 1984
, 1991
)], we continued to use this substance for the work presented here. As we will show, both peptides lower the resting K+ conductance, partly by reducing a previously described K+ conductance that slowly activates on hyperpolarization (Zittlau and Walther 1991
). This conductance is further characterized to provide a basis for analyzing the peptide effects as well as for comparing it with related conductances. We also exclude various potential second-messenger pathways for the peptide effects.
Generally the functional significance of lowering the resting K+ conductance is an increased membrane excitability. Although this effect is common to both peptides, its relevance to their overall effects is probably somewhat different because it combines with otherwise different or even opposite neuromuscular effects of the two peptides. Some of the results have been presented in abstract form (e.g., Murck et al. 1989
; Walther and Zittlau 1989
, 1991
).
 |
METHODS |
Preparation
Experiments were performed in the jumping muscle, i.e., M. extensor tibiae of the hindleg, of the locust Schistocerca gregaria. Data from >200 preparations were evaluated. Animals were obtained from University of Konstanz (Germany) or from Blades, Edenbridge (United Kingdom) and also were reared in our institute. Most of the exoskeleton of the femur was removed, and the proximal half of the muscle was discarded. The remaining part receives excitatory innervation from both the fast (FETi) and slow (SETi) motoneurons. It consists of comparatively small fibers partly suitable for voltage clamping and corresponds to region f of Hoyle's classification (1978; his Fig. 2). The preparation was fixed in such a position that the bundles became gently stretched and somewhat separated. The fibers to be investigated were located above the outlet of a superfusion line integrated into the recording chamber so that they were exposed directly to the incoming saline. Almost exclusively the second or third bundles counted backward from the distal end of the muscle were used.

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| FIG. 2.
Hyperpolarization-activated K+ current, IK,H, and the determination of EK. A1: voltage jump from 75 to 95 mV slowly activates an inward K+ current, IK,H ("on-relaxation"). A2: IK,H tail currents ("off-relaxations") recorded on termination of activating voltage jumps; as indicated on top of A1 and to the right of A2, the voltage was stepped back to various levels around 75 mV, the approximate reversal potential. B1: after an activating voltage jump from 55 to 75 mV (i.e., the approximate EK; hence no current relaxation is seen), an outward tail current at 55 mV is flowing due to deactivation of IK,H. B2: IK,H currents recorded, as indicated in on top of B1 and to the right of B2, at various potentials. Like the tail currents at A2 the on-relaxations reverse around 75 mV. Digitally smoothed records. Traces at A2 and B2 are arbitrarily spaced and start immediately after end of capacitive current.
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Only the ventral-most fibers, which appear to form a homogeneous population both in regard to electrical and mechanical properties, were used (fibers in the most distal bundle or in more dorsal layers seemed to have significantly greater resting K+ conductances than those in the ventral layer). The fibers had diameters of 70-100 µm and lengths of 1.6-1.9 mm. From a diameter of 90 µm and a length of 1.8 mm, i.e., dimensions representative of the comparatively large fibers used for recording, the calculated surface area is 5 × 10
3 cm2. This calculation accords with the figure of 4.7 × 10
3 cm2 obtained by dividing the average membrane capacitance of 34 nF (see RESULTS) by 7.3 µF/cm2, i.e., the specific membrane capacitance indicated by Fig. 2 of Kornhuber and Walther (1987)
for a fiber diameter of 90 µm. Measurements started only after superfusing the preparation for
1 h and could be extended over 5-10 h without signs of deterioration. Usually the dissection was performed the day before an experiment and the preparation stored in the refrigerator (6-8°C).
Solutions
The saline contained (in mM) 10 KCl, 150 NaCl, 2 CaCl2, 12 MgCl2, 90 sucrose (to achieve a physiological tonicity) and was buffered at pH 6.8 (at 30°C) with 10 mM 3-[N-Morpholino]propanesulfonic acid (MOPS). The comparatively high concentration of Mg2+ (free [Mg2+] in locust hemolymph is ~3 mM) (Clements and May 1974
) lowered the risk of contractions and strongly reduced a voltage-dependent Ca2+ current (cf. further). The bath temperature, 30°C unless stated otherwise, was maintained constant within ±0.1°C by means of a feedback-controlled heating system. The rate of superfusion, running continuously from a gravity fed device, was usually ~1 ml/min and exchange of solutions was complete within 1 min. Drugs were dissolved in saline and bath-applied through the perfusion system. When testing very low concentrations of peptides, 0.1% bovine serum albumin was added to the saline to prevent adsorption of peptide to tubes and vessels.
Electrophysiological techniques
A two-electrode voltage clamp (NPI Electronics; Tamm, Germany) was used. The current electrode contained 2 M K3 citrate with 10 mM KCl added; resistance 4-7 M
. The recording electrode contained 2 M K acetate plus 10 mM KCl unless specified otherwise; resistance was 6-10 M
. A broken pipette filled with 3 M KCl in 2% agar served as reference electrode. In some early experiments, recording electrodes filled with 3 M KCl were used, but this practice was discontinued after we discovered that on prolonged recording a hyperpolarization-activated Cl
current developed (Walther and Zittlau 1998
). To obtain a homogenous space clamp, the current electrode was inserted in the middle of the fiber and the recording electrode halfway between current electrode and one end of the fiber (Zittlau and Walther 1991
). With this arrangement of electrodes, it was essential to select thick fibers for clamping; slender fibers made voltage clamping difficult or even impossible. After adjustment of clamp, in response to a step command, the voltage settled within 0.5-1 ms, and the capacitive current was terminated within 4-6 ms. If the latter lasted considerably longer, the fiber was discarded. A prolonged capacitive current possibly indicated some electrical coupling to neighboring fibers, which occasionally occurs in this preparation (unpublished observations). For measurements of current-voltage relations (Fig. 8), the capacitive currents of the recordings were eliminated by subtraction of currents obtained from appropriate short voltage pulse commands (cf. Zittlau and Walther 1991
). Currents were low-pass filtered with a cutoff frequency of 1 kHz.

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| FIG. 8.
Instantaneous current-voltage characteristics of the K+ current suppressed by proctolin and of the K+ current activated by hyperpolarization, IK,H. A: instantaneous currents measured on stepping to voltages negative from Vh = 53 mV. Note that due to this holding potential IK,H was largely deactivated. Data points are from 2 pairs of successive measurements, performed before and during application of 4 × 10 10 M proctolin, and include the holding current (at 53 mV). Curves, drawn by eye, intersect at 73 mV, i.e., the approximate EK in this fiber estimated from the reversal of IK,H relaxations (as in Fig. 2). Current at 73 mV is accounted for by the leaks produced by the low-resistance electrodes used in this experiment. Subtraction of the 2 curves (control minus proctolin) yields the current suppressed by proctolin, IProct, which is shown at B on an enlarged scale (dotted curve). B: instantaneous I-V characteristic of IK,H in the same fiber. As in A, data from 2 series of measurements (from the same fiber) are shown to give an indication of the reproducibility. Data points for IK,H represent the differences of 2 instantaneous currents, 1 recorded on jumping to the test voltage (Vtest) after an activating prepulse, the other on jumping to Vtest after a deactivating prepulse. This is demonstrated, for one example, by the inset. Curve for IProct, derived from A, is also shown for comparison; note different calibration for the 2 currents. Inset: currents recorded during 2 voltage commands that 1st step from Vh = 70 mV either to 55 mV (for deactivation of IK,H) or to 115 mV (for activation of IK,H) and then both to Vtest, in this case 105 mV. Horizontal dotted line indicates 0 current level. Near the end of the deactivating prepulse (arrow head), a short segment of the record was omitted for clarity of presentation. At this time, a short jump to 115 mV was performed. This way, on digital subtraction of the 2 traces, the capacitive current elicited by the jump to Vtest was largely eliminated (for more details, cf. Zittlau and Walther 1991 , their Fig. 1 and legend). Voltage electrode contained K+ acetate so that the hyperpolarization-activated Cl current (ICl,H) was not present.
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To continuously monitor the resting conductance, 40 ms × 20 mV rectangular hyperpolarizing voltage commands provided by a Grass SD9 stimulator were applied every 6 s. Voltage jump sequences and voltage ramps were generated by a Hewlett Packard 9220 computer in conjunction with a TRC 4010 transient recorder (Krenz Electronics, Hirzenhain, Germany), which also served for data acquisition.
Measurements of membrane resting conductance, capacitance and the resting membrane potential
A pair of voltage ramps (Fig. 1; dV/dt: 100 or 250 mV/s) was applied for measuring resting conductance (Gr) and capacitance (C). This protocol was used frequently for studying effects of peptides and drugs. Subtracting ramp-induced currents measured before and during application yielded the magnitude and reversal potential of the affected conductance. Estimates of the specific membrane capacitance, Cm, derived from C and fiber geometry (4 measurements) closely agreed with those previously determined in a cable analysis based on current-clamp measurements (Kornhuber and Walther 1987
). For determination of the membrane resting potential (Vr), the voltage drop due to the leak conductance (gL) caused by the electrodes, was taken into account. gL/2 was derived from the current, Ileak, needed to clamp the fiber at the potential observed after inserting the first electrode (assuming that the leakage current induced by an electrode reverses at 0 mV). For optimal impalements, gL was ~0.03 µS, but in practice up to about four times larger leaks proved to be acceptable. Vr was assumed to be approximately equal to the holding potential at which the holding current was 2 × Ileak. Figures of the leak-corrected Vr were 2-6 mV more negative than those read after inserting only one electrode.

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| FIG. 1.
Muscle membrane currents recorded during voltage ramps in the absence and presence of a peptide. A: voltage recorded during double-ramp command; dV/dt = 250 mV/s; the holding potential, 74 mV, corresponded to EK determined for this fiber in the manner shown by Fig. 2. B: current elicited by the command shown at A; the cursors are positioned at the holding potential and mark the points where the slopes are measured for deriving the membrane resting conductance, Gr. In this fiber, Gr was 1.1 µS (mean from both ramps the slopes of which generally differed by only a few percentages). The difference of currents at the cursor positions equals the sum of the constant capacitive currents that flow during the de- and ascending branch of the ramp command according to IC = C × dV/dt. C of this cell was 30 nF. C: ramp currents in the presence of 2 × 10 7 M YGGFLRFamide. D: difference of the currents shown at B and C. Curves represent a rough I-V characteristic of the current suppressed by the peptide; they exhibit slight outward rectification and reverse at EK. All records are averages from 8 successive measurements. Transient capacitive currents are truncated. Recording electrode was filled with K acetate.
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K+ current measurements
Activation of IK,H was measured as previously described (Zittlau and Walther 1991
) from tail currents at a test potential which varied between
50 and
55 mV. After a negative going activating prepulse of 2 s duration to voltages between
60 and
135 mV, the voltage was stepped to the test potential. The amplitude of the tail current was measured 20 ms after this jump; the initial amplitude was ~8% larger. Prepulses more negative than
135 mV often led to membrane breakdown. With less negative jumps this also could happen, although it was not always immediately obvious (Zittlau and Walther 1991
). Test potentials more positive than
50 mV were not advisable since usually at around
40 mV a Ca2+ current and a Ca2+ activated K+ current (and above
40 mV a delayed rectifier current) became noticeable. We did not find potent selective blockers for these currents. In particular, divalent cations like Cd2+, Co2+, or Ni2+ were not very effective. Millimolar concentrations would have been required to completely block strongly activated Ca2+ current(s), which at the same time would have severely suppressed IK,H (cf. RESULTS).
When measuring activation curves or establishing instantaneous I-V characteristics, we not only kept to the above voltage range but, as an additional, precautionary measure, routinely had 50 µM 4-aminopyridine, 4 mM tetraethylammonium, and 0.2 mM Cd2+ in the saline. Experiments were discarded if the time courses of the tail currents varied with the voltage of the prepulses. Activating pulses of 2 s duration led to 80-90% of steady-state activation. To achieve complete activation, the pulses would have had to be extended to ~10 s, which would have been impractical particularly because at very negative voltages such long pulses would have led to frequent membrane breakdown. Activation measurements required ~8 min per run to allow enough time between voltage jumps so that cumulative effects were avoided. Many attempts to complete such measurements were frustrated due to various sources of trouble, particularly if repeated runs under different conditions in one fiber were required.
Considering membrane breakdown and K+ depletion (see RESULTS), the data for the most negative activation voltages (less than or equal to
115 mV) may not be very reliable. These data points might critically affect the calculated level of activation, which, according to the fits (cf. RESULTS, Eq. 1), is already present at the test potential. However, model calculations indicated that even an error of 20% would have had a negligible effect on this parameter. Accumulation of K+ ions due to the outward current was assumed to be minimal because the holding currents were between 5 and 10 nA.
Recording from collagenase-treated fibers
Investigation of the effects of collagenase was feasible only by first impaling a fiber and allowing some 20 min for optimal sealing of the electrodes before collagenase application. After application of collagenase, the recording was usually lost within 20-30 min due to breakdown-induced contractions of various fibers. Thereafter the preparation could no longer be used because collagenase-treated fibers were disrupted by attempts to impale them.
Microionophoresis
Ionophoretic injection of guanosine 5'-O-(3-thiotriphosphate) tetralithium salt (GTP-
-S), guanosine 5'-O-(2-thiodiphosphate) trilithium salt (GDP-
-S), and bis-(o-aminophenoxy)-N,N,N',N'tetraacetic acid (BAPTA) was performed by means of a WPI-160 microionophoresis apparatus. Current pulses of 60-120 nA, lasting 500 ms, were delivered at a rate of 1/s under voltage clamp (holding potentials around
70 mV).
Peptides and drugs
Proctolin (Arg-Tyr-Leu-Pro-Thr) and the FMRFamide peptides YGGFMRFamide and YGGFLRFamide [which also was used occasionally and which produced the same effects as YGGFMRFamide (Walther et al. 1984
, 1991
)] were obtained from Bachem, Switzerland. K acetate from Aldrich of the highest purity (99.98%; calcium content 5 ppm) was used for the filling solution of the recording electrodes. Some lots contained large quantities of acetic acid, which had to be neutralized (pH 7) because otherwise the K+ resting conductance of the impaled fiber exhibited a "run down" (i.e., a reduction by 10-30% within 1 h after impaling the fiber). K acetate of lower purity usually had the consequence that during prolonged recording the baseline became unstable, possibly due to minute contractions.
The following chemicals were obtained from Sigma: 4-aminopyridine, arachidonic acid, collagenase (Type IA), BAPTA, 4-bromo-phenacyl bromide, 8-bromoadenosine 3':5'-cyclic monophosphate (8-br-cAMP) and 8-bromoguanosine 3':5'-cyclic monophosphate (8-br-cGMP), GTP-
-S, and GDP-
-S, 1-(5-isoquinolinlylsulfonyl)-2-methylperazine dihydrochloride (H7), Nmethyl-D-glucamine (NMDG), phorbol-12,13-didecanoate (PDD), 4
-phorbol-12,13-didecanoate (4
-PDD), phorbol-12-myristate-13-acetate (PMA), D-sphingosine. Ionomycin, nonglycosidic indolcarbazole 1 (NGIG-1), and staurosporine (a kind gift from Dr. D. Swandulla) were from Calbiochem. Ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), quinine HCl, and quinidine HCl were from Serva.
Stock solutions of lipophilic drugs were prepared in either ethanol or dimethyl sulfoxide. In the final solutions the concentrations of these vehicles did not exceed 0.1% (0.2% could cause slight conductance changes).
Evaluation of data
Digitized recordings were evaluated by means of routines supplied by Krenz Electronics. Fits to mathematical expressions were performed using the software from Wavemetrics (Igor). Means are given ±SD; n = number of experiments.
 |
RESULTS |
Membrane resting potential and resting conductances
Resting properties of the muscle fibers are relevant to electrical events like synaptic potentials or action potentials. The cable constants have been previously investigated in the jumping muscle (Kornhuber and Walther 1987
). Here we present a brief account on its resting conductances. There are two main findings: the K+ resting conductance (GK,r) greatly dominates the resting conductance (Gr) so that there is practically no difference between the resting potential (Vr) and the K+ equilibrium potential (EK); and variability of the resting conductance under control conditions is largely accounted for by variation of GK,r.
The membrane resting potential, Vr, of isolated locust skeletal muscles in physiological saline is close to EK (e.g., Hoyle 1953
). However, at low [K+]o, (
5 mM), i.e., when GK,r is strongly reduced, contribution of a small Na+ conductance to Vr cannot be ignored (e.g., Leech 1986
). Such a conductance, in our preparation, however, may be largely due to the leaks introduced by the electrodes. The resting potentials, if corrected for leaks as described in the METHODS, agreed with EK (see further) within the limits of resolution (
1 mV) and were between
68 and
78 mV. Thus there seems to be practically no Na+ resting conductance. This conclusion also is supported by the fact that substituting Na+ for by NMDG or blocking the Na+/K+ pump by ouabain caused only small effects (Walther and Zittlau 1998
).
The resting Cl
-conductance (GCl), in a coxal muscle of this locust species (Lea and Usherwood 1973
), amounts to
40% of the resting conductance. However, in the muscle fibers investigated here, according to preliminary experiments with Cl
-free saline (n = 3), GCl seems to be only of the order of 10% of the resting conductance. Although ECl seems to be a few millivolts more negative than EK (Walther and Zittlau 1998
), the chloride conductance thus seems to have a negligible effect on the resting membrane potential.
The mean total resting conductance, Gr (at Vm
EK), as determined by voltage ramp measurements (Fig. 1; cf. METHODS), was 1.30 ± 0.40 µS (n = 64). The mean specific membrane conductance, derived from this figure and the fiber surface was ~270 µS/cm2. This is ~65% greater than that previously determined for more proximally situated fibers, albeit under somewhat different experimental conditions (extrapolated to 30°C) (Kornhuber and Walther 1987
). The membrane capacitance (C) was 34 ± 9.5 nF (n = 35; range: 18-52 nF).
The considerable variability of Gr (0.5-2.2 µS) might have been partly due to different fiber sizes. However, Gr divided by C, thus normalized to fiber surface area (0.04 ± 0.011 µS/nF), varied to a similar extent (range 0.025-0.068) as Gr. This indicates that the specific membrane conductance (Gm) differed considerably between fibers. This variation can be largely accounted for by variation of GK,r because Gr varied to a similar extent as the amount of conductance that was suppressed by 2 mM Ba2+, a selective blocker of the resting K+ conductance. On average 2 mM Ba2+ removed 76 ± 8.5% (n = 15) of Gr, which gives a lower limit for mean GK,r.
Hyperpolarization-activated K+ current, IK,H
IK,H is a K+ current that increases on hyperpolarization. As shown in Fig. 2A1, a jump to a voltage negative from EK leads to a slow, inward going "on-relaxation" while no relaxation is seen on jumping to the approximate EK (Fig. 2B1). Likewise, jumping to a voltage positive from EK leads to an "off-relaxation" due to deactivation of IK,H (Fig. 2B1). Basic properties of IK,H have been reported previously (Zittlau and Walther 1991
). The underlying conductance, GK,H, significantly contributes to the resting conductance. It is modulated both by octopamine (Walther and Zittlau 1998
) and the peptides investigated here. Before demonstrating this, we present a more comprehensive characterization of GK,H performed both for comparison of GK,H with other K+ conductances and to provide a framework for analyzing the peptide effects.
DETERMINATION OF EK.
Knowing EK was of considerable importance for conducting our investigations [including those on the actions of octopamine (Walther and Zittlau 1998
)]. In particular, EK was required for calculating K+ conductances from measured K+ currents. By holding a fiber at EK, it was also often immediately evident from the holding current whether or not a drug affected GK or another conductance.
Because IK,H is highly K+ selective (Zittlau and Walther 1991
), it offered the possibility to estimate EK "on-line" from the on- and off-relaxations of IK,H (Fig. 2, A2 and B2). The values of EK obtained ranged about from
68 to
78 mV. Another estimate of EK was the reversal potential of the current blocked by Ba2+. This figure rarely differed by >1 mV from that derived from IK,H relaxations in the same fiber (n > 10). Combining both methods, we obtained a mean EK of
73.7 ± 2.2 mV (n = 14).
According to this figure, the average internal K+ concentration ([K+]i) was 168 mM. Measurements with ion sensitive electrodes in another locust muscle (Leech 1986
), where the resting potential is the same as in jumping muscle (Kornhuber and Walther 1987
), indicated a [K+]i of ~140 mM. The difference in the estimates is probably due to the difference in temperature used (room temperature vs. 30°C in our present study) because [K+]i rises with warming (Leech 1986
).
LOCALIZATION OF GK,H.
The tail currents observed after hyperpolarizing pulses tended to reverse at slightly more negative values than the on-relaxations. The difference was ~1.5 mV on average. It was larger (
4 mV) in fibers having high normalized resting conductances (Gr/C), probably indicating that GK,r was particularly high. These observations suggest that a large fraction of the K+ channels is located within the transverse tubules. In that case, if inward K+ currents of sufficient strengths and durations are flowing, the tubules are expected to be depleted of K+ ions and EK to shift in the negative direction. Likewise, a K+ accumulation and a positive shift of EK may occur with outward K+ currents. Several lines of evidence support this notion.
The extent of transverse (T-)-tubular membrane can be inferred from measurements of the specific membrane capacitance, Cm, which increases with fiber diameter (Hodgkin and Nakajima 1972
). In our locust preparation, for a typical fiber diameter of 90 µm, Cm is ~7 µF/cm2 (Fig. 2 of Kornhuber and Walther 1987
), whereas Cm of plasmalemma (free of invaginations) is generally ~1 µF/cm2. The difference of these figures indicates that more than some 80% of the sarcolemma cannot be accounted for by the surface membrane but have to be attributed to T-tubular membrane. Thus if the channels are about equally distributed over the entire sarcolemma, >80% of them should reside within the T tubules.
Tubular K+ depletion due to inward rectifier currents has been shown previously and quantitatively modeled in frog muscle (Standen and Stanfield 1979
). There, with [K+]o =10 mM, an inward current density of ~60 µA/cm2 leads, within 1 s, to a reduction of [K+]o by as much as 70%. On the other hand, assuming that in our preparation a 20-mV hyperpolarizing jump of 2.7 s (like in Fig. 2A1) causes ~1 mV negative shift of EK, one can infer that this leads only to ~5% reduction of [K+]o. When, however, the K+ current produced by hyperpolarization was greatly upmodulated [by inhibition of protein kinase A (Walther and Zittlau 1998
)], the reversal potential of the tail currents shifted to rather negative values, indicating severe tubular K+ depletion.
ACTIVATION OF GK,H.
A previous study already indicated that this conductance was activated substantially at EK (Zittlau and Walther 1991
) (K+]o = 10 mM). It was, however, not investigated whether GK,H accounts for the entire resting K+ conductance and how the situation changes when [K+]o varies within a physiological range. One also would like to know how steeply GK,H declines when the membrane is depolarized. This is relevant to the effectiveness of depolarizing currents, e.g., on summation of excitatory synaptic potentials. We therefore attempted to characterize the steady-state activation of GK,H more thoroughly.
To establish activation curves for GK,H (Fig. 3) outward tail currents at
50 to
55 mV (as in Fig. 2B1) were recorded after negative going pulses. The amplitude of such a tail current represents the amount of activation that is obtained at a given potential in addition to that which already may be present at
55 mV (measurements at more positive voltages were not feasible, cf. METHODS). A simple and commonly used model for characterizing the activation of an ion conductance is a two-state process described by the Boltzmann equation. It assumes that the applied voltage change merely determines the fraction of channels that open and that, at sufficiently large voltages, all channels will be open. Although the measured relationship of current versus voltage therefore should saturate, this cannot always be clearly demonstrated, often for obvious technical reasons. This is the situation we are dealing with (cf. METHODS), and our approach, therefore, has to be regarded as tentative.

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| FIG. 3.
Steady-state activation of IK,H determined by tail current measurements in 1 fiber. After activating 2-s prepulses to the voltages indicated, the amplitudes of tail currents at 50 mV were measured (see METHODS). Data from 2 runs in 1 fiber and a fit according to Eq. 1 are shown. Inset: Boltzmann function I = Imax × (1/{1 exp[(V V0.5)/S]}) calculated by means of the fit parameters: Imax = 22.2 nA; V0.5 = 102.5 mV; and S = 19.8 mV. Note that at EK (cursor) there is ~20% activation.
|
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Data were fitted according to
|
(1)
|
where Imax is the maximal current, V0.5 the voltage at which activation is half-maximal, S the slope factor, and Vtest the potential at which the tail currents were measured, i.e.,
55 to
50 mV. The second term accounts for the fraction of current that already may be activated at this potential. With the parameters obtained from the fitting procedure the complete activation curve can be calculated (Fig. 3, inset; note that the original curve extends over a smaller range of voltages and, therefore, is only slightly sigmoidal). The average values from 17 fibers for which the routine indicated good fits were Imax = 25.7 ± 8.5 nA; V0.5 =
96.5 ± 5 mV; and S = 17.9 ± 3.15 mV. The values of V0.5 ranged between
85 and
105 mV, which probably indicates some variability between fibers and not just the limited accuracy of our measurements.
The steepest change of activation thus does not occur in the physiological voltage range. As the membrane is depolarized from EK, the decline of activation does not cause a major reduction of total resting K+ conductance, partly because the decline is counteracted by a slight outward-rectification of the instantaneous I-V characteristic of GK,H (Zittlau and Walther 1991
). That at physiological potentials a rather large fraction of this K+ conductance is not in use under control conditions might seem puzzling. There is in fact a huge reserve K+ conductance as becomes obvious when protein kinase A is inhibited. This leads to an enormous increase in resting K+ conductance, and this increase seems to be partly due to a positive shift of the curve on the voltage axis (Walther and Zittlau 1998
).
There was already substantial activation of GK,H at EK (on average 21%), and even at
55 mV activation was significant (cf. inset of Fig. 3). It seems, however, that GK,H activation at EK is insufficient to account for the entire resting K+ conductance. The magnitude of GK,H at EK can be estimated in the following manner: the mean tail current amplitude after stepping from EK to the mean test potential of
54 mV was 5.3 nA. Allowing for ~35% underestimation of steady-state activation (see METHODS) and converting to conductance yields 0.46 µS. From this figure, according to the instantaneous I-V characteristic (slightly outward rectifying; Fig. 8), at EK a conductance of 0.32 µS is derived. This is much smaller than GK,r, which in this sample of fibers was
1 µS because the mean total resting conductance was 1.33 µS of which
80% should consists of GK,r (cf. earlier text).
This discrepancy is probably too large to be simply accounted for by inherent inaccuracies of our procedures. Rather, we suggest as a working hypothesis that the underlying K+ channels do not completely deactivate on depolarization. Such kinetics have for example been demonstrated for certain chloride channels (e.g., Fahlke et al. 1995
). This would be the simplest interpretation of most of our findings. It accords with our experience that any treatment that affected GK,r also produced a similar change of GK,H. We therefore subsequently will consider GK,H as the voltage-dependent component of GK,r.
DEPENDENCE OF GK,H-ACTIVATION ON [K+]o.
The functioning of various K+ channels is affected by variation of the external K+ concentration, [K+]o. This can be relevant when [K+]o changes, e.g., on efflux of K+ from the muscle during electrical activity or due to K+ intake on feeding. We therefore investigated whether activation of GK,H is different at two K+ concentrations that are about the lower and upper limits of [K+]o in locust hemolymph (Hoyle 1954
). As will be seen, GK,H was greater at higher [K+]o. Based on outward tail current measurements as above, Fig. 4 and Table 1 show how activation of the conductance, GK,H, is affected by changing [K+]o from 7.5 to 15 mM (3 experiments). There was no clear change of V0.5 although a small effect might not have been resolved. However, the curve certainly did not shift on the voltage axis by as much as EK as for classical inward rectifiers (e.g., Leech and Stanfield 1981
). Gmax, however, was increased markedly when [K+]o was doubled. This seems to indicate that channel permeation was somehow facilitated. Permeation thus appears to be more complex than assumed by the Goldman-Hodgkin-Katz equation, which represents the most simple model relating current to the gradients of voltage and concentration.
[K+]o seems to affect the entire resting K+ conductance, not only the hyperpolarization-dependent component. This follows from the observation that the holding current at
55 mV (Ihold,
55) was only slightly affected by changing [K+]o and from the assumption that leak- and Cl
-currents should not have changed with [K+]o. Ihold,
55 was nearly the same at both K+ concentrations (9.8 ± 3.6 nA at 7.5 mM and 8.7 ± 6.0 nA at 15 mM; n = 6). Thus the change of the driving force for K+ and the change of total GK at
55 mV must have nearly cancelled each other on changing from 7.5 to 15 mM [K+]o. According to Ohm's law, it then follows from IK,
55 = GK,7.5 × (
55 + 80)
GK,15 × (
55 + 63) [µS × mV] that GK,7.5: GK,15
1: 2.5. This is similar to the twofold increase calculated for maximal GK,H (cf. Table 1). A clearly smaller effect would have been expected if solely GK,H had increased.
Although the physiological importance for the K+ sensitivity of GK,r remains to be shown, there is at least one obvious consequence of it. Whenever [K+]o rises, e.g., when the animal is feeding (Hoyle 1954
) or when K+ ions perhaps accumulate in the T tubules during high electrical activity of the muscle fiber, the driving force for K+ ions will be reduced. Depolarizing factors (cf. DISCUSSION) thus will become more effective. However, this tendency will be counterbalanced partly by the increase in K+ conductance on elevation of [K+]o.
PHARMACOLOGICAL CHARACTERIZATION OF GK,r, AND THE EFFECT OF COLLAGENASE.
Any blocker that reduced GK,r also reduced GK,H. As previously reported (Zittlau and Walther 1991
), Cs+ (if IK,H is flowing in the inward direction), Rb+, and Ba2+ are effective in the millimolar range. This also applies to quinine or quinidine (
50% reduction with 1 mM). In addition to Ba2+, other divalent cations investigated also blocked GK,r. The sequence of potency was Zn2+
Ni2+ > Cd2+ = Co2+ > Ba2+ (IC50 of Zn2+
10
5 M). Even Ca2+ and Mg2+ seem to be weak blockers because reduction of [Ca2+]o or [Mg2+]o below the levels in standard saline led to a increase in GK,H. Collagenase (Type IA; 1.5 mg/ml at 30°C), an enzyme frequently used to prepare cells for patch clamping (e.g., Gorczynska et al. 1996
), dramatically reduced GK,r within 7 min. By then GK,H
but not the hyperpolarization-activated Cl
current (Walther and Zittlau 1998
)
was reduced to <20% of the control (n = 5). There was no recovery from these effects during subsequent washing for 30 min.
Modulatory effects of YGGFMRFamide and proctolin
The findings obtained with YGGFMRFamide and proctolin are presented together because both peptides reduced GK,r in a nearly identical manner. Neither peptide, however, affected the hyperpolarization-activated Cl
current, ICl,H, or the Na+/K+ pump, in contrast to octopamine which modulated these currents as well as GK,r (Walther and Zittlau 1998
).
REDUCTION OF RESTING K+ CONDUCTANCE.
YGGFMRFamide or proctolin usually decreased the resting membrane conductance (cf. Fig. 5, A and B, chart recordings of repetitive pulses at slow time scale). For the standard concentrations used, i.e., 2 × 10
7 M and 2 × 10
10 M, respectively, the average reductions were
38 ± 11% (n = 39) and
46 ± 13% (n = 30). The time to half-maximal effect was ~1 min (cf. Fig. 5 and legend). Ihold remained practically constant if the holding potential (Vhold) was at EK, suggesting that the peptides acted on GK,r. IK,H, too, was reduced as is seen from the currents elicited by single prolonged voltage jumps also shown in Fig. 5. The average reduction (measured for 20 mV pulses as in Fig. 5) was
46 ± 13% (n = 12) and
52 ± 15% (n = 5) for YGGFMRFamide and proctolin, respectively. The time course of IK,H seemed to be slightly slower in the presence of peptide, a point not further investigated here. There was no indication of desensitization
30 min at standard concentrations. After application for 5-15 min, the recovery on washing usually took ~20 min for either peptide (time to 50% recovery:
4 min). Recovery often was not quite complete even with much longer periods of washing. The effects of YGGFMRFamide and proctolin were not additive and they were occluded when GK,r was strongly reduced by 2 mM Ba2+. Thus both peptides seem to affect the same K+ channels.

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| FIG. 5.
YGGFMRFamide and proctolin reduce the input conductance and the hyperpolarization-activated K+ current, IK,H. Chart recordings from one voltage clamped fiber. Holding potential, 73 mV, approximately corresponded to EK. Repetitive 20 mV × 40 ms hyperpolarizing commands were given at 6-s intervals. Top: holding current and miniature excitatory currents that render the lower edges of the traces noisy. Bottom: inward current levels attained during the short command pulses; the fast down- and upstrokes of the current pulses are not seen. Zero current levels are indicated (right). More rapid onset of the peptide effect in A compared with B may at least partly be due to the different peptide concentrations according to the law of mass action. Before and during applications of peptides single prolonged (2.7 s × 20 mV) voltage jumps were performed; the evoked currents, labeled by asterisks, are shown on an expanded time scale; short horizontal bars indicate the onset of IK,H. Gaps in the recordings last ~10 s; record B starts 8 min after the end of record A. Recording electrode was filled with 2 M K acetate.
|
|
Measurements performed with voltage ramps (cf. Fig. 1) demonstrated that the peptides exclusively affected GK. The currents suppressed reversed at
68 to
78 mV. These reversal potentials agreed within 1-2 mV with EK in the individual fibers (determined from IK,H; cf. earlier section). When [K+]o was increased to 20 mM or reduced to 5 mM the reversal potentials shifted in a manner close to that predicted by the Nernst equation for a K+-selective conductance (Fig. 6, A and B). Furthermore, if the peptide was applied in low [Na+]o saline (90% of Na+ substituted for by NMDG), the reversal potential of the peptide-sensitive current was not significantly changed.

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| FIG. 6.
Reversal potentials of the currents suppressed by peptides at different extracellular K+ concentrations. A: 2 × 10 7 M YGGFMRFamide, 4 different fibers; 25°C; B: 2 × 10 10 M proctolin, 3 different fibers; 22°C. ·····, slopes expected from the Nernst equation for a potassium current at the respective temperatures. Double-voltage ramp measurements as shown in Fig. 1 were performed at the K+ concentrations indicated. Reversal potentials were obtained by subtracting the currents measured in the presence of peptide from those recorded under control conditions. KCl-filled recording electrodes were used; chloride equilibrium potentials usually were in the range of 50 to 60 mV.
|
|
FURTHER CHARACTERIZATION OF THE EFFECTS ON IK,H.
Activation measurements for IK,H indicated that YGGFMRFamide consistently reduced the maximal current (Fig. 7). In some cases, the activation curve also was shifted to more negative values. Proctolin too reduced the maximal current, and in one example shifted the activation curve in the negative direction to a similar extent as YGGFMRFamide in the same fiber. Table 2 summarizes the mean effects from six experiments with YGGFMRFamide. These data also should give an indication whether or not the reduction of GK,r can be accounted for entirely by the reduction of GK,H. From the mean changes of the activation parameters of IK,H, an average reduction by only ~0.2 µS is estimated, whereas that observed for the resting conductance and thus for GK,r at EK was 0.33 µS (Table 2). Furthermore, the decrease of the holding current at
55 mV observed on application of YGGFMRFamide was more than twice that which would be expected from the change of the activation parameters of IK,H. Thus the voltage-insensitive component of GK,r probably also is reduced by YGGFMRFamide.

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| FIG. 7.
Effect of YGGFMRFamide on activation of the K+ current, IK,H. Tail currents were measured at 55 mV after activating prepulses to the voltages indicated (cf. METHODS and Fig. 3). Data points are averages from 2 runs in 1 fiber performed in the presence and absence of peptide. Curves indicate fits according to Eq. 1. Inset: Boltzmann function I = Imax × (1/{1 exp[(V V0.5)/S]}) calculated by means of the fit parameters obtained with Eq. 1. These were for control (and for peptide): Imax = 28.2 (12.8) nA; V0.5 = 105.1 ( 109.6) mV; and S = 20.6 (14.8) mV. Circles mark V0.5, i.e., the voltage at half-maximal activation; V0.5 was practically not affected by [K+]o in this experiment in conctrast to the maximal current, which was greatly reduced.
|
|
The instantaneous I-V relationship of the peptide-sensitive K+ current was established by means of voltage jumps performed in the absence and presence of peptide (Fig. 8A). The I-V characteristic was outwardly rectifying and indicated some conductance block at very negative potentials (Fig. 8B). When such measurements were performed from a holding potential close to EK (n = 4; not shown), the I-V curves resembled those of IK,H (outwardly rectifying) (Zittlau and Walther 1991
). Because in that condition
50% of the peptide effect was due to reduction of IK,H, such a similarity is not unexpected provided the I-V characteristic of the voltage sensitive and insensitive component of GK,r are not profoundly different. That this is the case is strongly supported by the nearly identical I-V curves of the peptide-blocked K+ current and IK,H demonstrated under conditions where GK,H was comparatively small (Fig. 8B; also observed in 1 further example).
CONCENTRATION DEPENDENCE OF PEPTIDE EFFECTS.
Figure 9 compares the concentration-response relationships for the reduction of GK,r by YGGFLRFamide (which is as potent as YGGFMRFamide) and proctolin based on ramp measurements. IC50, the concentration for half-maximal reduction of GK,r, was clearly much lower for proctolin (7.8 ± 4.3 × 10
11 M; n = 7) than for YGGFLRFamide (4.8 ± 2.3 10
9 M; n = 8). Proctolin probably also produces a larger maximal effect than YGGFLRFamide. However, our measurements do not allow us to specify the extent of this difference. Thus it was not possible to test saturating concentrations of proctolin. Usually at ~2 × 10
9 M it induced contractures. With YGGFLRFamide at concentrations
10
6 M often a decline in the response occurred, possibly due to desensitization.

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| FIG. 9.
Concentration dependencies of the peptide effects on resting K+ conductance. For both proctolin (circles) and YGGFMRFamide (triangles) data from 2 representative experiments (all from different fibers) are shown. Reduction of membrane resting conductance at V EK was measured by ramp commands as outlined in Fig. 1. It is normalized to the maximal response, which was extrapolated for saturating peptide concentrations by means of the equation: R = Rmax × C/(IC50 + C); R is the conductance-decrease and C is the concentration of peptide. Data from the experiments represented by filled symbols were fitted well by this relation, whereas those represented by open symbols deviate from the curves at low concentrations.
|
|
It should be pointed out that in about one-third of the experiments the peptide effects depended in a more complex manner on concentration than in the examples shown in Fig. 9. Both peptides also affected GK,r and GK,H in fibers from more proximal regions of the jumping muscle yet with a lower potency than in the distal bundles (data not shown). Occasional preparations exhibited an extremely low sensitivity to either peptide.
Intracellular mediation of the peptide effects
INVOLVEMENT OF G PROTEINS.
Many peptide receptors are coupled to G proteins but some are not (e.g., those for atrial natriuretic peptide) (Yuen and Garbers 1992
) and mediate their effects directly. To get some indication whether G proteins are involved in the actions of YGGFMRFamide and proctolin, ionophoretic injections of the nonhydrolyzable analogue GTP-
-S were performed. This nucleotide induces irreversible dissociation of the
subunit of a heterotrimeric G protein and thus renders the effect of agonist-binding more powerful and irreversible (e.g., Gilman 1987
). The latter indeed was observed in nine measurements performed with YGGFMRFamide and three with proctolin (examples are shown in Fig. 10).

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| FIG. 10.
Peptide effects become irreversible after injecting an activator of G proteins into cells. Membrane resting conductance was measured at various times by ramp commands as outlined in Fig. 1. Application of proctolin (2 × 10 10 M) or YGGFMRFamide (2 × 10 7 M), indicated by the thick horizontal bars, initially led to a reversible reduction of membrane conductance. After ionophoretic injection of guanosine 5'-O-(3-thiotriphosphate) tetralithium salt (GTP- -S) for the times indicated by the thin horizontal lines a 2nd application of either peptide lead to an irreversible conductance decrease. Reversal potentials of the peptide-sensitive ramp currents indicated that the peptides specifically reduced a K+ conductance. In 1 of these 2 examples application of GTP- -S by itself lead to a significant conductance decrease. Initial membrane conductance was 1.0 and and 1.4 µS in the cell treated with proctolin and YGGFMRFamide, respectively.
|
|
Injection of GTP-
-S by itself often introduced some reduction of Gr (on average roughly by 15% on injection of 60 nA, pulses for ~30 min; cf. METHODS). The sum of this conductance decrease plus that induced by peptide after GTP-
-S injection was generally greater (on average: by 97 ± 79%, n = 7, for YGGFMRFamide and by 45 ± 20%, n = 3, with proctolin) than the conductance decrease produced by peptide before GTP-
-S injection. Although the extent of this change varied considerably, the effect of both peptides always became irreversible after injection of GTP-
-S (Fig. 10; no recovery during
40 min of washing). In a few experiments, we also tested GDP-
-S, which stabilizes the 

-complex of G proteins. The effects of both peptides were reduced by 30-50% in its presence.
ARE SECOND MESSENGERS INVOLVED IN THE ACTIONS OF THE PEPTIDES?
We screened a variety of drugs known to interfere with second-messenger pathways in other preparations. Although we did not yet arrive at an answer to the question of second messenger involvement, it seems of interest to briefly summarize our findings (in each case based on
2 experiments) and those of others where immediately relevant.
Intracellular Ca2+ is important for both peptides to be effective. We injected the chelator BAPTA ionophoretically up to the point that the fiber could no longer contract (50-nA pulses delivered for 90 min). Although this did not significantly change GK,r, it clearly reduced the effect on GK,r of both proctolin (by 56 ± 15%; n = 4) and YGGFMRFamide (by 88 ± 5.5%; n = 3). There was, however, no requirement for extracellular Ca2+. If Ca2+ was substituted for by Mg2+ and 1 mM EGTA was present, both peptides acted with seemingly undiminished efficiency.
Membrane-permeant analogues of both cAMP and cGMP reduced GK,H (Walther and Zittlau 1998
), but the following observations indicate that these nucleotides do not mediate the actions of either peptide (see also Evans and Myers 1986b
): Both cAMP and cGMP have the additional effect of enhancing the hyperpolarization-activated Cl
conductance (GCl,H) (Walther and Zittlau 1998
). However, as already mentioned, this was not observed with YGGFMRFamide or proctolin, whereas it is a characteristic effect of octopamine (Walther and Zittlau 1998
) that is known to raise the level of cAMP (Evans and Myers 1986a
).
Application of arachidonic acid (e.g., Di-Marzo 1995
) at 10
5 M was without effect on GK,r. Also it was not possible to block the effect of either peptide by 10
5 M 4-bromo-phenacyl bromide, which in other preparations inhibits liberation of arachidonic acid from membrane lipids (e.g., Piomelli et al. 1987
). Thus there is so far no evidence for involvement of eicosanoids, but more work is required to settle this point.
Two activators of protein kinase C (PKC), PMA and PDD, reduced GK,r and GK,H. An analogue, 4
-PDD, which should not activate protein kinase C, acted similarly but was only 10-20% as effective as PDD. The effects of these phorbol esters (2.5 × 10
7 M; n = 6) started 1-2 min after application and built up rather slowly, i.e., within 20-30 min. After this time, the membrane conductance was reduced by some 40%. When GK,r was already reduced by prior application of phorbol esters, the effects on GK,r of YGGFMRFamide and proctolin as well as of Ba2+ were clearly smaller than usual.
There was virtually no recovery within 2 h of washing after such long applications. In the continuous presence of 10
7 M PDD for up to 20 h, there was no fading of the effect as might be expected from a downregulation of PKC (e.g., Crow et al. 1991
). Although phorbol esters thus affect the same conductance as the peptides, it seems unlikely that the peptides act via PKC because their effects were neither blocked nor significantly reduced by kinase blockers H7 (10 µM), sphingosine (10 µM), NGIG-1 [1 µM (Kleinschroth et al. 1993
); this compound blocks PKC in tissue of the bee (U. Müller, personal communication)] or staurosporine (0.1 µM). There are precedents for phorbol ester-effects not mediated via PKC (e.g., Zoukri et al. 1993
).
Proctolin in this preparation, as in various other locust muscles, raises the level of inositol-trisphosphate (IP3) (e.g., Baines et al. 1996
). As a consequence, one may assume, [Ca2+]i rises (e.g. Berridge 1995
). To produce a rise of [Ca2+]i, we applied the Ca2+ ionophore ionomycin (1 µM), which, however, did not lead to a reduction of GK,r (there was a gradual conductance increase and an inward shift of holding current; not analyzed). Furthermore, caffeine, generally known to elevate [Ca2+]i, consistently hyperpolarized the fibers by a few millivolts and increased GK,r (n = 11) (Schiebe and Walther 1989
). Therefore an involvement of IP3 is not very likely.
Thus, although a subpopulation of proctolin receptors is linked to IP3, neither branch of phospholipase C dependent signaling seems to be responsible for the peptide effects on K+ conductance. This conclusion is also strongly supported by the fact that a proctolin analogue, [Afb(p-NO2)2]-proctolin, and proctolin are equipotent in their ability to raise IP3 but differ in their ability to reduce GK,r, the former being ~1,000-fold less effective than the latter (Baines et al. 1996
).
 |
DISCUSSION |
We have further characterized the hyperpolarization-activated K+ conductance, GK,H (Zittlau and Walther 1991
), within limits imposed both by the preparation and the lack of potent, selective blockers for other currents yet under close to physiological conditions. We have shown that both a FMRFamide-related peptide and proctolin reduce the hyperpolarization-activated K+ conductance (GK,H) and the resting K+ conductance (GK,r). Their actions involve G proteins and are mimicked by phorbol esters. However, tests performed with a variety of drugs have not yet answered the question which second messenger(s), if any, provide the links to the K+ channels. No differences between the effects of the two peptides were found except that proctolin acts considerably more potently than YGGFMRFamide.
Comparison of the hyperpolarization-activated K+ current, IK,H, with related currents
There is a hyperpolarization-activated K+ current in crayfish muscle, termed IA,B (Araque and Buño 1991
, 1994
), that shares several properties with IK,H: the underlying conductance, GA,B, like locust GK,H, is highly selective for K+ ions; both currents activate and deactivate with time constants in the range of hundreds (locust) or tens of milliseconds (crayfish) and do not inactivate during maintained hyperpolarization; and their instantaneous I-V relationships do not show inward rectification. However, there are also several dissimilarities: particularly striking is the fact that IA,B is not blocked by Ba2+, Cs+, and Rb+ ions, which effectively block IK,H and fast inward rectifier K+ currents and, with different potencies, also mixed Na+/K+ currents activating slowly on hyperpolarization (Ih-type currents) (Pape 1996
); with physiological [K+]o, GK,H is already activated at EK, whereas activation of GA,B only starts near EK; and if [K+]o is varied, the activation curve of GA,B shifts on the voltage axis along with EK (like in classical inward rectifier K+ currents) (e.g., Leech and Stanfield 1981
), whereas GA,B max is not changed. The voltage dependence of GK,H, however, is not shifted if [K+]o is raised, but GK,H max increases like in classical inward rectifier K+ currents (e.g., Leech and Stanfield 1981
) and Ih-type currents (e.g., Angstadt and Calabrese 1989
; Pape 1996
).
In vertebrates, K+ currents with properties similar to those of IK,H and IA,B have so far only been described in collecting ducts of rat kidney (Wang et al. 1994
; personal communication) and in a neuro-glial-cell line (Wischmeyer and Karschin 1997
). In plant plasma membranes, phenomenologically similar currents flow through K+ channels closely related to the shaker K+ channel superfamily (Schroeder et al. 1994
). In vertebrate skeletal muscles, "classical" K+ inward rectifiers (e.g., Leech and Stanfield 1981
) are present instead of currents such as IK,H and IA,B, whereas in arthropod muscles, to our knowledge, fast inward rectifier K+ currents so far have not been detected although they do exist, for example, in molluscan muscles (Brezina et al. 1994
).
What accounts for the resting K+ conductance?
Little is known about the K+ channels that are responsible for the resting K+ conductance (GK,r) in arthropod skeletal muscle. In Drosophila, however, a recently detected K+ "leak" channel with a purely ohmic characteristic probably plays this role in neuromuscular tissues (Goldstein et al. 1996
). In a marine crustacean, Idothea, again nonvoltage-dependent K+ channels were found to contribute to GK,r (Erxleben et al. 1995
). In the locust preparation, quantitative comparisons of total resting conductance and GK,H led us to propose that GK,H accounts only for part of GK,r and that there is a substantial amount of K+ conductance, which, except for the voltage dependence, is similar to GK,H. Interestingly in crayfish muscle, there also seems to be a substantial fraction of the resting K+ conductance, which may not be accounted for by GA,B (cf. preceding section) although it is blocked, like GA,B, by Cd2+ ions (Araque et al. 1995
).
Modulation of GK,H by (YGG)FMRFamide and proctolin
A multiple modulation of the resting K+ conductance by peptides or biogenic amines, often involving more than one type of internal signaling pathway, is not uncommon in vertebrates and invertebrates, e.g., in inward rectifiers (Benson and Levithan 1983
; Nakajima et al. 1988
; Tatsumi et al. 1990
), background K+ currents (e.g., Brezina et al. 1987
) or agonist-gated K+ currents (e.g., Brown 1988
; Sasaki and Sato 1987
). The peptides (YGG)FMRFamide and proctolin as well as the biogenic amine octopamine (Walther and Zittlau 1998
) all cause a reduction of GK,r. Their efficacy differs according to the sequence proctolin > YGGFMRFamide > octopamine. They all affect the activation curve by a consistent reduction of Gmax and by a variable shift on the voltage axis. Interestingly in muscle of the crustacean Idothea, proctolin was found to lower the resting conductance by reducing the number of functionally active, voltage-independent K+ channels, an effect mimicked by cAMP (Erxleben et al. 1995
). FMRFamide and related peptides also reduce a background conductance in a shrimp muscle (Meyrand and Marder 1991
), whereas in snail neurons, by contrast, it enhances a background K+ current (Brezina et al. 1987
).
Physiological consequences of the modulation of resting K+ conductance
A reduction of resting K+-conductance raises membrane excitability. The amplitudes of excitatory junction potentials (ejps) should be somewhat enhanced. In addition, as a consequence of the longer membrane time constant and as previously demonstrated with YGGFMRFamide (Walther and Schiebe 1987
), the decay phases of the ejps are prolonged. Obviously this will lead to more efficient summation of ejps. In turn, action potentials are more likely to be generated by activation of Ca2+ currents (Pichon and Ashcroft 1985
). These, however, seem also to be potentiated due to a direct modulation by both proctolin and YGGFMRFamide (as well as by octopamine) (Walther and Zittlau, unpublished findings).
It is uncertain whether the decrease of GK,r by itself leads to a depolarization. In vitro this is certainly not the case because the resting potential is practically at EK. However, should there be some tonic depolarizing current in vivo, its impact of course would increase on reduction of GK,r. One substance that might be considered to cause such a current would be glutamate, the excitatory neuromuscular transmitter (Usherwood and Cull-Candy 1975
), although reports that glutamate is present in locust hemolymph at a sufficiently high concentration (e.g., Clements and May 1974
) were contradicted by a subsequent investigation (Irving et al. 1976
).
Although octopamine, in the jumping muscle, seems to be mainly involved in supporting vigorous fast contractions produced by activity of the fast excitatory motoneuron, FETi (e.g., Burrows and Pflüger 1995
), FMRFamide-peptides and proctolin may be more relevant for tuning the effects produced by the slow excitatory neuron, SETi. Both peptides lead to an increased force of single contractions (e.g., Evans and Myers 1986a
), yet they have opposite effects on the time course of contractions. Proctolin prolongs their decay and supports tonic force development at least in other locust muscles (ventral abdominal muscle, Facciponte et al. 1996
; mandibular closer muscle, Baines, personal communication) like first demonstrated in cockroach coxal muscle (Adams and O'Shea 1983
), while FMRFamide-peptides shorten the decay and thus improve performance of the muscle in walking (Evans and Myers 1986b
). These alterations of the contractions seem to be largely due to direct effects on excitation-contraction coupling.
Recently, for a muscle of Aplysia, Brezina et al. (1996)
modeled the whole range of contraction-forms observed in the presence of varying ratios of two neuropeptides. These peptides modulate both a K+ current and the relaxation rate though not to the same extent. In locust jumping muscle, there is not yet the large body of data required for such an approach and the situation is probably more complicated. It may, however, be hypothesized that the reduction of GK,r in the case of proctolin is suitable to support the described prolongation of contractions because its main effect on the ejps is their prolongation (as observed on recording from weakly contracting fibers at reduced extracellular [Ca2+]) (Walther, unpublished observations). On the other hand, FMRFamide peptides in addition enhance the amplitudes of excitatory synaptic potentials through a presynaptic effect not observed with proctolin (e.g., Walther et al. 1991
). Hence FMRFamide peptides should increase the likelihood that a Ca2+ current will be activated, which in turn will lead to immediate activation of a powerful Ca2+ activated K+ current [unpublished findings, similar to those in crayfish muscle (Araque and Buño 1995
)]. Thus the total duration of a depolarization initiated in a muscle fiber by one presynaptic action potential should be considerably shortened, an effect which may contribute to the more phasic contractions observed in the presence of FMRFamide peptides.