A cyclic-AMP-gated conductance in cochlear hair cells

AbstractThe patch clamp technique was used to record cAMP-dependent currents of the guinea pig cochlear hair cell plasma membrane. Data obtained indicate that the channels passing this current are moderately selective for monovalent cations and are effectively blocked by L-cis-diltiazem and reversibly blocked by 1 mM Mg2+ or Ca2+. The single-channel unit conductance estimated in the absence of divalent cations is about 16 pS. The results demonstrate that cyclic nucleotide-dependent channels of cochlear hair cells are virtually identical to the photoreceptor and olfactory ones

Single-channel currents of a peptide-gated sodium channel expressed in Xenopus oocytes

Single-channel recordings were made from outside-out membrane patches of Xenopus oocytes injected with the cDNA clone FaNaCh, which encodes a peptide-gated Na+ channel from Helix aspersa.The natural peptides FMRFamide and FLRFamide only activated unitary currents in oocytes injected with FaNaCh; the EC50 values were 1.8 and 11.7 μm, respectively.The slope conductance of the channel was 9.2 pS for both peptides.With FMRFamide, the open probability (Po) of the channel was 0.06 at 0.3 μm and 0.76 at 30 μm, whereas for FLRFamide the open probability increased from 0.04 at 1.8 μm to 0.49 at 50 μm. The Hill coefficient was greater than 1 for both peptides.High concentrations of each peptide evoked very fast flickering between open and closed states which led to decreased unitary current amplitude.At low doses, brief single openings and bursts of longer openings occurred. With higher doses, the occurrence of the brief openings declined and the number of longer openings increased; the duration of the longer openings was shorter with FLRFamide than with FMRFamide.For each peptide, frequency distribution histograms of open events were best fitted by the sum of two exponential components, suggesting the existence of two open states of the channel. Closed events were fitted by the sum of three components, suggesting the existence of three closed states.The data were analysed according to a five-state model in which the brief openings correspond to a single liganded open form of the channel and the longer openings to a doubly liganded open form. According to this interpretation, the greater whole-cell response observed with FMRFamide than with FLRFamide results mostly from a slower closing rate constant for the longer (doubly liganded) channel openings

Molecular mechanism for sodium-dependent activation of G protein-gated K+ channels

G protein-gated inwardly rectifying K+ (GIRK) channels are activated independently by Gβγ and internal Na+ via mechanisms requiring phosphatidylinositol phosphates. An aspartate (Asp) at position 226 in GIRK2 is crucial for Na+-dependent activation of GIRK1–GIRK2 heteromeric channels. We expressed wild-type and mutant GIRK1–GIRK2 channels in Xenopus oocytes and tested the effects of Na+ and neutralizing Asp226 on the functional interactions of the channels with phosphatidylinositol 4,5-bisphosphate (PIP2).The rate of inhibition of GIRK1–GIRK2 currents by application of anti-PIP2 antibody to inside-out membrane patches was slowed > 2-fold by the D226N mutation in GIRK2 and by increasing internal [Na+]. The reverse mutation in GIRK1 (N217D) increased the rate of inhibition.The dose–response relationship for activation by purified PIP2 was shifted to lower concentrations in the presence of 20 mM Na+.Three synthetic isoforms of PIP2, PI(4,5)P2, PI(3,4)P2 and PI(3,5)P2, activated GIRK channels with similar potencies.We conclude that Na+ directly interacts with Asp226 of GIRK2 to reduce the negative electrostatic potential and promote the functional interaction of the channels with PIP2

Serum fractions from amyotrophic lateral sclerosis patients depress voltage-activated Ca2+ of rat cerebellar granule cells in culture

Whole-cell patch clamp recording from rat cerebellar granule cells in culture was used to study the effect of immune protein fractions extracted from the serum of amyotrophic lateral sclerosis (ALS) patients on voltage-activated Ca2+ currents. The inward currents, carried by Ba2+, were induced by depolarizing step commands positive to -50 mV and showed typical voltage-dependent inactivation. Application of immunoprotein fractions obtained from the serum of ALS patients produced a strong depression of the inward current amplitude without changing its threshold potential at which the maximum was attained, or its time course. These data support the hypothesis that the serum of ALS patients contains an immunoprotein capable of interacting with high threshold Ca2+ channels of central neurones

Cloning and expression of a FMRFamide-gated Na+ channel from Helisoma trivolvis and comparison with the native neuronal channel

We have cloned a cDNA encoding a Phe-Met-Arg-Phe-NH2 (FMRFamide)-gated Na+ channel from nervous tissue of the pond snail Helisoma trivolvis (HtFaNaC) and expressed the channel in Xenopus oocytes. The deduced amino acid sequence of the protein expressed by HtFaNaC is 65 % identical to that of the FMRFamide-gated channel cloned from Helix aspersa (HaFaNaC).HtFaNaC expressed in oocytes was less sensitive to FMRFamide (EC50 = 70 μM) than HaFaNaC (EC50 = 2 μM). The two had a similar selectivity for Na+. The amplitude of the FMRFamide response of HtFaNaC was increased by reducing the extracellular concentration of divalent cations.The conductance of the two channels was similar, but the mean open time of unitary events was shorter for expressed HtFaNaC compared to expressed HaFaNaC. Each channel was susceptible to peptide block by high agonist concentrations.In marked contrast to HaFaNaC and other amiloride-sensitive Na+ channels, amiloride, and the related drugs benzamil and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), enhanced the FMRFamide response in oocytes expressing HtFaNaC cRNA. The potentiating effects of EIPA and benzamil were greater than those of amiloride. Unitary current analysis showed that with such drugs, there was channel blockade as well as an increased probability of channel opening.The similar permeability of the oocyte-expressed HtFaNaC and the Helisoma neuronal channel, and the susceptibility of both to agonist blockade and blockade by divalent cations, suggest that the channels are the same. However, neuronal channels were less susceptible to enhancement by amiloride analogues and in some patches were more sensitive to FMRFamide than expressed HtFaNaC

Adaptation of the odour-induced response in frog olfactory receptor cells

Receptor current and spiking responses were recorded simultaneously from isolated frog olfactory receptor cells using the suction pipette technique. Cells were stimulated with the odour cineole by rapid exchange of the solution bathing the olfactory cilia.The receptor current response to a 1 s odour stimulus increased in a graded manner over a 300-fold range of odour concentration without clear saturation, and was accompanied by a train of action potentials. As the concentration of the odour stimulus increased, the frequency of firing increased also, until it saturated at the highest concentrations. The number of spikes evoked by the stimulus first increased and then decreased with increasing concentration, reaching a maximum at intermediate odour concentrations. The dose-response relation for spike firing rose at lower odour concentrations than the dose-response relation for the receptor current response.Adaptation to steady odour stimuli was investigated by exposing the cilia to a 4 s odour pre-pulse and then to a 1 s odour test pulse. As the pre-pulse concentration was increased the dose-response relations derived from the receptor current and spiking responses shifted to higher absolute test pulse concentrations. However the number of spikes fired in response to a given test pulse was little affected by the pre-pulse until, at the highest pre-pulse concentrations spike firing was abolished despite the continued presence of a receptor current response.The sensitivity of the receptor-current response to incremental stimuli fell with increasing pre-pulse concentration, declining with a limiting slope of 2.4 in double logarithmic co-ordinates. The sensitivity determined from the spiking responses declined to zero at a lower pre-pulse concentration, reflecting the abolition of spike firing at pre-pulse concentrations which still evoked a graded receptor-current response