Hodgkin-Huxley analysis of a GCAC1 anion channel in the plasma membrane of guard cells

A quantitative analysis of the time- and voltage-dependent kinetics of the guard cell anion channel (GCAC1) current in guard cell protoplasts from Vicia faba was analyzed using the whole-cell patch clamp technique. The voltage-dependent steady-state activation of GCAC1 current followed a Boltzmann distribution. For the corresponding steady-state value of the activation variable a power of two was derived which yielded suitable fits of the time course of voltage-dependent current activation. The GCAC1 mediated chloride current could successfully be described in terms of the Hodgkin-Huxley equations commonly evoked for the Na channel in nerve. After step depolarizations from a potential in the range of the resting potential to potentials above the equilibrium potential for chloride an activation and also an inactivation could be described. The gating of both processes exhibited an inverse relationship on the polarity of the applied step potentials in the order of milliseconds. Deactivating tail currents decline exponentially. The presented analysis contributes to the understanding of the rising phase of the observed action potentials in guard cells of V. faba. Evidence is presented that the voltage-dependent kinetic properties of the GCAC1 current are different from those properties described for the excitable anion currents in the plasmalemma of Chara corallina (Beilby & Coster, 1979a).The authors gratefully acknowledge the encouragement of Dr. David Colquhoun to apply the Hodgkin-Huxley model to the GCAC1 channel. The work was in part supported by a grant of the Deutsche Forschungsgemeinschaft to R.H. and a grant of the Herman and Lilly Schilling Stiftung to H.-A.K.     

Thiamine triphosphate activates an anion channel of large unit conductance in neuroblastoma cells

In neuroblastoma cells, the intracellular thiamine triphosphate (TTP) concentration was found to be about 0.5 m, which is several times above the amount of cultured neurons or glial cells. In inside-out patches, addition of TTP (1 or 10) m to the bath activated an anion channel of large unit conductance (350–400 pS) in symmetrical 150 mm NaCl solution. The activation occurred after a delay of about 4 min and was not reversed when TTP was washed out. A possible explanation is that the channel has been irreversibly phosphorylated by TTP. The channel open probability (P _o) shows a bell-shaped behavior as a function of pipette potential (V _p). P _o is maximal for –25 mV<V _p<10 mV and steeply decreases outside this potential range. From reversal potentials, permeability ratios of P_Cl/ P_Na = 20 and P_Cl/P_gluconate = 3 were estimated. ATP (5 mm) at the cytoplasmic side of the channel decreased the mean single channel conductance by about 50%, but thiamine derivatives did not affect unit conductance; 4,4 -diisothiocyanostilbene-2,2-disulfonic acid (0.1 mm) increased the flickering of the channel between the open and closed state, finally leading to its closure. Addition of oxythiamine (1 mm), a thiamine antimetabolite, to the pipette filling solution potentiates the time-dependent inactivation of the channel at V _p=–20 mV but had the opposite effect at +30 mV. This finding corresponds to a shift of P _o towards more negative resting membrane potentials. These observations agree with our previous results showing a modulation of chloride permeability by thiamine derivatives in membrane vesicles from rat brain.We would like to thank the National Funds for Scientific Research (Belgium) for financially supporting the stay of L.B. in Konstanz. We wish to thank A. Ngezahayo, F. Mendez and Dr. P. Wins for helpful discussions. This work was in part supported by a research grant from the Fonds special pour la Recherche à l'Université de Liege to L.B., the SFB 156 of the DFG and a grant of the Hermann and Lilly Schilling Stiftung to H.-A.K. Neuroblastoma, PC-12 and glioma cell lines were a gift from Prof. G. Moonen (Department of Human Physiology, University of Liège).     

Mutations in the S4 region isolate the final voltage-dependent cooperative step in potassium channel activation

Charged residues in the S4 transmembrane segment play a key role in determining the sensitivity of voltage-gated ion channels to changes in voltage across the cell membrane. However, cooperative interactions between subunits also affect the voltage dependence of channel opening, and these interactions can be altered by making substitutions at uncharged residues in the S4 region. We have studied the activation of two mutant Shaker channels that have different S4 amino acid sequences, ILT (V369I, I372L, and S376T) and Shaw S4 (the S4 of Drosophila Shaw substituted into Shaker), and yet have very similar ionic current properties. Both mutations affect cooperativity, making a cooperative transition in the activation pathway rate limiting and shifting it to very positive voltages, but analysis of gating and ionic current recordings reveals that the ILT and Shaw S4 mutant channels have different activation pathways. Analysis of gating currents suggests that the dominant effect of the ILT mutation is to make the final cooperative transition to the open state of the channel rate limiting in an activation pathway that otherwise resembles that of Shaker. The charge movement associated with the final gating transition in ILT activation can be measured as an isolated component of charge movement in the voltage range of channel opening and accounts for 13% ( approximately 1.8 e0) of the total charge moved in the ILT activation pathway. The remainder of the ILT gating charge (87%) moves at negative voltages, where channels do not open, and confirms the presence of Shaker-like conformational changes between closed states in the activation pathway. In contrast to ILT, the activation pathway of Shaw S4 seems to involve a single cooperative charge-moving step between a closed and an open state. We cannot detect any voltage-dependent transitions between closed states for Shaw S4. Restoring basic residues that are missing in Shaw S4 (R1, R2, and K7) rescues charge movement between closed states in the activation pathway, but does not alter the voltage dependence of the rate-limiting transition in activation.     

Characterization of ion channels in the plasma membrane of epidermal cells of expanding pea (Pisum sativum arg) leaves

Ion channels in isolated patches of the plasma membrane of pea (Pisum sativum arg) epidermal cells were studied with the patch-clamp technique. One anion and one cation channel were dominantly present in most trials. The anion channel conducts nitrate, halides and malate, with a conductance in symmetrical 100 mm Cl^– of 300 pS and can be blocked by SITS when applied to the cytoplasmic side of the membrane. The cation channel poorly discriminates between potassium, sodium and lithium, is not blocked by either TEA or Ba²⁺, and has a conductance of 35 pS in symmetrical 100 mm K⁺. The open probability of the cation channel increases with increase of the Ca²⁺ concentration on the cytoplasmic side of the membrane from 0.1 to 1 m. The possible role of these two channels in the physiology of epidermal cells is discussed.This work was supported by NSF grant DCB-890 3744 to E.V.     

Patch clamp analysis of the dominant plasma membrane K+ channel in root cell protoplasts of Plantago media L. Its significance for the P and K state

Ion channels in the plasma membrane of root cell protoplasts of Plantago media L. were studied with the patch clamp technique in the cell-attached patch and outside-out patch configuration. An outward rectifying potassium channel was dominantly present in the plasma membrane. It appears responsible for the diffusional part, dominated by the K⁺ diffusion potential, of the cell membrane potential, in vivo. This channel is activated at potentials near to and more positive than the K⁺ diffusion potential. The dependence of this ion channel on K⁺ activity and voltage has been characterized. The current-voltage relationships of the open channel at various K⁺ concentrations are described by a four-state model. The membrane potential of intact protoplasts appears either dominated by the K⁺ diffusion potential, the protoplast is then said to be in the K state, or by the pump potential generated by the plasma membrane-bound proton pump/H⁺ ATPase, the P state. An experimental procedure is described to determine in cell-attached patch mode the state of the protoplast, either K or P state.Institution paper no.: ECOTRANS publication no. 45.     

Demonstration and characterization of Ca2+ channel in tonoplast-free cells ofNitellopsis obtusa

Summary The presence of a Ca²⁺ channel in the plasmalemma of tonoplast-freeNitellopsis obtusa cells was demonstrated and its characteristics were studied using current- and voltage-clamp techniques. A long-lasting inward membrane current (I _m ), recorded using a step voltage clamp, consisted of a single component without time-dependent inactivation. Increasing either [Ca²⁺] _o or [Cl^–] _o 1) enhanced the maximum amplitude of inwardI _m ((I _m ) _p ) and 2) shifted the peak voltage ((V _m ) _p ) at(I _m ) _p to more positive values under ramp-shaped voltage clamping and 3) depolarized the peak value of action potentials. This behavior is consistent with predictions based on the Nernst equation for Ca²⁺ but not for Cl^–. DIDS (4,4-diisothiocyano-2,2-disulfonic acid stilbene) did not suppress(I _m ) _p in tonoplast-free cells, in contrast with its effect on normal cells. La³⁺ and nifedipine blocked(I _m ) _p irreversibly. On the other hand, Ca²⁺ channel agonist, BAY K 8644 irreversibly enhanced(I _m ) _p . Both Sr²⁺ influx and K⁺ efflux increased upon excitation. The charge carried by Sr²⁺ influx was compensated for by K⁺ efflux. It is concluded that only the Ca²⁺ channel is activated during plasmalemma excitation in tonoplast-free cells. In terms of the magnitude of(I _m ) _p , Sr²⁺ could replace Ca²⁺, but Mn²⁺, Mg²⁺ and Ba²⁺ could not. External pH affected(I _m ) _p and the membrane conductance (g _m ) at(I _m ) _p ((g _m ) _p ). Increasing the external ionic strength caused increases in both(I _m ) _p and(g _m ) _p , and shifted(V _m ) _p to positive values. At the same time, Sr²⁺ influx increased. Thus Ca²⁺ channel activation seems to be enhanced by increasing external ionic strength. The possible involvement of surface potential is discussed.     

Characterization of a voltage-dependent Ca2+-selective channel from wheat roots

A new mechanism for calcium flux in wheat (Triticum aestivum L.) root cells has been characterized. Membrane vesicles were enriched in plasma membrane using aqueous-polymer two-phase partitioning and incorporated into artificial lipid bilayers, allowing characterization of single channels under voltage-clamp conditions. Membrane marker activities showed 74% and 83% purity in plasma membrane when expressed in terms of membrane area and activity, respectively. Since membrane vesicles obtained by aqueous-polymer two-phase partitioning yield a population of membrane vesicles of regular orientation, and vesicle fusion into planar lipid bilayers occurs in a defined manner, the orientation of the channel upon vesicle incorporation could be determined. Thus ionic activities and potentials could be controlled appropriately on what we propose to be the cytosolic (trans) and extracellular (cis) faces of the channel. The unitary conductance in symmetrical 1 mM CaCl₂ was 27±0.4 (pS). The correlation between the theoretical and observed reversal potentials in asymmetrical conditions showed that the channel was highly selective for Ca²⁺ over Cl^–. Experiments simulating physiological ionic conditions showed a P_Ca ²⁺/P_K ⁺ of 17–26, decreasing in this range as the extracellular CaCl₂ concentration increased from 0.1 to 1 mM. The channel was also permeable to the essential nutrient ions, Mg²⁺ and Mn²⁺. The open probability of the channel was strongly dependent on the membrane potential. Inactivation with time was observed at more negative membrane potentials, and was immediately reversed as soon as the membrane potential was decreased. At membrane potentials more negative than -130mV, the channel remained mainly in the closed state, suggesting that in vivo the channel would remain largely closed and would open only upon membrane depolarization. The channel was blocked by micromolar concentrations of extracellular verapamil and trivalent cations, Al³⁺ being the most effective of those tested. Exposure of the cytosolic and extracellular sides of the channel to inositol 1,4,5-trisphosphate had no effect on the channel activity. We suggest a plasma-membrane origin for the channel as shown by biochemical and electrophysiological evidence, and discuss possible physiological roles of this channel, both in Ca²⁺ uptake into roots and in signal transduction.Abbreviations IP₃ 1,4,5-trisphosphate - PM plasma membrane We wish to thank Dr. Christa Niemietz, Dr. Robert Reid and Prof. Andrew Smith for valuable discussions. This work was supported by the Australian Research Council and an OPRS award to M.P.     

Properties of the inner membrane anion channel in intact mitochondria

The mitochondrial inner membrane possesses an anion channel (IMAC) which mediates the electrophoretic transport of a wide variety of anions and is believed to be an important component of the volume homeostatic mechanism. IMAC is regulated by matrix Mg²⁺ (IC₅₀=38 µM at pH 7.4) and by matrix H⁺ (pIC₅₀=7.7). Moreover, inhibition by Mg²⁺ is pH-dependent. IMAC is also reversibly inhibited by many cationic amphiphilic drugs, including propranolol, and irreversibly inhibited byN,N-dicyclohexylcarbodiimide. Mercurials have two effects on its activity: (1) they increase the IC₅₀ values for Mg²⁺, H⁺, and propranolol, and (2) they inhibit transport. The most potent inhibitor of IMAC is tributyltin, which blocks anion uniport in liver mitochondria at about 1 nmol/mg. The inhibitory dose is increased by mercurials; however, this effect appears to be unrelated to the other mercurial effects. IMAC also appears to be present in plant mitochondria; however, it is insensitive to inhibition by Mg²⁺, mercurials, andN,N-dicyclohexylcarbodiimide. Some inhibitors of the adenine nucleotide translocase also inhibit IMAC, including Cibacron Blue, agaric acid, and palmitoyl CoA; however, atractyloside has no effect.     

Ion channels in the plasma membrane ofAmaranthus protoplasts: One cation and one anion channel dominate the conductance

Summary This report details preliminary findings for ion channels in the plasma membrane of protoplasts derived from the cotyledons ofAmaranthus seedlings. The conductance properties of the membrane can be described almost entirely by the behavior of two types of ion channel observed as single channels in attached and detached patches. The first is a cation-selective outward rectifier, and the second a multistate anion-selective channel which, under physiological conditions, acts as an inward rectifier.The cation channel has unit conductance of approx. 30 pS (symmetrical 100 K⁺) and relative permeability sequence K⁺>Na⁺>Cl^– (10.160.03); whole-cell currents activate in a time-dependent manner, and both activation and deactivation kinetics are voltage dependent. The anion channel opens for hyperpolarized membrane potentials, has a full-level conductance of approx. 200 pS and multiple subconductance states. The number of sub-conductances does not appear to be fixed. When activated the channel is open for long periods, though shuts if the membrane potential (V _m ) is depolarized; at millimolar levels of [Ca²⁺]_cyt this voltage dependency disappears. Inward current attributable to the anion channel is not observed in whole-cell recordings when MgATP (2mm) is present in the intracellular solution. By contrast the channel is active in most detached patches, whether MgATP is present or not on the cytoplasmic face of the membrane. The anion channel has a significant permeability to cations, the sequence being NO ₃ ^– >Cl^–>K⁺>Aspartate (2.0410.18 to 0.090.04). The relative permeability for K⁺ decreased at progressively lower conductance states. In the absence of permeant anions this channel could be mistaken for a cation inward rectifier. The anion and cation channels could serve to clampV _m at a preferred value in the face of events which would otherwise perturbV _m .     

Chloroplast thylakoid membrane fluidity and its sensitivity to temperature

In order to investigate membrane fluidity, the hydrophobic probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), has been incorporated into intact isolated thylakoids and separated granal and stromal lamellae obtained from the chloroplasts of Pisum sativum. The steady-state polarization of DPH fluorescence was measured as a function of temperature and indicated that at physiological values the thylakoid membrane is a relatively fluid system with the stromal lamellae being less viscous than the lamellae of the grana. According to the DPH technique, neither region of the membrane, however, showed a sharp phase transition of its bulk lipids from the liquid-crystalline to the gel state for the temperature range -20° to 50° C. Comparison of intact thylakoids isolated from plants grown at cold (4°/7°C) and warm (14°/17° C) temperatures indicate that there is an adaptation mechanism operating which seems to maintain an optimal membrane viscosity necessary for growth. Using a modified Perrin equation the optimal average viscosity for the thylakoid membrane of the chill-resistant variety used in the study (Feltham First) is estimated to be about 1.8 poise.Abbreviations DPH 1,6-diphenyl-1,3,5-hexatriene - Hepes N-(2-hydroxyethyl)-1-piperazineethanesulphonic acid     

A technical consideration concerning the removal of oocyte vitelline membranes for patch clamp recording

We have developed an efficient method for removing the vitelline membrane of Xenopus oocytes for patch clamp recording. Functional studies using oocytes as models provide insights into the biological profiles and physiological properties of ion channels. A methodological modification is described in this paper. The important feature of this modification is that protease treatment is used to remove the oocyte's vitelline membrane. This method is simple and the oocytes produced remain in a healthy state during the recording process.     

Identification of a cationic channel in synaptosomal membranes

Synaptosomal membranes were fused with liposomes using the hydration technique to produce giant proteoliposomes amenable to patch clamp recordings. Single channel currents of a cationic channel with particular properties were detected. In a solution of 150 mM NaCl, the channel displayed a unit conductance of 136 pS and a mean open state lifetime of 1.1 ms. The gating of the channel was shown to be voltage as well as calcium dependent. Pharmacological studies revealed that the channel was insensitive to a variety of channel blockers, but was inactivated by ruthenium red. Presumably, this channel may play a role in regulating the evoked release of neurotransmitters. Offprint requests to: H. Breer     

Characterization of a high-conductance,voltage-dependent cation channel from the plasma membrane of rye roots in planar lipid bilayers

Plasma-membrane vesicles were purified by aqueous-polymer two-phase partitioning of a microsomal membrane fraction from rye (Secale cereale L.) roots and incorporated into planar 1-palmitoyl-2-oleoyl phosphatidylethanolamine bilayers. A high-conductance cation channel (a maxi cation channel) was characterized from single-channel electrical recordings. The channel was incorporated into the bilayer with its cytoplasmic surface facing the trans compartment and voltages were referenced cis with respect to trans. The channel was permeable to both monovalent and divalent cations. The unitary conductance was 451 pS in symmetrical 100 mM KCl and 213 pS in symmetrical 100 mM BaCl₂. The permeability ratio P_KP_Ba was 1.002.56. Unitary conductances declined in the order K⁺Rb⁺>Cs⁺>Na⁺> Li⁺ (monovalent cations) and Ba²⁺>Sr²⁺>Ca²⁺> Mg²⁺>Co²⁺>Mn²⁺ (divalent cations). The relative permeabilities of monovalent cations mirrored their conductivity sequence, whereas the permeabilities of all divalent cations were similar. The maxi cation channel showed complex kinetics, exhibiting both voltage- and time-dependent inactivation and voltage-dependent gating. The voltage dependence of the kinetics shifted in parallel with changes in the reversal potential of the channel. In symmetrical 100 mM KCl, following a voltage step from zero to the test voltage, the channel inactivated and the active-channel lifetime ( _i) shortened exponentially as the test voltage was increased. The channel always opened immediately upon depolarization to zero volts, indicating that inactivation of the channel did not result from the loss of any intrinsic factor. The probability of finding an active channel in the open state (P₀) exhibited a bell-shaped relationship with membrane potential. At voltages between -40 and 80 mV, P₀ exceeded 0.99, but p₀ declined abruptly at more extreme voltages. Under ionic conditions which approximated physiological conditions, in the presence of 100 mM KCl on the trans (cytoplasmic) side and 1 mM KCl plus 2 mM CaCl₂ on the cis (extracellular) side, the reversal potential was 15.6 mV and the kinetics approximated those observed in symmetrical 100 mM KCl. Thus, the channel would open upon depolarization of the plasma membrane in vivo. If the channel functioned physiologically as a Ca²⁺ channel it might be involved in intracellular signalling: the channel could open in response to a variety of environmental, developmental and pathological stimuli which depolarize the plasma membrane, allowing Ca²⁺ into the cytoplasm and thereby initiating a physiological response.Abbreviations E_K Nernst (equilibrium) potential for potassium - E_rev zero-current (reversal) potential - I/V current/voltage - _c apparent mean lifetime of the activated-channel closed state - _i apparent mean lifetime of the activated channel following a voltage step from zero volts - ₀ apparent mean lifetime of the activated-channel open state - PE 1-palmitoyl-2-oleoyl phosphatidylethonlamine - P₀ probability of finding the activated channel in an open state - TEA⁺ tetraethylammonium This work was supported by the Agriculture and Food Research Council and by a grant from the Science and Engineering Research Council Membrane Initiative (GR/F 33971) to Prof. E.A.C. MacRobbie (University of Cambridge, UK).     

Further investigation on the high-conductance ion channel of the inner membrane of mitochondria

By use of the patch-clamp technique, the inner membrane of mouse liver and heart mitochondria is shown to contain a highly conductive (around 100 pS in symmetrical 150 mM KCl) and voltage-dependent ion channel. This channel closely resembles that previously found in cuprizone-treated mouse liver inner mitochondrial membrane. The paper discusses the electrical properties of the channel and its possible physiological function. The reconstitution in giant liposomes of a partially purified ox heart inner membrane fraction containing the channel and the use of various inhibitors are also presented.     

The ultrastructure of plasma and thylakoid membrane vesicles from the fresh water cyanobacteriumAnacystis nidulans adapted to salinity

Summary Photoautotrophically growing cultures of the fresh water cyanobacteriumAnacystis nidulans adapted to the presence of 0.4–0.5 M NaCl (about sea water level) with a lag phase of two days after which time the growth rate reassumed 80–90% of the control. Plasma and thylakoid membranes were separated from cell-free extracts of French pressure cell treatedAnacystis nidulans by discontinuous sucrose density gradient centrifugation and purified by repeated recentrifugation on fresh gradients. Identity of the plasma and thylakoid membrane fractions was confirmed by labeling of intact cells with impermeant protein markers prior to breakage and membrane isolation. Electron microscopy revealed that each type of membrane was obtained in the form of closed and perfectly spherical vesicles. Major changes in structure and function of the plasma membranes (and, to a much lesser extent, of the thylakoid membranes) were found to accompany the adaptation process. On the average, diameters of plasma membrane vesicles from salt adapted cells were only one-third of the diameters of corresponding vesicles from control cells. By contrast, the diameters of thylakoid membrane vesicles were the same in both cases.Freeze-etching the cells and counting the number of membrane-intercalating particles on both protoplasmic and exoplasmic fracture faces of plasma and thylakoid membranes indicated a roughly 50% increase of the particle density in plasma membranes during the adaptation process while that in thylakoid membranes was unaffected. Comparison between particle densities on isolated membranes and those on corresponding whole cell membranes permitted an estimate as to the percentage of inside-out and right-side-out vesicles. Stereometric measurement of particle sizes suggested that two distinct sub-populations of the particles in the plasma membranes increased during the adaptation process, tentatively correlated to the cytochrome oxidase and sodium-proton antiporter, respectively. The effects of salt adaptation described in this paper were fully reversed upon withdrawal of the additional NaCl from the growth medium (deadaptation). Moreover, they were not observed when the NaCl was replaced by KCl.Abbreviations CM cytoplasmic or plasma membrane - ICM intracytoplasmic or thylakoid membrane - EF exoplasmic fracture face - PF protoplasmic fracture face - DABS diazobenzosulfonate; Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sulfonate - PMSF phenylmethylsulfonylfluoride Dedicated to the memory of Professor Oswald Kiermayer     

Isolation of protoplasts and ion channel recording of plasma membrane patches and whole-cell recordings of the marine alga, <Emphasis Type="Italic">Ulva pertusa</Emphasis> (Chlorophyta)

Whole-cell patch-clamp techniques were used to study ion channels of a marine alga. High quality protoplasts suitable for electrophysiological studies were isolated from the green marine alga, Ulva pertusa, using enzyme mixtures consisting of cellulase and abalone power and identified by calcofluor fluorescence. The vitality of protoplasts varied depending on the alga growth stage, and those isolated from younger tissue in March maintained a high vitality with high sealing success rate compared with protoplasts isolated from mature or non-growing plants in August or November. In the whole-cell configuration, large inward currents were elicited by negative voltage pulses. The voltage-dependent component was predominantly carried by Cl⁻, as confirmed by the use of the Cl⁻ channel inhibitor DIDS and reversal potential of current-voltage plots. This evidence suggests that hyperpolarization-activated Cl⁻ permeable channels are responsible for the influx of Cl⁻ into U. pertusa cells. Voltage-dependent outward currents were also recorded in several protoplasts, and their properties need further investigation.     

Patch clamp analysis of chemically activated and modulated ionic channels in isolated mammalian cardiomyocytes

Techniques routinely utilized in this laboratory for recording currents through single ionic channels of isolated atrial and ventricular rat cardiomyocytes are described. Emphasis is placed in two main areas: first, on methods for obtaining a sufficient yield of Ca⁺⁺-tolerant myocytes suitable for patch clamp experiments, and secondly, on methods for analyzing the temporal characteristics of patched ionic channels. These methods were used on acetylcholine activated K⁺ channels in isolated atrial myocytes and on an inwardly-rectifying K⁺ channel in ventricular myocytes. The latter is an example of a hormonally modulated K⁺ channel, since its activity could be substantially increased by norepinephrine. Analysis of the closed and open time distributions suggested that one of the closed states of this channel is markedly abbreviated by norepinephrine, whereas the open state is nearly unaffected. Norepinephrine was effective when channel activity was recorded from on-cell patches and the hormone was added to the solution bathing the cell membrane outside of the patched area. This indicates that a second messenger substance is probably mediating the action of norepinephrine.