Understanding factors associated with continuation of intrauterine device use in Gujarat and Rajasthan, India: a cross-sectional household study.

The Government of India has promoted the expansion of access to and uptake of intrauterine devices (IUDs), during both the interval (IIUD) and postpartum (PPIUD) periods, as part of its Family Planning 2020 initiative. This study, conducted by EngenderHealth as part of the Expanding Access to IUD Services in India project, examines IIUD and PPIUD continuation rates over time and investigates factors associated with IUD continuation. We recruited respondents (N = 5024) through a repeated cross-sectional household study between February and December 2019. We identified respondents using IUD client data from public health facility registers in 20 districts of Gujarat and Rajasthan. We compared continuation rates for IIUD and PPIUD adopters and used regression analyses to measure the association between continuation and demographic, quality of care, and counselling variables. IIUD continuation rates decreased from 85.6% to 78.3% and PPIUD rates decreased from 78.5% to 70.7% between month 3 and month 12. Clients experiencing side effects or other problems were 15 times more likely to discontinue IUD use than clients who did not. Clients who received IUD counselling prior to insertion were more likely to continue than those who did not. IUD continuation increased significantly in cases where both partners jointly selected the method compared to situations where women decided alone. Several sociodemographic factors were associated with continuation. Our study demonstrates the value and benefits of programmes offering IUD services emphasising quality counselling and client-centred care to increase access, uptake, and continuation.

L-pGlu-(2-propyl)-L-His-L-ProNH2 attenuates 4-aminopyridine-induced epileptiform activity and sodium current: a possible action of new thyrotropin-releasing hormone analog for its anticonvulsant potential.

L-PGlu-(2-propyl)-L-His-L-ProNH2 (NP-647) is a CNS active thyrotropin-releasing hormone (TRH) analog with potential application in various CNS disorders including seizures. In the present study, mechanism of action for protective effect of NP-647 was explored by studying role of NP-647 on epileptiform activity and sodium channels by using patch-clamp methods. Epileptiform activity was induced in subicular pyramidal neurons of hippocampal slice of rat by perfusing 4-aminopyridine (4-AP) containing Mg⁺²-free normal artificial cerebrospinal fluid (nACSF). Increase in mean firing frequency was observed after perfusion of 4-AP and zero Mg⁺² (2.10±0.47 Hz) as compared with nACSF (0.12±0.08 Hz). A significant decrease in mean firing frequency (0.61±0.22 Hz), mean frequency of epileptiform events (0.03±0.02 Hz vs. 0.22±0.05 Hz of 4-AP+0 Mg), and average number of action potentials in paroxysmal depolarization shift-burst (2.54±1.21 Hz vs. 8.16±0.88 Hz of 4-AP+0 Mg) was observed. A significant reduction in peak dV/dt (246±19 mV ms⁻¹ vs. 297±18 mV ms⁻¹ of 4-AP+0 Mg) and increase (1.332±0.018 ms vs. 1.292±0.019 ms of 4-AP+0 Mg) in time required to reach maximum depolarization were observed indicating role of sodium channels. Concentration-dependent depression of sodium current was observed after exposure to dorsal root ganglion neurons to NP-647. NP-647 at different concentrations (1, 3, and 10 µM) depressed sodium current (15±0.5%, 50±2.6%, and 75±0.7%, respectively). However, NP-647 did not show change in the peak sodium current in CNa18 cells. Results of present study demonstrated potential of NP-647 in the inhibition of epileptiform activity by inhibiting sodium channels indirectly.

Inhibition of human two-pore domain K+ channel TREK1 by local anesthetic lidocaine: negative cooperativity and half-of-sites saturation kinetics.

TWIK-related K+ channel TREK1, a background leak K+ channel, has been strongly implicated as the target of several general and local anesthetics. Here, using the whole-cell and single-channel patch-clamp technique, we investigated the effect of lidocaine, a local anesthetic, on the human (h)TREK1 channel heterologously expressed in human embryonic kidney 293 cells by an adenoviral-mediated expression system. Lidocaine, at clinical concentrations, produced reversible, concentration-dependent inhibition of hTREK1 current, with IC(50) value of 180 muM, by reducing the single-channel open probability and stabilizing the closed state. We have identified a strategically placed unique aromatic couplet (Tyr352 and Phe355) in the vicinity of the protein kinase A phosphorylation site, Ser348, in the C-terminal domain (CTD) of hTREK1, that is critical for the action of lidocaine. Furthermore, the phosphorylation state of Ser348 was found to have a regulatory role in lidocaine-mediated inhibition of hTREK1. It is interesting that we observed strong intersubunit negative cooperativity (Hill coefficient = 0.49) and half-of-sites saturation binding stoichiometry (half-reaction order) for the binding of lidocaine to hTREK1. Studies with the heterodimer of wild-type (wt)-hTREK1 and Delta119 C-terminal deletion mutant (hTREK1(wt)-Delta119) revealed that single CTD of hTREK1 was capable of mediating partial inhibition by lidocaine, but complete inhibition necessitates the cooperative interaction between both the CTDs upon binding of lidocaine. Based on our observations, we propose a model that explains the unique kinetics and provides a plausible paradigm for the inhibitory action of lidocaine on hTREK1.

Glutamate transporter blockade affects Ca(2+) responses in astrocytes.

Brief pretreatment of astrocytes in culture with glutamate (500 microM for 20 min), was earlier shown to significantly enhance the Ca(2+) responses to a depolarizing pulse. It is known that malfunction of glutamate transporters increases extracellular glutamate concentration. We hypothesized that pretreatment of astrocytes with glutamate in conditions where the glutamate transporter activity is blocked should cause further elevation of the Ca(2+) responses to a depolarizing pulse. To test the hypothesis we pretreated astrocytes in culture (primary rat astrocyte cultures) with glutamate (500 microM) and glutamate transport inhibitor, threo-beta-hydroxy-aspartate (200 microM, TBHA) or glutamate (500 microM) in Na(+) free extracellular solution for 20 min. The Ca(2+) responses were elicited by depolarization of the astrocyte to evoke voltage-gated Ca(2+) currents. Paradoxical attenuation of the Ca(2+) transients was observed when the glutamate pretreatment was done in conditions that blocked glutamate transport, accompanied by faster rise and decay times. When the experiments were done on astrocyte pairs that were pretreated with glutamate and TBHA, we observed attenuated Ca(2+) responses in the adjoining cell when compared with the depolarized cell. The results were contrary to our earlier observation of heightened responses in the adjoining cell of the astrocyte pair, in cells pretreated with glutamate alone. The attenuated Ca(2+) responses in astrocytes would imply decrease in the vesicular release of glutamate and ATP. Extracellular glutamate concentration dependent regulation of the Ca(2+) signaling mechanism thus seems to operate in astrocytes, which may be important in regulating the neurotoxic accumulation of glutamate in the extracellular space and the synapse.

Time-dependent molecular memory in single voltage-gated sodium channel.

Excitability in neurons is associated with firing of action potentials and requires the opening of voltage-gated sodium channels with membrane depolarization. Sustained membrane depolarization, as seen in pathophysiological conditions like epilepsy, can have profound implications on the biophysical properties of voltage-gated ion channels. Therefore, we sought to characterize the effect of sustained membrane depolarization on single voltage-gated Na+ channels. Single-channel activity was recorded in the cell-attached patch-clamp mode from the rNa(v)1.2 alpha channels expressed in CHO cells. Classical statistical analysis revealed complex nonlinear changes in channel dwell times and unitary conductance of single Na+ channels as a function of conditioning membrane depolarization. Signal processing tools like weighted wavelet Z (WWZ) and discrete Fourier transform analyses attributed a "pseudo-oscillatory" nature to the observed nonlinear variation in the kinetic parameters. Modeling studies using the hidden Markov model (HMM) illustrated significant changes in kinetic states and underlying state transition rate constants upon conditioning depolarization. Our results suggest that sustained membrane depolarization induces novel nonlinear properties in voltage-gated Na+ channels. Prolonged membrane depolarization also induced a "molecular memory" phenomenon, characterized by clusters of dwell time events and strong autocorrelation in the dwell time series similar to that reported recently for single enzyme molecules. The persistence of such molecular memory was found to be dependent on the duration of depolarization. Voltage-gated Na+ channel with the observed time-dependent nonlinear properties and the molecular memory phenomenon may determine the functional state of the channel and, in turn, the excitability of a neuron.

Characterization of contryphans from Conus loroisii and Conus amadis that target calcium channels.

Distinctly different effects of two closely related contryphans have been demonstrated on voltage-activated Ca(2+) channels. The peptides Lo959 and Am975 were isolated from Conus loroisii, a vermivorous marine snail and Conus amadis, a molluscivore, respectively. The sequences of Lo959 and Am975 were deduced by mass spectrometric sequencing (MALDI-MS/MS) and confirmed by chemical synthesis. The sequences of Lo959, GCP(D)WDPWC-NH(2) and Am975, GCO(D)WDPWC-NH(2) (O: 4-trans-hydroxyproline: Hyp), differ only at residue 3; Pro in Lo959, Hyp in Am975, which is identical to contryphan-P, previously isolated from Conus purpurascens, a piscivore; while Lo959 is a novel peptide. Both Lo959 and Am975 undergo slow conformational interconversion under reverse-phase chromatographic conditions, a characteristic feature of all contryphans reported thus far. Electrophysiological studies performed using dorsal root ganglion neurons reveal that both peptides target high voltage-activated Ca(2+) channels. While Lo959 increases the Ca(2+) current, Am975 causes inhibition. The results establish that subtle sequence effects, which accompany post-translational modifications in Conus peptides, can have dramatic effects on target ion channels.

Astrocytes in 17beta-estradiol treated mixed hippocampal cultures show attenuated calcium response to neuronal activity.

Glial cells in the brain are capable of responding to hormonal signals. The ovarian steroid hormone 17beta-estradiol, in addition to its actions on neurons, can directly affect glial cells. Estrogen receptors have been described on both neurons and astrocytes, suggesting a complex interplay between these two in mediating the effects of the hormone. Astrocytes sense and respond to neuronal activity with a rise in intracellular calcium concentration ([Ca(2+)](i)). Using simultaneous electrophysiology and calcium imaging techniques, we monitored neuronal activity evoked astrocyte ([Ca(2+)](i)) changes in mixed hippocampal cultures loaded with fluo-3 AM. Action potential firing in neurons, elicited by injecting depolarizing current pulses, was associated with ([Ca(2+)](i)) elevations in astrocytes, which could be blocked by 200 microM MCPG and also 1 microM TTX. We compared astrocytic ([Ca(2+)](i)) transients in control and 24-hour estradiol treated cultures. The amplitude of the ([Ca(2+)](i)) transient, the number of responsive astrocytes, and the ([Ca(2+)](i)) wave velocity were all significantly reduced in estradiol treated cultures. ([Ca(2+)](i)) rise in astrocytes in response to local application of the metabotropic glutamate receptor (mGluR) agonist t-ACPD was attenuated in estradiol treated cultures, suggesting functional changes in the astrocyte mGluR following 24-h treatment with estradiol. Since astrocytes can modulate synaptic transmission by release of glutamate, the attenuated ([Ca(2+)](i)) response seen following estradiol treatment could have functional consequences on astrocyte-neuron signaling.

FM1-43 measurements of local exocytotic events in rat melanotrophs.

We have explored the existence of fusion- and secretion-competent sites on the plasma membrane of peptide secreting rat pituitary melanotrophs at rest, and following stimulation with glutamate. We monitored changes in fluorescence of FM1-43, a styryl dye which labels plasma membrane. The results show spontaneous local increases in FM1-43 reporting changes in membrane surface area due to cumulative exocytosis. Addition of glutamate, further increased the occurrence of these events. Statistical analysis of local FM1-43 fluorescence changes suggests that this is due to the recruitment of inactive exocytotic domains and due to the stimulation of already active exocytotic domains.

Inhibition of human TREK-1 channels by caffeine and theophylline.

Caffeine (1,3,7-trimethylxanthine) and theophylline (1,3-dimethylxanthine) are used for therapeutic purposes and can cause life-threatening convulsive seizures due to systemic toxicity. The mechanisms for the epileptogenicity of caffeine and theophylline are not clear. TWIK-related K(+) channels (TREK-1) are highly expressed in the human central nervous system and have a major role in the control of neuronal excitability by regulating the resting membrane potential. In view of their physiological significance, inhibition of TREK-1 channels may be implicated in caffeine- and theophylline-induced seizures. We thus investigated, using whole-cell patch-clamp technique, modulation of hTREK-1 channels expressed in Chinese hamster ovary (CHO) cells by caffeine and theophylline. Caffeine and theophylline produced reversible inhibition of TREK-1 channels in a concentration-dependent manner. The half-maximal inhibitory concentrations (IC(50)) for caffeine and theophylline were 377+/-54microM and 486+/-76microM, respectively. Caffeine and theophylline depolarized the membrane potential of CHO(TREK-1) cells in a reversible and concentration-dependent manner. Inhibition by caffeine (5mM) and theophylline (2mM) was attenuated in TREK-1 channels with mutation of the PKA consensus sequence at serine 348, suggesting the involvement of cAMP/PKA pathway in the inhibitory process. Inhibition of TREK-1 channels and consequent membrane depolarization may contribute to the convulsive seizures induced by toxic levels of caffeine and theophylline.

Fast pseudo-periodic oscillation in the rat brain voltage-gated sodium channel alpha subunit.

In the work reported here, we have investigated the changes in the activation and fast inactivation properties of the rat brain voltage-gated sodium channel (rNa(v) 1.2a) alpha subunit, expressed heterologously in the Chinese Hamster Ovary (CHO) cells, by short depolarizing prepulses (10-1000 ms). The time constant of recovery from fast inactivation (tau(fast)) and steady-state parameters for activation and inactivation varied in a pseudo-oscillatory fashion with the duration and amplitude of a sustained prepulse. A consistent oscillation was observed in most of the steady-state and non-inactivating current parameters with a time period close to 225 ms, although a faster oscillation of time period 125 ms was observed in the tau(fast). The studies on the non-inactivating current and steady-state activation indicate that the phase of oscillation varies from cell to cell. Co-expression of the beta1 subunit with the alpha subunit channel suppressed the oscillation in the charge movement per single channel and free energy of steady-state inactivation, although the oscillation in the half steady-state inactivation potential remained unaltered. Incidentally, the frequencies of oscillation in the sodium channel parameters (4-8 Hz) correspond to the theta component of network oscillation. This fast pseudo-oscillatory mechanism, together with the slow pseudo-oscillatory mechanism found in these channels earlier, may contribute to the oscillations in the firing properties observed in various neuronal subtypes and many pathological conditions.

Estradiol-induced changes in the activity of hippocampal neurons in network culture are suppressed by co-incubation with gabapentin.

The ovarian steroid hormone estradiol, in addition to its function in the maintenance and regulation of reproductive capacity, can alter neuronal excitability. Estradiol is proconvulsant, increases neuronal excitability and decreases the threshold for seizure activity. Over one-third to one-half of women with epilepsy experience catamenial seizures, which are seizures influenced by cyclical hormone changes. These hormone-sensitive seizures respond to the anti-epileptic drug gabapentin, which is a structural analogue of the inhibitory amino acid neurotransmitter GABA. We studied the effects of 17-beta-estradiol alone and estradiol co-incubated with gabapentin on neuronal activity in network cultures of rat hippocampal neurons using a fluorescent calcium binding dye fluo-3 AM, FM 1-43 labeling of synaptic vesicles and electrophysiological recordings. Significant changes in the neuronal network activity were observed in the estradiol-treated neuronal cultures; the reactivity of the neurons to KCl depolarization induced intracellular calcium changes, and FM 1-43 destaining was increased as was the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSC). All these excitatory effects of estradiol were nullified by co-incubating the neurons with a combination of estradiol and gabapentin. This suggests that gabapentin can indeed affect the estradiol-induced changes in neuronal network hyperexcitability by influencing the neuronal calcium levels, exocytosis and synaptic activity. Our findings could provide an understanding of the cellular basis of hormone-sensitive seizure control by gabapentin.

Trichloroethanol enhances the activity of recombinant human TREK-1 and TRAAK channels.

Human TREK-1 and TRAAK (hTREK-1 and hTRAAK) are the recently cloned tandem pore-domain potassium channels that are highly expressed in the central nervous system (CNS). The roles of 2P domain K+ channels in general anesthesia and neuroprotection have been proposed recently. We have investigated the ability of 2,2,2-trichloroethanol (an active metabolite of the general anesthetic chloral hydrate (CH)) to modulate the activity of hTREK-1 and hTRAAK channels expressed heterologously in Chinese hamster ovary cells by using whole-cell patch-clamp recording. Trichloroethanol potentiated hTREK-1 and hTRAAK channel activity in a reversible, concentration-dependent manner. The parent compound CH also augmented the activity of both the channels reversibly. CH activation of hTREK-1 was transient followed by a rapid inhibition, whereas hTRAAK activation was not followed by inhibition. Deletions of the carboxy terminal domain (Delta89, Delta100 and Delta119) of hTREK-1 did not abolish sensitivity to TCE (20 mM) suggesting that C-terminal tail is not essential for the activation of hTREK-1 by TCE. The hTREK-1 currents consisted of an instantaneous and a time-dependent component. The time-dependent current was reduced by trichloroethanol (20 mM). Our findings identify TREK-1 and TRAAK channels as molecular targets for trichloroethanol and suggest that activation of these channels might contribute to the CNS depressant effects of CH.

Functional characterization of the pentapeptide QYNAD on rNav1.2 channels and its NMR structure.

The endogenous pentapeptide QYNAD (Gln-Tyr-Asn-Ala-Asp) is present in human cerebrospinal fluid (CSF), and its concentration is increased in demyelinating diseases. QYNAD was synthesized and its action on the rNav1.2 voltage-gated sodium channel alpha-subunit was studied using whole-cell recordings in a heterologous expression system. The effects were seen only upon equilibration of the peptide in the external bath solution for at least 10 min before the commencement of whole-cell experiments. The steady-state activation curve showed a rightward shift of 10 mV, while the steady-state inactivation curve showed a leftward shift of 5 mV. Frequency-dependent inhibition of the sodium current amplitude was observed at 2-10 Hz, in the presence of external QYNAD, but was not seen when applied internally. Fits of the whole-cell sodium current traces by Hodgkin-Huxley equations revealed subtle changes in the voltage-dependent rate constants governing the transition of the activation and the inactivation gates. Two dimensional NMR spectroscopy revealed the absence of medium and long-range Nuclear Overhauser effects (NOEs), which indicates that the peptide does not adopt any canonical secondary structure in solution. In summary, our studies show that although the pentapeptide QYNAD does not have a defined structure in solution, it has defined actions on the rNav1.2 voltage-gated sodium channel isoform.

Sodium channel modulating activity in a delta-conotoxin from an Indian marine snail.

A 26 residue peptide (Am 2766) with the sequence CKQAGESCDIFSQNCCVG-TCAFICIE-NH(2) has been isolated and purified from the venom of the molluscivorous snail, Conus amadis, collected off the southeastern coast of India. Chemical modification and mass spectrometric studies establish that Am 2766 has three disulfide bridges. C-terminal amidation has been demonstrated by mass measurements on the C-terminal fragments obtained by proteolysis. Sequence alignments establish that Am 2766 belongs to the delta-conotoxin family. Am 2766 inhibits the decay of the sodium current in brain rNav1.2a voltage-gated Na(+) channel, stably expressed in Chinese hamster ovary cells. Unlike delta-conotoxins have previously been isolated from molluscivorous snails, Am 2766 inhibits inactivation of mammalian sodium channels.

Studies on the aggregation and possible channel formation in membranes of a cyclic hexapeptide, cyclo (D-Ala-L-Pro-L-Ala)2.

The interaction of zwitterionic lipid DMPC and DPPC with cyclic hexapeptide, cyclo (D-Ala-L-Pro-L-Ala)2 was studied using circular dichroism (CD) and differential scanning calorimetry (DSC). Preliminary membrane conductance results showed that the peptide has a tendency to form channels inside the lipid bilayer. CD studies indicated that as the lipid/peptide (L/P) ratio (DMPC/peptide) was increased, the magnitude of the negative CD band having a lambda(max) around 200 nm decreased. At a L/P ratio of 210:1, this band disappeared completely, indicating dramatic conformational changes in the peptide on interaction with the lipid bilayer. Reduction of the phase transition temperature and the maximum heat capacity of the lipid bilayer (DPPC) for gel-to-liquid crystalline phase transition indicates a strong interaction of the peptide with the lipid bilayer.

Polycysteine and other polyamino acid functionalized microfiltration membranes for heavy metal capture.

Polycysteine and other polyamino acid functionalized microfiltration membrane sorbents work exceptionally well for the removal and recovery of toxic heavy metals from aqueous streams. These are high capacity sorbents (0.3-3.7 mg/cm2) with excellent accessibility and selectivity for heavy metals, such as Hg(II), Pb(II), and Cd(II) over nontoxic components such as calcium. Polycysteine functionalized membranes work particularly well for metals such as Hg(II) and Cd(II), even in high total dissolved solids containing streams. Parameters such as permeate flow rate,feed metal concentration, and counterion (for Hg(II)) have also been found to influence sorbent behavior. For multicomponent systems, polyglutamic acid functionalized membranes have been found to selectively sorb Pb(II) versus Cd(II). Selective sorption of Cr(III) has also been observed with actual waste streams containing several heavy metals, hardness, and high sodium (2,000 mg/L). The high capacity, site accessibility, and ease of regeneration of these membrane-based sorbents make them ideal for environmental separations when volume reduction or selective recovery is required.

Tetrapentylammonium block of chloramine-T and veratridine modified rat brain type IIA sodium channels.

Tetrapentylammonium (TPeA) block of rat brain type IIA sodium channel alpha subunit was studied using whole cell patch clamp. Results indicate that TPeA blocks the inactivating brain sodium channel in a potential and use-dependent manner similar to that of the cardiac sodium channel. Removal of inactivation using chloramine-T (CT) unmasks a time-dependent block by TPeA consistent with slow blocking kinetics. On the other hand, no time dependence is observed when inactivation is abolished by modification with veratridine. TPeA does not bind in a potential-dependent fashion to veratridine-modified channels and does not significantly affect gating of veratridine-modified channels suggesting that high affinity binding of TPeA to the brain sodium channel is lost after veratridine modification.

The kinetics of a non-inactivating K(+) current in alphaT3-1 pituitary gonadotropes is not affected by holding potential.

The non-inactivating K(+) currents in alphaT3-1, a gonadotroph cell line, were recorded in the presence of low intracellular free calcium concentration. The activation kinetics of the whole-cell currents and the gating charge measured from holding potential (V(HOLD)) of -10 mV, V(HOLD)=-80 mV in presence of 4-AP (4-aminopyridine), and V(HOLD)=-10 mV with a hyperpolarizing prepulse to -80 mV were similar. No difference was observed in the onset of currents elicited from the hyperpolarizing potentials, suggesting deviation from the Cole-Moore prediction of increase in the delay of current onset with increasing hyperpolarization. The data suggests that the channel opens with at least one rate-limiting voltage-dependent step, which may imply that the position of the voltage sensor is unaffected by hyperpolarization.

Rapid regulated dense-core vesicle exocytosis requires the CAPS protein.

Although many proteins essential for regulated neurotransmitter and peptide hormone secretion have been identified, little is understood about their precise roles at specific stages of the multistep pathway of exocytosis. To study the function of CAPS (Ca(2+)-dependent activator protein for secretion), a protein required for Ca(2+)-dependent exocytosis of dense-core vesicles, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. Flash photolysis of caged Ca(2+) elicited biphasic capacitance increases consisting of rapid and slow components with distinct Ca(2+) dependencies. A threshold of approximately 10 microM Ca(2+) was required to trigger the slow component, while the rapid capacitance increase was recorded already at a intracellular Ca(2+) activity < 10 microM. Both kinetic membrane capacitance components were abolished by botulinum neurotoxin B or E treatment, suggesting involvement of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent vesicle fusion. The rapid but not the slow component was inhibited by CAPS antibody. These results were further clarified by immunocytochemical studies that revealed that CAPS was present on only a subset of dense-core vesicles. Overall, the results indicate that dense-core vesicle exocytosis in melanotrophs occurs by two parallel pathways. The faster pathway exhibits high sensitivity to Ca(2+) and requires the presence of CAPS, which appears to act at a late stage in the secretory pathway.

Modification of alpha subunit of RIIA sodium channels by aconitine.

The effect of aconitine (AC), an alkaloid toxin, on the electrophysiological properties of the rat brain type IIA alpha subunit expressed heterologously in the Chinese Hamster Ovarian (CHO) cell line was studied under the whole-cell patch-clamp configuration. The activation threshold of modified channels shifted by about -40 mV. As the number of depolarizations increased, the transient current at 0 mV decreased and, in proportion, the AC-modified current at -50 mV increased. This suggests a transition of channels to an AC-modified state. The rate of modification was nearly four times faster when 50 microM AC was applied internally than when applied in the bath solution. This supports the idea that the AC-binding site is located close to the cytoplasmic mouth of the channel pore. The AC-modified sodium currents inactivated completely, although with slower kinetics. The steady-state inactivation followed a simple Boltzmann function. AC-modified currents activated without a sigmoidal delay. The permeability of the NH4+ ion was enhanced such that its permeability ratio increased from an initial value of 0. 18 to 0.95 and for Cs+ it was enhanced from 0.03 to 0.07. These studies show that the AC-binding site resides at the pore region of the alpha subunit of the Na+ channel, and that the presence of beta subunit/s is not essential for AC binding.