Statistical analysis of single sodium channels. Effects of N-bromoacetamide

Currents were obtained from single sodium channels in outside-out excised patches of membrane from the cell line GH3. The currents were examined in control patches and in patches treated with N- bromoacetamide ( NBA ) to remove inactivation. The single-channel current-voltage relationship was linear over the range -60 to + 10 mV, and was unaffected by NBA . The slope conductance at 9.3 degrees C was 12 pS, and the Q10 for single channel currents was about 1.35. The currents in both control and NBA -treated patches showed evidence of a slow process similar to desensitization in acetylcholine-receptor channels. This process was especially apparent at rapid rates of stimulation (5 Hz), where openings occurred in clusters of records. The clustering of records with and without openings was analyzed by runs analysis, which showed a statistically significant trend toward nonrandom ordering in the responses of channels to voltage pulses. NBA made this nonrandom pattern more apparent. The probability that an individual channel was "hibernating" during an activating depolarization was estimated by a maximum likelihood method. The lifetime of the open state was also estimated by a maximum likelihood method, and was examined as a function of voltage. In control patches the open time was mildly voltage-dependent, showing a maximum at about -50 mV. In NBA -treated patches the open time was greater than in the control case and increased monotonically with depolarization; it asymptotically approached that of the control patches at hyperpolarized potentials. By comparing channel open times in control and NBA -treated patches, we determined beta A and beta I, the rate constants for closing activation gates and fast inactivation gates. Beta I was an exponential function of voltage, increasing e-fold for 34 mV. beta A had the opposite voltage dependence. The probability of an open channel closing its fast inactivation gate, rather than its activation gate, increased linearly with depolarization from -60 to -10 mV. These results indicate that inactivation is inherently voltage dependent.     

Modulation of single cardiac sodium channels by DPI 201-106

Single sodium channel currents were analysed in cell attached patches from single ventricular cells of guinea pig hearts in the presence of a novel cardiotonic compound DPI 201-106. The mean single channel conductance of DPI-treated Na channels was not changed by DPI (20.8 +/- 4 pS, control, 3 patches; 21.3 +/- 1 pS with DPI, 5 mumol/1,3 patches). DPI voltage-dependently prolongs the cardiac sodium channel openings by removal of inactivation at potentials positive to -40 mV. At potentials negative to -40 mV a clustering of short openings at the very beginning of the depolarizing voltage steps can be observed causing a transient time course of the averaged currents. Long openings induced an extremely slow inactivation. Short openings, long openings and nulls appeared in groups referring to a modal gating behaviour of DPI-treated sodium channels. DPI-modified Na channels showed a monotonously prolonged mean open time with increased depolarizing voltage steps, e.g. the open state probability within a sweep was increased. However, the number of non-empty sweeps was decreased with the magnitude of the depolarizing steps, e.g. the probability of the channel being open as calculated from the averaged currents was voltage-dependently decreased by DPI (50% decrease at -50.7 +/- 9 9 mV, 3 patches). Short and long openings of DPI-modified channels could be separated by variation of the holding potential. The occurrence of long Na channel openings was much more suppressed by reducing the holding potential (half maximum inactivation at -112 +/- 8 mV, 4 patches) than that of short openings (half maximum inactivation at -88 +/- 8 mV, 4 patches). Otherwise, short living openings completely disappeared at potentials positive to -40 mV where the occurrence of long openings was favoured. The differential voltage dependence of blocking and activating effects of DPI on cardiac Na channels as well as the differential voltage dependence of the appearance of short and long openings refers to a modal gating behaviour of cardiac Na channels.     

Kinetic properties of single sodium channels in rat heart and rat brain

Single Na channel currents were compared in ventricular myocytes and cortical neurons of neonatal rats using the gigaseal patch-clamp method to determine whether tissue-specific differences in gating can be detected at the single-channel level. Single-channel currents were recorded in cell-attached and excised membrane patches at test potentials of -70 to -20 mV and at 9-11 degrees C. In both cell-attached and excised patches brain Na channel mean open time progressively increased from less than 1 ms at -70 mV to approximately 2 ms at -20 mV. Near threshold, single openings with dispersed latencies were observed. By contrast, in cell-attached patches, heart Na channel mean open time peaked near -50 mV, was three times brain Na channel mean open time, and declined continuously to approximately 2 ms at -20 mV. Near threshold, openings occurred frequently usually as brief bursts lasting several milliseconds and rarely as prolonged bursts lasting tens of milliseconds. Unlike what occurs in brain tissue where excision did not change gating, in excised heart patches both the frequency of prolonged bursting and the mean open time of single units increased markedly. Brain and cardiac Na channels can therefore be distinguished on the basis of their mean open times and bursting characteristics.     

Inactivation viewed through single sodium channels

Recordings of the sodium current in tissue-cultured GH3 cells show that the rate of inactivation in whole cell and averaged single channel records is voltage dependent: tau h varied e-fold/approximately 26 mV. The source of this voltage dependence was investigated by examining the voltage dependence of individual rate constants, estimated by maximum likelihood analysis of single channel records, in a five-state kinetic model. The rate constant for inactivating from the open state, rather than closing, increased with depolarization, as did the probability that an open channel inactivates. The rate constant for closing from the open state had the opposite voltage dependence. Both rate constants contributed to the mean open time, which was not very voltage dependent. Both open time and burst duration were less than tau h for voltages up to -20 mV. The slowest time constant of activation, tau m, was measured from whole cell records, by fitting a single exponential either to tail currents or to activating currents in trypsin-treated cells, in which the inactivation was abolished. tau m was a bell-shaped function of voltage and had a voltage dependence similar to tau h at voltages more positive than -35 mV, but was smaller than tau h. At potentials more negative than about -10 mV, individual channels may open and close several times before inactivating. Therefore, averaged single channel records, which correspond with macroscopic current elicited by a depolarization, are best described by a convolution of the first latency density with the autocorrelation function rather than with 1 - (channel open time distribution). The voltage dependence of inactivation from the open state, in addition to that of the activation process, is a significant factor in determining the voltage dependence of macroscopic inactivation. Although the rates of activation and inactivation overlapped greatly, independent and coupled inactivation could not be statistically distinguished for two models examined. Although rates of activation affect the observed rate of inactivation at intermediate voltages, extrapolation of our estimates of rate constants suggests that at very depolarized voltages the activation process is so fast that it is an insignificant factor in the time course of inactivation. Prediction of gating currents shows that an inherently voltage-dependent inactivation process need not produce a conspicuous component in the gating current.     

Calcium- and voltage-activated potassium channels in adrenocortical cell membranes

Current flowing through single Ca- and voltage-activated K channels has been recorded from cell-attached and inside-out excised membrane patches of cultured Y-1 adrenocortical cells. In intact cells, single-channel current amplitude and the time a channel stays in the open state increase with membrane depolarization. In excised patches bathed in symmetrical 130 mM K solutions, single-channel conductance is 170 pS. This value is constant in the membrane potential range of +/- 50 mV but decreases at larger hyper- and depolarizations. Channel open probability is heavily influenced by the concentration of ionic Ca at the inner surface of the membrane in the range between 0.01 and 10 microM. When internal Ca concentration is close to 0.01 microM, channels are usually closed even at large depolarizing voltages. With larger Ca concentrations, channel open probability increases and its voltage dependence is greater. These channels are uniformly distributed in the plasma membrane, since one to four channels were seen in more than 99% of the patches isolated in this study. There are previous reports suggesting a role for calcium ions in the secretory response of adrenocortical cells to ACTH. Therefore, it is possible that, as in other endocrine cells, these K channels modulate Ca influx across the plasma membrane and thus contribute to regulate steroid biosynthesis and release.     

Single-channel recordings from cultured human retinal pigment epithelial cells

We have applied patch-clamp techniques to on-cell and excised-membrane patches from human retinal pigment epithelial cells in tissue culture. Single-channel currents from at least four ion channel types were observed: three or more potassium-selective channels with single-channel slope conductances near 100, 45, and 25 pS as measured in on-cell patches with physiological saline in the pipette, and a relatively nonselective channel with subconductance states, which has a main-state conductance of approximately 300 pS at physiological ion concentrations. The permeability ratios, PK/PNa, measured in excised patches were 21 for the 100-pS channels, 3 for the 25-pS channels, and 0.8 for the 300-pS nonselective channel. The 45-pS channels appeared to be of at least two types, with PK/PNa's of approximately 41 for one type and 3 for the other. The potassium-selective channels were spontaneously active at all potentials examined. The average open time for these channels ranged from a few milliseconds to many tens of milliseconds. No consistent trend relating potassium-selective channel kinetics to membrane potential was apparent, which suggests that channel activity was not regulated by the membrane potential. In contrast to the potassium-selective channels, the activity of the nonselective channel was voltage dependent: the open probability of this channel declined to low values at large positive or negative membrane potentials and was maximal near zero. Single-channel conductances observed at several symmetrical KCl concentrations have been fitted with Michaelis-Menten curves in order to estimate maximum channel conductances and ion-binding constants for the different channel types. The channels we have recorded are probably responsible for the previously observed potassium permeability of the retinal pigment epithelium apical membrane.     

Sodium channels in planar lipid bilayers. Channel gating kinetics of purified sodium channels modified by batrachotoxin

Single channel currents of sodium channels purified from rat brain and reconstituted into planar lipid bilayers were recorded. The kinetics of channel gating were investigated in the presence of batrachotoxin to eliminate inactivation and an analysis was conducted on membranes with a single active channel at any given time. Channel opening is favored by depolarization and is strongly voltage dependent. Probability density analysis of dwell times in the closed and open states of the channel indicates the occurrence of one open state and several distinct closed states in the voltage (V) range-120 mV less than or equal to V less than or equal to +120 mV. For V less than or equal to 0, the transition rates between stages are exponentially dependent on the applied voltage, as described in mouse neuroblastoma cells (Huang, L. M., N. Moran, and G. Ehrenstein. 1984. Biophysical Journal. 45:313-322). In contrast, for V greater than or equal to 0, the transition rates are virtually voltage independent. Autocorrelation analysis (Labarca, P., J. Rice, D. Fredkin, and M. Montal. 1985. Biophysical Journal. 47:469-478) shows that there is no correlation in the durations of successive open or closing events. Several kinetic schemes that are consistent with the experimental data are considered. This approach may provide information about the mechanism underlying the voltage dependence of channel activation.     

Statistical properties of single sodium channels

Single channel currents were obtained from voltage-activated sodium channels in outside-out patches of tissue-cultured GH3 cells, a clonal line from rat pituitary gland. In membrane patches where the probability of overlapping openings was low, the open time histograms were well fit by a single exponential. Most analysis was done on a patch with exactly one channel. We found no evidence for multiple open states at -25 and -40 mV, since open times, burst durations, and autocorrelation functions were time independent. Amplitude histograms showed no evidence of multiple conductance levels. We fit the gating with 25 different time-homogeneous Markov chain models having up to five states, using a maximum likelihood procedure to estimate the rate constants. For selected models, this procedure yielded excellent predictions for open time, closed time, and first latency density functions, as well as the probability of the channel being open after a step depolarization, the burst duration distribution, autocorrelation, and the distribution of number of openings per record. The models were compared statistically using likelihood ratio tests and Akaike's information criterion. Acceptable models allowed inactivation from closed states, as well as from the open state. Among the models eliminated as unacceptable by this survey were the Hodgkin-Huxley model and any model requiring a channel to open before inactivating.     

Single channel currents in adrenocortical cells

Single channel currents have been recorded from cell-attached patches of tumoral adrenocortical cells. Our experiments suggest the existence of three sets of potassium channels in the surface membrane of these cells. All channel types can be recorded in a given membrane patch but some patches have only one type of single channel currents. One channel type has a unitary conductance of about 103 pS. The other two channels have smaller conductances and opposite voltage dependence. In one case channels open on depolarization and have a single channel conductance of 31.6 pS. In the other case the probability of being in the open state increases on hyperpolarization and the single channel conductance is of 21 pS. These channels seem to be similar to the delayed and anomalous rectifying potassium channels seen in other preparations. The role of membrane ionic permeability in steroid release induced by ACTH is discussed.     

The calcium-activated potassium channels of turtle hair cells

A major factor determining the electrical resonant frequency of turtle cochlear hair cells is the time course of the Ca-activated K current (Art, J. J., and R. Fettiplace. 1987. Journal of Physiology. 385:207- 242). We have examined the notion that this time course is dictated by the K channel kinetics by recording single Ca-activated K channels in inside-out patches from isolated cells. A hair cell's resonant frequency was estimated from its known correlation with the dimensions of the hair bundle. All cells possess BK channels with a similar unit conductance of approximately 320 pS but with different mean open times of 0.25-12 ms. The time constant of relaxation of the average single- channel current at -50 mV in 4 microM Ca varied between cells from 0.4 to 13 ms and was correlated with the hair bundle height. The magnitude and voltage dependence of the time constant agree with the expected behavior of the macroscopic K(Ca) current, whose speed may thus be limited by the channel kinetics. All BK channels had similar sensitivities to Ca which produced half-maximal activation for a concentration of approximately 2 microM at +50 mV and 12 microM at -50 mV. We estimate from the voltage dependence of the whole-cell K(Ca) current that the BK channels may be fully activated at -35 mV by a rise in intracellular Ca to 50 microM. BK channels were occasionally observed to switch between slow and fast gating modes which raises the possibility that the range of kinetics of BK channels observed in different hair cells reflects a common channel protein whose kinetics are regulated by an unidentified intracellular factor. Membrane patches also contained 30 pS SK channels which were approximately 5 times more Ca-sensitive than BK channels at -50 mV. The SK channels may underlie the inhibitory synaptic potential produced in hair cells by efferent stimulation.     

Glutamate receptor-channel gating. Maximum likelihood analysis of gigaohm seal recordings from locust muscle.

Gigaohm recordings have been made from glutamate receptor channels in excised, outside-out patches of collagenase-treated locust muscle membrane. The channels in the excised patches exhibit the kinetic state switching first seen in megaohm recordings from intact muscle fibers. Analysis of channel dwell time distributions reveals that the gating mechanism contains at least four open states and at least four closed states. Dwell time autocorrelation function analysis shows that there are at least three gateways linking the open states of the channel with the closed states. A maximum likelihood procedure has been used to fit six different gating models to the single channel data. Of these models, a cooperative model yields the best fit, and accurately predicts most features of the observed channel gating kinetics.     

Modification of single cardiac Na+ channels by DPI 201-106

Summary In inside-out patches from cultured neonatal rat heart cells, single Na⁺ channel currents were analyzed under the influence of the cardiotonic compound DPI 201-106 (DPI), a putative novel channel modifier. In absence of DPI, normal cardiac single Na⁺ channels studied at –30 mV have one open state which is rapidly left with a rate constant of 826.5 sec^–1 at 20°C during sustained depolarization., Reconstructed macroscopic currents relax completely with 7 to 10 msec. The current decay fits a single exponential. A considerable percentage of openings may occur during relaxation of the macroscopic current. In patches treated with 3×10^–6 m DPI in the pipette solution, stepping to –30 mV results in drastically prolonged and usually repetitive openings. This channel activity mostly persists over the whole depolarization (usually 160 msec in duration) but is abruptly terminated on clamping back the patch to the holding potential. Besides these modified events, apparently normal openings occur. The open time distribution of DPI-treated Na⁺ channels is the sum of two exponentials characterized by time constants of 0.85 msec (which is close to the time constant found in the control patches, 1.21 msec) and 12 msec. Moreover, DPI-modified Na⁺ channels exhibit a sustained high, time-independent open probability. Similar to normal Na⁺ channels, the mean number of open DPI-modified Na⁺ channels is voltage-dependent and increases on shifting the holding potential in the hyperpolarizing direction. These kinetic changes suggest an elimination of Na⁺ channel inactivation as it may follow from an interaction of DPI with Na⁺ channels.     

Voltage-dependent open-state inactivation of cardiac sodium channels: gating current studies with Anthopleurin-A toxin

The gating charge and voltage dependence of the open state to the inactivated state (O-->I) transition was measured for the voltage- dependent mammalian cardiac Na channel. Using the site 3 toxin, Anthopleurin-A (Ap-A), which selectively modifies the O-->I transition (see Hanck, D. A., and M. F. Sheets. 1995. Journal of General Physiology. 106:601-616), we studied Na channel gating currents (Ig) in voltage-clamped single canine cardiac Purkinje cells at approximately 12 degrees C. Comparison of Ig recorded in response to step depolarizations before and after modification by Ap-A toxin showed that toxin-modified gating currents decayed faster and had decreased initial amplitudes. The predominate change in the charge-voltage (Q-V) relationship was a reduction in gating charge at positive potentials such that Qmax was reduced by 33%, and the difference between charge measured in Ap-A toxin and in control represented the gating charge associated with Na channels undergoing inactivation by O-->I. By comparing the time course of channel activation (represented by the gating charge measured in Ap-A toxin) and gating charge associated with the O-->I transition (difference between control and Ap-A charge), the influence of activation on the time course of inactivation could be accounted for and the inherent voltage dependence of the O-->I transition determined. The O-->I transition for cardiac Na channels had a valence of 0.75 e-. The total charge of the cardiac voltage-gated Na channel was estimated to be 5 e-. Because charge is concentrated near the opening transition for this isoform of the channel, the time constant of the O-->I transition at 0 mV could also be estimated (0.53 ms, approximately 12 degrees C). Prediction of the mean channel open time-voltage relationship based upon the magnitude and valence of the O- ->C and O-->I rate constants from INa and Ig data matched data previously reported from single Na channel studies in heart at the same temperature.     

A slow anion channel in guard cells, activating at large hyperpolarization, may be principal for stomatal closing.

Slowly activating anion channel currents were discovered at micromolar 'cytoplasmic' Ca2+ during patch-clamp measurements on guard-cell protoplasts of Vicia faba and Xanthium strumarium. They activated at potentials as low as -200 mV, with time constants between 5 and 60 s, and no inactivation. The broad voltage dependence exhibited a current maximum near -40 mV. The single-channel open time was in the order of seconds, and the unitary conductance was 33 ps, similar to that of the already described 'quick' anion channel of guard cells. Because of its activity at low potentials, the slow anion channel may be essential for the depolarization of the plasmalemma that is required for salt efflux during stomatal closing.     

Cl- channels in intact human T lymphocytes.

We recently described a large, multiple-conductance Cl- channel in excised patches from normal T lymphocytes. The properties of this channel in excised patches are similar to maxi-Cl- channels found in a number of cell types. The voltage dependence in excised patches permitted opening only at nonphysiological voltages, and channel activity was rarely seen in cell-attached patches. In the present study, we show that Cl- channels can be activated in intact cells at physiological temperatures and voltages and that channel properties change after patch excision. Maxi-Cl- channels were reversibly activated in 69% of cell-attached patches when the temperature was above 32 degrees C, whereas fewer than 2% of patches showed activity at room temperature. Upon excision, the same patches displayed large, multiple-conductance Cl- channels with characteristics like those we previously reported for excised patches. After patch excision, warm temperatures were not essential to allow channel activity; 37% (114/308) of inside-out patches had active channels at room temperature. The voltage dependence of the channels was markedly different in cell-attached recordings compared with excised patches. In cell-attached patches, Cl- channels could be open at cell resting potentials in the normal range. Channel activation was not related to changes in intracellular Ca2+ since neither ionomycin nor mitogens activated the channels in cell-attached patches, Ca2+ did not rise in response to warming and the Cl- channel was independent of Ca2+ in inside-out patches. Single-channel currents were blocked by internal or external Zn2+ (100-200 microM), 4-acetamido-4' isothiocyanostilbene-2,2'-disulfonate (SITS, 100-500 microM) and 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS, 100 microM). NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) reversibly blocked the channels in inside-out patches.     

Gating kinetics of batrachotoxin-modified Na+ channels in the squid giant axon. Voltage and temperature effects.

The gating kinetics of batrachotoxin-modified Na+ channels were studied in outside-out patches of axolemma from the squid giant axon by means of the cut-open axon technique. Single channel kinetics were characterized at different membrane voltages and temperatures. The probability of channel opening (Po) as a function of voltage was well described by a Boltzmann distribution with an equivalent number of gating particles of 3.58. The voltage at which the channel was open 50% of the time was a function of [Na+] and temperature. A decrease in the internal [Na+] induced a shift to the right of the Po vs. V curve, suggesting the presence of an integral negative fixed charge near the activation gate. An increase in temperature decreased Po, indicating a stabilization of the closed configuration of the channel and also a decrease in entropy upon channel opening. Probability density analysis of dwell times in the closed and open states of the channel at 0 degrees C revealed the presence of three closed and three open states. The slowest open kinetic component constituted only a small fraction of the total number of transitions and became negligible at voltages greater than -65 mV. Adjacent interval analysis showed that there is no correlation in the duration of successive open and closed events. Consistent with this analysis, maximum likelihood estimation of the rate constants for nine different single-channel models produced a preferred model (model 1) having a linear sequence of closed states and two open states emerging from the last closed state. The effect of temperature on the rate constants of model 1 was studied. An increase in temperature increased all rate constants; the shift in Po would be the result of an increase in the closing rates predominant over the change in the opening rates. The temperature study also provided the basis for building an energy diagram for the transitions between channel states.     

Gating of the squid sodium channel at positive potentials: II. Single channels reveal two open states.

Single-channel recordings from squid axon Na+ channels were made under conditions of reverse sodium gradient. In the range of potentials studied, +40-(+)120 mV, channels opened promptly after depolarization, closed and reopened several times during the pulse. In patches containing only one channel, the distributions of open dwell times showed two components showing the existence of a second open state. The ensemble average of single-channel records showed incomplete inactivation that became more pronounced at more positive potentials, showing that the maintained phase of the current is the result of only one type of sodium channel with two open states. Analysis of bursts indicated that the dwell times of the events at the onset of the depolarization are longer than those later in the pulse. The dwell open times of the first events could be fitted with a single exponential. This indicated that the channels open preferentially through the first open state, the access to the second open state happening subsequently. Maximum likelihood analysis was used to evaluate several possible kinetic schemes incorporating a second open state. The best model to fit the data from single channels, and consistent with the data from macroscopic and gating currents, has a second open state evolving from the inactivated state. A kinetic model is proposed that incorporates information obtained from dialyzed axons.     

Depolarization increases the single-channel conductance and the open probability of crayfish glutamate channels.

We have studied the voltage sensitivity of glutamate receptors in outside-out patches taken from crayfish muscles. We found that single-channel conductance, measured directly at the single-channel level, increases as depolarization rises. At holding potentials from -90 mV to approximately 20 mV, the conductance is 109 pS. At holding potentials positive to 20 mV, the conductance is 213 pS. This increase in single-channel conductance was also observed in cell-attached patches. In addition, desensitization, rise time, and the dose-response curve were all affected by depolarization. To further clarify these multifaceted effects, we evaluated the kinetic properties of single-channel activity recorded from cell-attached patches in hyperpolarization (membrane potential around -75 mV) and depolarization (membrane potential approximately 105 mV). We found that the glutamate dissociation rate constant (k_) was affected most significantly by membrane potential; it declined 6.5-fold under depolarization. The rate constant of channel closing (k(c)) was also significantly affected; it declined 1.8-fold. The rate constant of channel opening (k(o)) declined only 1.2-fold. The possible physiological significance of the depolarization-mediated changes in the above rate constants is discussed.     

Voltage- and cold-dependent gating of single TRPM8 ion channels

Transient receptor potential (TRP) channels play critical roles in cell signaling by coupling various environmental factors to changes in membrane potential that modulate calcium influx. TRP channels are typically activated in a polymodal manner, thus integrating multiple stimuli. Although much progress has been made, the underlying mechanisms of TRP channel activation are largely unknown. The TRPM8 cation channel has been extensively investigated as a major neuronal cold sensor but is also activated by voltage, calcium store depletion, and some lipids as well as by compounds that produce cooling sensations, such as menthol or icilin. Several models of TRPM8 activation have been proposed to explain the interaction between these diverse stimuli. However, a kinetic scheme is not yet available that can describe the detailed single-channel kinetics to gain further insight into the underlying gating mechanism. To work toward this goal, we investigated voltage-dependent single-channel gating in cell-attached patches at two different temperatures (20 and 30 °C) using HEK293 cells stably expressing TRPM8. Both membrane depolarization and cooling increased channel open probability (P(o)) mainly by decreasing the duration of closed intervals, with a smaller increase in the duration of open intervals. Maximum likelihood analysis of dwell times at both temperatures indicated gating in a minimum of five closed and two open states, and global fitting over a wide range of voltages identified a seven-state model that described the voltage dependence of P(o), the single-channel kinetics, and the response of whole-cell currents to voltage ramps and steps. The major action of depolarization and cooling was to accelerate forward transitions between the same two sets of adjacent closed states. The seven-state model provides a general mechanism to account for TRPM8 activation by membrane depolarization at two temperatures and can serve as a starting point for further investigations of multimodal TRP activation.     

N-bromoacetamide removes a calcium-dependent component of channel opening from calcium-activated potassium channels in rat skeletal muscle

Calcium-activated potassium channels from cultured rat skeletal muscle were treated with the protein-modifying reagent N-bromoacetamide (NBA) (0.3-1 mM) and studied in excised patches using patch-clamp techniques. After NBA treatment, channels opened only occasionally, and, in contrast to untreated channels, the open probability was no longer sensitive to intracellular surface calcium ions (1 nM to 100 microM). Channel activity did, however, exhibit a voltage dependence similar in direction and magnitude to that shown before NBA treatment (increasing e-fold with 19 mV depolarization). Distributions of open channel lifetimes revealed that NBA treatment virtually abolished openings of long duration, which suggests that this class of openings requires calcium sensitivity. These effects were not reversed by subsequent washing. Quantitatively similar open probability, voltage dependence, and open-interval distributions were observed in untreated channels in calcium-free medium. These results suggest that NBA removed a calcium-dependent component of channel opening, and that normal channels are able to open in the absence of significant intracellular calcium concentrations.