Robert W Meech

The sensitivity of Helix aspersa neurones to injected calcium ions

1. When calcium chloride was injected into Helix aspersa neurones there was a fall in membrane resistance and the membrane potential became hyperpolarized.

The effect of calcium injection on the intracellular sodium and pH of snail neurones

1. Ion‐sensitive glass micro‐electrodes were used to measure the intracellular pH (pHi) and the intracellular sodium ion concentration, [Na+]i, in identified Helix aspersa neurones. 2. The injection of small volumes of 0‐1 McaCl2, which increased the membrane potential by 10‐15 mV for 1‐2 min, had little or no effect on [Na+]i. Increases of up to 1 mM in [Na+]i could be reversibly induced by larger injections. 3. Calcium injection caused an immediate decrease in pHi, which appeared to be directly proportional to the amount of calcium injected. Injections causing hyperpolarizations of 10‐20 mV which recovered in 2‐5 min caused pHi decreases of 0‐04‐0‐15 units. After each of these injections both pHi and the membrane potential recovered exponentially but with different time constants. 4. The injection of calcium at a low rate could decrease pHi without affecting the membrane potential. 5. Neither membrane potential nor pHi were affected by the injection of small volumes of 0‐1 M‐MgCl2, Injection of CoCl2 produced a large transient decrease in pHi but no significant change in membrane potential. 6. Exposure of the cell to saline equilibrated with 2‐5% CO2 greatly reduced the pHi decrease caused by calcium injection but had only small effects on the membrane potential response. 7. It is concluded that most of the injected calcium is exchanged for protons inside the cell.

Fast Ca2+ signals at mouse inner hair cell synapse: a role for Ca2+‐induced Ca2+ release

Inner hair cells of the mammalian cochlea translate acoustic stimuli into ‘phase‐locked’ nerve impulses with frequencies of up to at least 1 kHz. Little is known about the intracellular Ca2+ signal that links transduction to the release of neurotransmitter at the afferent synapse. Here, we use confocal microscopy to provide evidence that Ca2+‐induced Ca2+ release (CICR) may contribute to the mechanism. Line scan images (2 ms repetition rate) of neonatal mouse inner hair cells filled with the fluorescent indicator FLUO‐3, revealed a transient increase in intracellular Ca2+ concentration ([Ca2+]i) during brief (5–50 ms) depolarizing commands under voltage clamp. The amplitude of the [Ca2+]i transient depended upon the Ca2+ concentration in the bathing medium in the range 0–1.3 mm. [Ca2+]i transients were confined to a region near the plasma membrane at the base of the cell in the vicinity of the afferent synapses. The change in [Ca2+]i appeared uniform throughout the entire basal sub‐membrane space and we were unable to observe hotspots of activity. Both the amplitude and the rate of rise of the [Ca2+]i transient was reduced by external ryanodine (20 μm), an agent that blocks Ca2+ release from the endoplasmic reticulum. Intracellular Cs+, commonly used to record at presynaptic sites, produced a similar effect. We conclude that both ryanodine and intracellular Cs+ block CICR in inner hair cells. We discuss the contribution of CICR to the measured [Ca2+]i transient, the implications for synaptic transmission at the afferent synapse and the significance of its sensitivity to intracellular Cs+.

Nitric oxide regulates swimming in the jellyfish Aglantha digitale.

The cnidarian nervous system is considered by many to represent neuronal organization in its earliest and simplest form. Here we demonstrate, for the first time in the Cnidaria, the neuronal localization of nitric oxide synthase (NOS) in the hydromedusa Aglantha digitale (Trachylina). Expression of specific, fixative‐resistant NADPH‐diaphorase (NADPH‐d) activity, characteristic of NOS, was observed in neurites running in the outer nerve ring at the base of the animal and in putative sensory cells in the ectoderm covering its tentacles. At both sites, diphenyleneiodonium (10‐4 M) abolished staining. Capillary electrophoresis confirmed that the NO breakdown products NO2‐ and NO3‐ were present at high levels in the tentacles, but were not detectable in NADPH‐d–negative areas. The NADPH‐d–reactive neurons in the tentacles send processes to regions adjacent to the inner nerve ring where swimming pacemaker cells are located. Free‐moving animals and semi‐intact preparations were used to test whether NO is involved in regulating the swimming program. NO (30–50 nM) and its precursor L‐arginine (1 mM) stimulated swimming, and the effect was mimicked by 8‐Br‐cGMP (50–100 μM). The NO scavenger PTIO (10–100 μM) and a competitive inhibitor of NOS, L‐nitroarginine methyl ester (L‐NAME, 200 μM), significantly decreased the swimming frequency in free‐moving animals, while its less‐active stereoisomer D‐nitroarginine methyl ester (D‐NAME, 200 μM) had no such effect. 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxaline‐1‐one (ODQ, 5–20 μM), a selective inhibitor of soluble guanylyl cyclase, suppressed spontaneous swimming and prevented NO‐induced activation of the swimming program. We suggest that an NO/cGMP signaling pathway modulates the rhythmic swimming associated with feeding in Aglantha, possibly by means of putative nitrergic sensory neurons in its tentacles. J. Comp. Neurol. 471:26–36, 2004.

A conductive pathway generated from fragments of the human red cell anion exchanger AE1

Human red cell anion exchanger AE1 (band 3) is an electroneutral Cl–HCO3− exchanger with 12–14 transmembrane spans (TMs). Previous work using Xenopus oocytes has shown that two co‐expressed fragments of AE1 lacking TMs 6 and 7 are capable of forming a stilbene disulphonate‐sensitive 36Cl‐influx pathway, reminiscent of intact AE1. In the present study, we create a single construct, AE1Δ(6: 7), representing the intact protein lacking TMs 6 and 7. We expressed this construct in Xenopus oocytes and evaluated it employing a combination of two‐electrode voltage clamp and pH‐sensitive microelectrodes. We found that, whereas AE1Δ(6: 7) has some electroneutral Cl–base exchange activity, the protein also forms a novel anion‐conductive pathway that is blocked by DIDS. The mutation Lys539Ala at the covalent DIDS‐reaction site of AE1 reduced the DIDS sensitivity, demonstrating that (1) the conductive pathway is intrinsic to AE1Δ(6: 7) and (2) the conductive pathway has some commonality with the electroneutral anion‐exchange pathway. The conductance has an anion‐permeability sequence: NO3−≈ I− > NO2− > Br− > Cl− > SO42−≈ HCO3−≈ gluconate−≈ aspartate−≈ cyclamate−. It may also have a limited permeability to Na+ and the zwitterion taurine. Although this conductive pathway is not a usual feature of intact mammalian AE1, it shares many properties with the anion‐conductive pathways intrinsic to two other Cl–HCO3− exchangers, trout AE1 and mammalian SLC26A7.

Endogenous Na+-K+ (or NH4+)-2Cl- cotransport in Rana oocytes ; anomalous effect of external NH4+ on pHi

1. In Rana oocytes, measurements with chloride‐sensitive microelectrodes show that the mean intracellular chloride activity (34.8 +/‐ 6.3 mM, n = 79) is three times higher than that expected for the passive distribution of chloride ions across the outer membrane (12.4 mM, mean membrane potential ‐43 +/‐ 8.8 mV, n = 79). 2. Reuptake of chloride into oocytes depleted by prolonged exposure to chloride‐free saline takes place against the electrochemical gradient. 3. Chloride reuptake does not take place in sodium‐free solution or in a sodium‐substituted potassium‐free solution. It is inhibited by bumetanide (10(‐5) M) in the bathing medium. 4. The overall stoichiometry of the transport mechanism deduced from simultaneous measurements of intracellular sodium and chloride using ion‐selective electrodes is 1Na+:1K+:2Cl‐. 5. Ammonium ions substitute for potassium on the cotransporter. 6. In oocytes smaller than 0.9 mm in diameter, exposure to external ammonium causes an alkaline shift in intracellular pH as the NH3 enters and takes up H+ to form NH4+. We propose that chloride‐dependent NH4+ transport contributes to the accumulation of NH4+ and causes the ‘postexposure’ acidification as the intracellular NH4+ releases H+ to form NH3 which is then lost from the cell. 7. In larger oocytes ammonium exposure produces a rapid reduction in pHi which may be explained in part by cotransport‐mediated uptake of NH4+. Evidence is also provided for a second chloride‐dependent NH4+ transport mechanism and a chloride‐independent process.

Potassium inhibition of sodium‐activated potassium (KNa) channels in guinea‐pig ventricular myocytes

1 Na+‐activated potassium channels (KNa channels) were studied in inside‐out patches from guinea‐pig ventricular myocytes at potentials between ‐100 and +80 mV. External K+ (K+o) was set to 140 mM. For inwardly directed currents with 105 mM internal K+ (K+i), the unitary current‐voltage relationship was fitted by the constant field equation with a potassium permeability coefficient, PK, of 3.72 × 10−13 cm3 s−1. The slope conductance (‐100 to ‐10 mV) was 194 ± 4.5 pS (mean ± s.d., n= 4) with 105 mM K+i (35 mM Na+i) but it decreased to 181 ± 5.6 pS (n= 5) in 70 mM K+i (70 mM Na+i). 2 KNa channels were activated by internal Na+ in a concentration‐dependent fashion. With 4 mM K+i, maximal activation was recorded with 100 mM Na+i (open probability, Po, about 0.78); half‐maximal activation required about 35 mM Na+i. When K+i was increased to 70 mM, half‐maximal activation shifted to about 70 mM Na+i. 3 With Na+i set to 105 mM, channel activity was markedly inhibited when K+i was increased from 35 to 105 mM. Channel openings were abolished with 210 mM K+i. 4 The inhibitory effect of internal K+ was also observed at more physiological conditions of osmolarity, ionic strength and chloride concentration. With 35 mM Na+i and 4 mM K+i, Po was 0.48 ± 0.10 (n= 6); when K+i was increased to 35 mM, Po was reduced to 0.04 ± 0.05 (n= 7, P= 0.001). 5 The relationship between Po and Na+i concentration at different levels of K+i is well described by a modified Michaelis‐Menten equation for competitive inhibition; the Hill coefficients were 4 for the Po‐Na+i relationship and 1.2 for the Po‐K+i relationship. It is suggested that Na+ and K+ compete for a superficial site on the channels permeation pathway. 6 KNa channels would be most likely to be activated in vivo when an increase in Na+i is accompanied by a decrease of K+i.

A contribution to the history of the proton channel

The low numbers of hydrogen ions in physiological solutions encouraged the assumption that H+ currents flowing through conductive pathways would be so small as to be unmeasurable even if theoretically possible. Evidence for an H+-based action potential in the luminescent dinoflagellate Noctiluca and for an H+-conducting channel created by the secretions of the bacterium Bacillus brevis, did little to alter this perception. The clear demonstration of H+ conduction in molluscan neurons might have provided the breakthrough but the new pathway was without an easily demonstrable function, and escaped general attention. Indeed the extreme measures that must be taken to successfully isolate H+ currents meant that it was some years before proton channels were identified in mammalian cells. However, with the general availability of patch-clamp techniques and evidence for an important role in mammalian neutrophils, the stage was set for a series of structure/function studies with the potential to make the proton channel the best understood channel of all. In addition, widespread genomic searches have established that proton channels play important roles in processes ranging from fertilization of the human ovum to the progression of breast cancer.

Evolution of excitability in lower metazoans

All forms of life exhibit excitability; it is one of the characteristics by which living creatures can be recognized. In this chapter, we examine the different manifestations of excitability exhibited by the Metazoa and show how elements present in the Bacteria come together in the Protozoa, Porifera, Cnidaria, and Ctenophora (see Fig. 1) to form the patterns of excitability known as “behavior.” We consider the role of excitation in fertilized eggs and conducting epithelia, as well as the origins of signaling in nerves and muscles. We describe different forms of all-or-nothing signaling, as well as ways of generating graded responsiveness. This study attempts to provide a practical approach to understanding the limitations of excitable systems. We believe that defining these limits is more useful than glorifying their seemingly endless sophistication. THE NATURE OF EXCITABILITY Excitability Defined Excitability is easy to recognize; less easy to define. We recognize excitability when we see it, by the way an organism responds to an external stimulus. For there to be a response, stimulus and organism must interact and the organism must “receive” the stimulus. Inevitably, the stimulus site, or receptor, and the response site, or effector, will be at different locations even in single cells. Thus, excitability depends on the transmission of signals from receptor to effector. The signals may be chemical and spread by passive diffusion, electrical and spread by the transfer of ionic charge, or mechanical and spread by a physical disturbance. We focus in this section on the links between chemical...

Nerves in the endodermal canals of hydromedusae and their role in swimming inhibition.

Neoturris breviconis (Anthomedusae) has a nerve plexus in the walls of its endodermal canals. The plexus is distinct from the ectodermal nerve plexuses supplying the radial and circular muscles in the ectoderm and no connections have been observed between them. Stimulation of the endodermal plexus evokes electrical events recorded extracellularly as “E” potentials. These propagate through all areas where the plexus has been shown by immunohistology to exist and nowhere else. When Neoturris is ingesting food, trains of “E” potentials propagate down the radial canals to the margin and cause inhibition of swimming. This response is distinct from the inhibition of swimming associated with contractions of the radial muscles but both may play a part in feeding and involve chemoreceptors. Preliminary observations suggest that the “E” system occurs in other medusae including Aglantha digitale (Trachymedusae) where the conduction pathway was previously thought to be an excitable epithelium.