SUMMARY1. Whole-cell or single-channel currents through acetylcholine (ACh) receptor channels were studied in voltage-clamped rat myoballs or in excised membrane patches from myoballs. The recording pipette contained CsC1 to suppress outward currents, and tetrodotoxin was used to help suppress Na+ currents.2. To minimize problems associated with bath applied agonists, myoballs were bathed in a solution containing the inactive (cis) isomer of the photo-isomerizable azobenzene derivative, Bis-Q. Calibrated light flashes of varying intensity were presented to produce concentration jumps of agonist, trans-Bis-Q. The resulting whole-cell current relaxations through ACh channels approach a steady state along an exponential time course, then decline as the newly created agonist diffuses away over the next few seconds.3. The dose-response relationship was inferred from Hill (double-log) plots for myoballs bathed in 500 nM-cis-Bis-Q at three membrane potentials. At low agonist concentrations (< 300nM-trans-Bis-Q), the slope of the Hill plot averaged 1'62 at -150 mV, 1-89 at -100 mV, and 2-05 at + 80 mV. These results are consistent with an apparent agonist affinity constant that decreases with membrane depolarization and shifts the responses further down on the dose-response curve.4. When the myoballs were bathed in higher concentrations ofcis-Bis-Q (1 -5-20 /LM), the slope of the Hill plot was reduced at all membrane potentials, although it was still closer to two at positive potentials. This is expected from the known sigmoid shape of the dose-response relation. The shallow dependence of the Hill slope on agonist concentration suggests the presence of negative cooperativity in the over-all binding of agonist molecules.5. Following treatment of the membrane with dithiothreitol to reduce disulphide groups, the Hill slope for the reversibly bound agonist, trans-Bis-Q, remained near two.6. The kinetics of currents at hyperpolarized membrane potentials became complicated at higher agonist concentrations in a manner that was consistent with L. D. CHABALA, A. M. GURNEY AND H. A. LESTERopen-channel block by tranw-Bis-Q; the currents showed a slow secondary increase in conductance.7. Averaged single-channel recordings at higher agonist concentrations resemble macroscopic relaxations under comparable conditions. Furthermore, those recordings also suggested that open channels are blocked by trans-Bis-Q at concentrations > 2,um; the block depends strongly on membrane potential and increases with hyperpolarization. Currents at positive membrane potentials showed no evidence of open-channel block.8. In the presence of 20 tM-Ci8-Bis-Q, the probability of a channel being open was constant from one episode to the next for up to several hundred flash-induced agonist concentration jumps (0 to 10 6/tM-trans-Bis-Q).9. Fractional activation of single ion channels at the highest agonist concentration tested (20 1M-cis-Bis-Q) was small, which agrees with the observation that whole-cell currents, under similar conditions, are still in the low...

SUMMARY1. K+ currents were studied at a normal (-69 mV) and at a depolarized (-49 mV) membrane potential in voltage-clamped squid giant axons perfused with 350 mM-K+ and bathed in K+-free artificial sea water containing tetrodotoxin to block the Na+ channels.2. Steady-state and instantaneous K+ currents were reduced by over 50 % at corresponding voltages at the depolarized membrane potential. Instantaneous chord conductance-voltage curves showed that the depolarized membrane potential caused a uniform reduction of K+ conductance across the voltage range under study.3. The driving force for K+ ions was comparable at both membrane potentials when a short (2 ms) pre-pulse was used to open the K+ channels. When a longer (7-5 ms) pre-pulse was used, the driving force was actually larger at the depolarized membrane potential.4. The depolarized membrane potential did drive some K+ ions into the periaxonal space. The amount of K+ ions driven into the periaxonal space was estimated by two independent methods, with similar results. The resulting increase of K+ ions in the periaxonal space (10 mM) was about 40 times too small to account for the large reduction in currents in terms of a reduced driving force for K+ ions.5. The kinetics of recovery and development of inactivation were monitored by repeatedly applying a 7-5 ms test pulse followed by a long conditioning potential. Both recovery and development of inactivation, from the depolarized membrane potential, were described by the sum of two exponential terms plus a constant.6. The time constant-voltage curves for both phases of inactivation peaked at about -54 mV at 10 'C. The time constant of the slow phase of inactivation at -54 mV was about 12-4 s, while the corresponding time constant for the fast phase was about 2-3 s. The slow relaxation had an apparent plateau of about 11 s at more depolarized membrane potentials. Recovery from inactivation was rapid at hyperpolarized membrane potentials. where aij = exp (Aij Vm + B0l). If the K+ channels form a homogeneous population of identical and independent channels, then the asymmetry in the steady-state inactivation curve suggests that all three states are open and that S2 has a low relative conductance associated with it. In the second version, two populations of K+ channels are assumed, one with and one without an inactivation mechanism. In this latter instance, the shape of the steady-state inactivation curve suggests that only the first two states are open, and the non-inactivating population of K+ channels accounts for the incomplete steady-state inactivation curve.

A B S T R A C TVoltage-dependent K + channels control repolarization of action potentials and help establish firing patterns in nerve cells. To determine the nature and role of molecular components that modulate K + channel function in vivo, we coinjected Xenopus oocytes with cRNA encoding a cloned subthreshold A-type K + channel (mShall, also referred to as mKv4.1) and a low molecular weight (LMW) fraction (2-4 kb) of poly(A) ÷ mRNA (both from rodent brain). Coinjected oocytes exhibited a significant (fourfold) increase in the surface expression of mShall K ÷ channels with no change in the open-channel conductance. Coexpression also modified the gating kinetics of mShall current in several respects. Macroscopic inactivation of whole oocyte currents was fitted with the sum of two exponential components. Both fast and slow time constants of inactivation were accelerated at all membrane potentials in coinjected oocytes (rf = 47.2 ms vs 56.5 ms at 0 mV and % = 157 ms vs 225 ms at 0 mV), and the corresponding ratios of amplitude terms were shifted toward domination by the fast component (Af/As = 2.71 vs 1.17 at 0 mV). Macroscopic activation was characterized in terms of the time-to-peak current, and it was found to be more rapid at all membrane potentials in coinjected oocytes (9.9 ms vs 13.5 ms at 0 mV). Coexpression also leads to more rapid recovery from inactivation ( ~ 2.4-fold faster at -100 mV). The coexpressed K + currents in oocytes resemble currents expressed in mouse fibroblasts (NIH3T3) transfected only with mShall cDNA. These results indicate that mammalian regulatory subunits or enzymes encoded by LMW mRNA species, which are apparently missing or expressed at low levels in Xenopus oocytes, may modulate gating in some native subthreshold A-type K ÷ channels.

SUMMARY1. Kinetic and equilibrium aspects ofreceptor activation by two irreversibly bound ('tethered') agonists, QBr and bromoacetylcholine (BrACh), were examined in cultured embryonic rat muscle. Myoballs were treated with dithiothretitol (2 mM), washed, exposed to BrACh or QBr, and then washed again. Voltage-clamp recordings were made both in the whole-cell mode and with excised outside-out patches at 15 'C.2. Whole-cell voltage-jump relaxations resembled those observed with reversibly bound agonists. The relaxation time constants were 5 ms for tethered QBr and 10 ms for tethered BrACh (-100 mV, 15 TC). At more positive membrane potentials, the relaxation rate constants increased and the conductance decreased.3. Whole-cell light-flash relaxations with tethered QBr were also studied. The conductance was increased and decreased, respectively, by cis--etrans and trans--cis photoisomerizations. The relaxation time constants equalled those for voltage jumps.4. The functional stoicheiometry of tethered QBr was investigated by studying the relaxations in response to light flashes that produced known changes in the mole fractions of the two isomers. It is concluded that the open state of each receptor channel is controlled by the isomeric state of a single tethered QBr molecule.5. In single-channel recordings, tethered agonists opened channels with the same conductance as reversibly bound agonists (30 pS at 15 'C and -100 mV). More than 80 % of the conductance was contributed by a population of openings with an average burst duration (lifetime) of5 ms for QBr and 10 ms for BrACh. Thus the single-channel and macroscopic currents seem to be dominated by the same type of channel; these are presumably monoliganded receptors.6. About 300 of the openings belonged to a population with an average lifetime of about 0 5 ms. This population contributed less than 50 of the conductance. There were also more long openings (> 50 ms) than expected from a simple exponential distribution. A few patches from BrACh-treated cells showed openings with a conductance of 45 pS (-100 mV) and an average duration of -2 ms.7. These data allow one to assess whether the agonist-receptor binding step plays

The photochemical properties of the azobenzene derivative, Bis-Q, were exploited to carry out an agonist concentration jump followed by a molecular rearrangement of bound agonist molecules at acetylcholine (ACh) receptor channels of voltage-clamped rat myoballs. Myoballs were bathed in solutions containing low concentrations of cis-Bis-Q, the inactive isomer. Whole-cell current relaxations were studied following a light flash that produced a concentration jump of agonist, trans-Bis-Q, followed by a second flash that produced net trans----cis photoisomerizations of Bis-Q molecules. The concentration-jump relaxation provided a measure of the mean burst duration for ACh receptor channels occupied by trans-Bis-Q (7.7 ms, 22 degrees C). The second current relaxation was a more rapid conductance decrease (phase 1, tau = 0.8 ms). Phase 1 may represent either the burst duration for receptors initially occupied by a single cis- and a single trans-Bis-Q molecule or that for unliganded receptors. Single-channel current recordings from excised outside-out membrane patches showed that single channels open following an agonist concentration jump comparable to that used in the whole-cell experiments; when many such records were averaged, a synthetic macroscopic relaxation was produced. Individual open channels closed faster following a flash that promoted trans----cis photoisomerizations of the bound ligand, thus confirming the whole-cell observations of phase 1.