In freshly dispersed guinea pig taenia coli myocytes the activity of the large conductance Ca(2+)-activated K+ channel (maxi-K+ channel) predominates. The open probability (Po) of this channel is increased by micromolar concentrations of the beta-adrenergic agonist isoproterenol (ISO). Low concentrations of cholera toxin (CTX, 1 pM) and guanosine 5'-O-2-thiodiphosphate (GDP beta S, 0.5 mM) suppress the ISO-induced increase of Po. Higher concentrations of CTX (e.g., 0.5 nM) as well as forskolin and dibutyryl cAMP increase the Po. 1,9-Dideoxyforskolin, the forskolin analogue, which lacks the adenylate cyclase-stimulating effect, does not. A specific protein kinase A inhibitor (Wiptide), applied intracellularly via diffusion from the patch electrode, suppresses the ISO-induced increase of whole-cell outward K+ current during step depolarization. In contrast, intracellularly applied protein kinase C (19-36), a specific protein kinase C inhibitor, has no effect on the whole-cell current. TMB-8, an inhibitor of intracellular calcium mobilization, does not affect either the whole-cell outward K+ current during step depolarization or the Po. These observations show that ISO increases the Po of the maxi-K+ channels in the guinea pig taenia coli myocytes through the G protein-adenylate cyclase-protein kinase A system.

Chiriquitoxin (CqTX) from the Costa Rican frog Atelopus chiriquensis differs from tetrodoxin (TTX) only in that a glycine residue replaces a methylene hydrogen of the C-11 hydroxymethyl function. On the voltage-clamped frog skeletal muscle fiber, in addition to blocking the sodium channel and unrelated to such an action, CqTX also slows the activation of the fast potassium current in approximately 40% of the muscle fiber population. At pH 7.25, CqTX is as potent as TTX in blocking the sodium channel, with an ED50 of 3.8 nM. Its ED50's at pH 6.50 and 8.25 are 6.8 and 2.3 nM, contrasted with 3.8 and 4.3 nM for TTX. These differences are attributable to changes in the chemical states in the glycine residue. The equipotency of CqTX with TTX at pH 7.25 is explainable by an intramolecular salt bridge between the amino and carboxyl groups of the glycine function, all other surface groups in TTX and CqTX being the same. From available information on these groups and those in saxitoxin (STX), the TTX/STX binding site is deduced to be in a pocket 9.5 A wide, 6 A high, and 5 A deep. The glycine residue of CqTX probably projects out of the entrance to this pocket. Such a view of the binding site could also account for the actions of STX analogues, including the C-11 sulfated gonyautoxins and the 21-sulfocarbamoyl analogues. In the gonyautoxins the sulfate groups are equivalently placed as the glycine in CqTX, whereas in the sulfocarbamoyl toxins the sulfate groups extend the carbamoyl side-chain, leading to steric hinderance to productive binding.

Neosaxitoxin (neoSTX) differs structurally from saxitoxin (STX) in that the hydrogen on N-1 is replaced by a hydroxyl group. On single frog skeletal muscle fibers in the vaseline-gap voltage clamp, the concentrations for reducing the maximum sodium current by 50% (ED50) at pH's 6.50, 7.25, and 8.25 are, respectively, 4.9, 5.1, and 8.9 nM for STX and 1.6, 2.7, and 17.2 nM for neoSTX. The relative potencies of STX at the different pH's closely parallel the relative abundance of the protonated form of the 7,8,9 guanidinium function, but the relative potencies of neoSTX at the same pH's vary with the relative abundance of the deprotonated N-1 group. In constant-ratio mixtures of the two toxins, the observed ED50's are consistent with the notion that the two toxins compete for the same site. At pH's 6.50 and 7.25, the best agreement between observed and computed values is obtained when the efficacy term (epsilon) for either toxin is 1. At pH 8.25 the best agreement is obtained if the efficacy is 1 for STX but 0.75 for neo-STX. The marked pH dependence of the actions of neoSTX probably reflects the presence of a site in the receptor that interacts with the N-1 -OH, in addition to those interacting with the 7,8,9 guanidinium and the C-12 hydroxyl groups. Considering the three-dimensional structure of the STX and neoSTX molecules, the various site points are probably located in a fold or a crevice of the channel protein, where the extracellular orifice of the sodium channel is located.

Currents through single potassium channels were studied in cell-attached or inside-out patches from collagenase-dispersed smooth muscle cells of the guinea pig taenia coli. Under conditions mimicking the physiological state with [K+]i = 135 mM: [K+]o = 5.4 mM, three distinct types of K+ channel were identified with conductances around 0 mV of 147, 94, and 63 pS. The activities of the 94- and 63-pS channel were observed infrequently. The 147-pS channel was most abundant. It has a reversal potential of approximately -75 mV. It is sensitive to [Ca2+]i and to membrane potential. At -30 mV, the probability of a channel being open is at a minimum. At more positive voltages, the probability follows Boltzman distribution. A 10-fold change in [Ca2+]i causes a 25-mV negative shift of the voltage where half of the channels are open; an 11.3-mV change in membrane potential produces an e-fold increase in the probability of the channel being open when P is low. At voltages between -30 and -50 mV, the open probability increases in an anomalous manner because of a large decrease of the channel closed time without much change in the channel open time. This anomalous activity may play a regulatory role in maintaining the resting potential. The histograms of channel open and closed time fit well, respectively, with single and double exponential distributions. Upon step depolarizations by 100-ms pulses, the 147-pS channel opens with a brief delay. The delay shortens and both the number of open channels and the open time increase with increasing positivity of the potential. The averaged currents during the step depolarizations closely resemble the delayed rectifying outward K+ currents in whole-cell recordings.

The permeation properties of the 147-pS Ca2+-activated K+ channel of the taenia coli myocytes are similar to those of the delayed rectifier channel in other excitable membranes. It has a selectivity sequence of K+ 1.0 greater than Rb+ 0.65 greater than NH4+ 0.50. Na+, Cs+, Li+, and TEA+ (tetraethylammonium) are impermeant. Internal Na+ blocks K+ channel in a strongly voltage-dependent manner with an equivalent valence (zd) of 1.20. Blockade by internal Cs+ and TEA+ is less voltage dependent, with d of 0.61 and 0.13, and half-blockage concentrations of 88 and 31 mM, respectively. External TEA+ is about 100 times more effective in blocking the K+ channel. All these findings suggest that the 147-pS Ca2+-activated K+ channel in the taenia myocytes, which functions physiologically like the delayed rectifier, is the single-channel basis of the repolarizing current in an action potential.

Using the tight-seal voltage-clamp method, the ionic currents in the enzymatically dispersed single smooth muscle cells of the guinea pig taenia coli have been studied. In a physiological medium containing 3 mM Ca2+, the cells are gently tapering spindles, averaging 201 (length) x 8 microns (largest diameter in center of cell), with a volume of 5 pl. The average cell capacitance is 50 pF, and the specific membrane capacitance 1.15 microF/cm2. The input impedance of the resting cell is 1-2 G omega. Spatially uniform voltage-control prevails after the first 400 microseconds. There is much overlap of the inward and outward currents, but the inward current can be isolated by applying Cs+ internally to block all potassium currents. The inward current is carried by Ca2+. Activation begins at approximately -30 mV, maximum ICa occurs at +10-+20 mV, and the reversal potential is approximately +75 mV. The Ca2+ channel is permeable to Sr2+ and Ba2+, and to Cs+ moving outwards, but not to Na+ moving inwards. Activation and deactivation are very rapid at approximately 33 degrees C, with time-constants of less than 1 ms. Inactivation has a complex time course, resolvable into three exponential components, with average time constants (at 0 mV) of 7, 45, and 400 ms, which are affected differently by voltage. Steady-state inactivation is half-maximal at -30 mV for all components combined, but -36 mV for the fast component and -26 and -23 mV for the other two components. The presence of multiple forms of Ca2+ channel is inferred from the inactivation characteristics, not from activation properties. Recovery of the fast channel occurs with a time-constant of 72 ms (at +10 mV). Ca2+ influx during an action potential can transfer approximately 9 pC of charge, which could elevate intracellular Ca2+ concentration adequately for various physiological functions.

In single myocytes of the guinea pig taenia coli, dispersed by enzymatic digestion, the late outward current is carried by K+. It has both a Ca2+-activated component and a voltage-dependent component which is resistant to external Co2+. The reversal potential is -84 mV, and the channel(s) for it are highly selective to K+. At 33 degrees C, the activation follows n2 kinetics, with a voltage-dependent time constant of 10.6 ms at 0 mV, which shortens to 1.7 ms at +70 mV. Deactivation follows a single-exponential time course, with a voltage-dependent time constant of 11 ms at -50 mV, which lengthens to 33 ms at -20 mV. During a 4.5-s maintained depolarization, IK inactivates, most of it into two exponential components, but there is a small noninactivating residue. It is surmised that during an action potential under physiological conditions, there is sufficient IK to cause repolarization.

With the use of a point voltage-clamp technique, the effects of Zn 2+ , UO 2 2+ , tetraethylammonium, and several other homologous quaternary ammonium ions on the electrical properties of the frog sartorius muscle and its mechanical threshold were studied. None of the agents separated the voltage thresholds for mechanical activation and delayed rectification. However, Zn 2+ , UO 2 2+ , and TEA, which are known to potentiate the twitch, caused some inhibition of the normal increase in potassium conductance during delayed rectification. Zn 2+ and UO 2 2+ also slowed the rate of development of the outward current. A strength-duration relation was studied for depolarization pulses capable of initiating contraction. With a depolarizing pulse of 2.5 msec the mechanical threshold is about -13 mv at about 20°C. UO 2 2+ , 0.5 µ M , which markedly reduced the outward current produced by such a short pulse, did not raise the mechanical threshold. All findings indicate that there is no direct causal relation between delayed rectification and mechanical activation.

The electrical activities of myometrial cells of the pregnant rabbit uterus have been studied by means of sucrose-gap and intracellular micro-electrode recording techniques. The resting potential of the myometrial cell was about -50 mv, and it is unaffected by the duration of pregnancy or placental attachment. Action potentials of the myometrium, although dependent on external Na + , were not always of the regenerative type; preparations from nonparturient uteri often produce mainly small spikes. The mean spike amplitude was 35 mv, rising at a mean maximum rate of 3 v/sec. Oxytocin, in concentrations less than 500 µU/ml, increased the mean spike amplitude to 48 mv and the mean maximum rate of rise to 7 v/sec, without affecting the resting potential. The relation between membrane potential and dV/dt of the spike was steepened by oxytocin, suggesting that oxytocin increased the number of normally sparse sodium gates in the myometrial membrane. By this action, oxytocin is believed to increase the probability of successful regenerative spikes and thereby initiate electrical activity in quiescent preparations, increase the frequency of burst discharges, the number of spikes in each burst, and the amplitude of spikes in individual cells.

Tarichatoxin, isolated from California newt eggs, has been found to selectively block the increase of sodium conductance associated with excitation in lobster giant axons at nanomolar concentrations. This resulted from a reduction in the amplitude of the conductance increase rather than a change in its temporal characteristics. The normal potassium conductance increase with depolarization is not altered. A high concentration of calcium applied concomitantly with the toxin significantly improves the reversibility of the sodium blocking. This toxin has recently been identified as chemically identical with tetrodotoxin from the puffer fish. Toxins from the two sources are equally effective and are shown to have an action which is distinctly different from that of procaine.

Electrical constants of the plasma membrane of the Fundulus egg have been measured with microelectrodes by the transient method. No consistent and significant membrane potential was measured. Membrane capacity averages 0.63 µF/cm. 2 for both unactivated and activated eggs. Membrane resistance averages 3450 ohm-cm. 2 in the unactivated eggs, but increases 2 to 7 times to an average of 13,290 ohm-cm. 2 in the fully activated state. In a hypertonic sucrose solution, the swelling of the egg proper is accompanied by a rapid fall of membrane resistance towards that in the unactivated state. The changes of the membrane resistance are interpreted as probably caused by alterations in the effective pore size in the plasma membrane.

Upon activation, an internal hydrostatic pressure develops within the Fundulus egg, and compresses the egg proper to a reduced volume. When the perivitelline pressure is abolished by a highly hypertonic sucrose solution, the egg volume increases. As sucrose penetrates the chorion, the volume again decreases. The relation between P and V in these conditions is inverse, and approximates a rectangular hyperbola. The limiting factor causing most of the deviation is shown to be the incompressible fraction. It is concluded that the volume of the egg proper is controlled by the perivitelline pressure, and that the effect of hypertonic sucrose solution is exerted by lowering the pressure and thereby increasing membrane permeability non-specifically. It is also shown that some permanent alterations occur within the plasma membrane during activation that reduce the permeance, and thereby, increase the incompressible fraction.

1. A technique is described for recording the bioelectric activity of the squid giant axon during and following alteration of the internal axonal composition with respect to ions or other substances. 2. Experimental evidence indicates that the technique as described is capable of measuring changes in local bioelectric activity with an accuracy of 10 to 15 per cent or higher. 3. Alterations of the internal K + or Cl - concentrations do not cause the change in resting potential expected on the basis of a Donnan mechanism. 4. The general effect of microinjection of K + Rb + , Na + , Li + , Ba ++ , Ca ++ , Mg ++ , or Sr ++ is to cause decrease in spike amplitude, followed by propagation block. 5. The resting potential decreases when the amplitude of the spike becomes low and block is incipient. 6. The decrease in resting potential and spike amplitude may be confined to the immediate vicinity of the injection. 7. At block, the resting potential decreases up to 50 per cent, but injection of small quantities of divalent cations may cause much larger localized depolarization. 8. The blocking effectiveness of K + , Na + , and Ca ++ expressed as reciprocals of the relative amounts needed to cause block is approximately 1:5:100. Rb + has the same low effectiveness as does K + . Li + resembles Na + . Ba ++ and Mg ++ are approximately as effective as Ca ++ . 9. Microinjection of Na + may cause marked prolongation of the spike at the injection site as well as decrease in its amplitude. 10. The anions used (Cl - , HCO 3 - , NO 3 - , SO 4 - , aspartate, and glutamate) do not seem to exert specific effects. 11. A tentative explanation is offered for the insensitivity of the resting potential to changes in the axonal ionic composition. 12. New data are presented on the range of variation, in a large sample, of the magnitude of the resting potential and spike amplitude.