Voltage-independent Transition Research Articles

Hv1 Proton Channel Opening Is Preceded by a Voltage-independent Transition

The voltage sensing domain (VSD) of the voltage-gated proton channel Hv1 mediates a H+-selective conductance that is coordinately controlled by the membrane potential (V) and the transmembrane pH gradient (ΔpH). Allosteric control of Hv1 channel opening by ΔpH (V-ΔpH coupling) is manifested by a characteristic shift of approximately 40 mV per ΔpH unit in the activation. To further understand the mechanism for V-ΔpH coupling in Hv1, H+ current kinetics of activation and deactivation in excised membrane patches were analyzed as a function of the membrane potential and the pH in the intracellular side of the membrane (pHI). In this study, it is shown for the first time to our knowledge that the opening of Hv1 is preceded by a voltage-independent transition. A similar process has been proposed to constitute the step involving coupling between the voltage-sensing and pore domains in tetrameric voltage-gated channels. However, for Hv1, the VSD functions as both the voltage sensor and the conduction pathway, suggesting that the voltage independent transition is intrinsic to the voltage-sensing domain. Therefore, this article proposes that the underlying mechanism for the activation of Hv1 involves a process similar to VSD relaxation, a process previously described for voltage-gated channels and voltage-controlled enzymes. Finally, deactivation seemingly occurs as a strictly voltage dependent process, implying that the kinetic event leading to opening of the proton conductance are different than those involved in the closing. Thus, from this work it is proposed that Hv1 activity displays hysteresis.

Opening and Closing of the HCN2 Channel Pore is Voltage-Independent

Recent studies have suggested that the opening and closing of Hyperpolarization-activated Cyclic Nucleotide-gated 'HCN' channels involve a step that is voltage-independent, which depends upon a region that resides within the S4 and S6 transmembrane domains of the channels. In the current study, we use two electrode voltage clamp of the HCN2 channel expressed in Xenopus oocytes to show that mutation of a residue site in the S6 segment, in region that likely corresponds to the pore, dramatically slows channel closing, without large effects on the rate of channel opening, and decreases the dependence of channel closing on voltage. A 6-state,but not a 4-state, cyclic allosteric model incorporating voltage-dependent transitions moving the channels between resting and active states and voltage-independent transitions between closed and open states was best able to describe the complex gating behaviour of both the wild type and pore mutant channels in response to changes in voltage. Modification of the voltage-independent closing transition recapitulates both the very slow and voltage-independent closing of mutant channel. In summary, our data supports a cyclic allosteric model of HCN2 channel gating in which the opening and closing of the pore is voltage-independent.

Ph Sensitivity of Voltage Sensing Domain Relaxation

Voltage sensing domain (VSD) relaxation is a process involving a voltage-independent transition from the activated state into the relaxed state. This process has been shown to shift the voltage dependence for sensing charge movement (sensing current) in the voltage sensitive phosphatase (VSP) known as Ci-VSP and to slow down the deactivation of potassium currents in Shaker and Kv1.2. Given the effect of VSD relaxation on the dynamics of these voltage sensitive proteins, it is thought that the relaxed and the active states are comprised of different set of conformations. Thus, it is possible that these states are sensitive to different physiological parameters. Here, it is shown that VSD activation and relaxation in Ci-VSP display different sensitivity to external pH (pHEXT). Sensing currents of Ci-VSP recorded during activation slowed down as pHEXT was decreased. Noteworthy, the kinetic of deactivating sensing currents was less affected, suggesting that the initial activation of the VSD involved a pHEXT-sensitive, rate-limiting step. In spite of these observations, the voltage dependence of activating sensing currents was almost unaltered by pHEXT (6.5 to 9.0). In contrast, voltage dependence was strongly modulated by pHEXT after VSD relaxation. In fact, the shift in voltage dependence for sensing current was 30 mV larger at pHEXT 6.5 with respect to 9.0. These observations indicated that the equilibrium between the resting and active states was pHEXT insensitive. Conversely, the equilibrium between the relaxed and the relaxed-resting states was unambiguously pHEXT sensitive. Furthermore, given that decreasing pHEXT slowed sensing currents, this observation suggests that the energy barrier between the resting and active states, rather than the difference in energy between these states, was modulated by pHEXT. In contrast, for the relaxed VSD, pHEXT strongly modulated the difference in energy between the relaxed and the relaxed-resting states.

Voltage Sensor Trapping in the Relaxed State

The voltage sensitive phosphatase from Ciona intestinalis (Ci-VSP) has constituted an intriguing model for the study of the dynamic of voltage sensing domains (VSD). Four of the five arginines in the fourth (S4) segment of the VSD function as charges sensing the difference in electrical potential across the membrane. The voltage-driven movement of the S4 segment towards the extracellular space triggers relaxation which is characterized by a shift in voltage dependence for the movement of charges to more negative values. The mechanism for relaxation remains unclear. However, it is thought to encompass the rearrangement of the VSD to satisfy the new position of the S4 segment following activation. In this view, changing the membrane potential from negative to positive voltages drives the VSD from the resting to the active state. As the S4 segment moves, the VSD gains potential energy, part of which is dissipated during a voltage-independent transition leading the VSD to the relaxed state. Replacement of the fourth arginine to a histidine (R232H) causes the VSD of Ci-VSP to display a “pump-like” behavior, which differs from the “transporter-like” behavior observed in the mutant R371H of Shaker. The cycling of this “pump” is driven by relaxation. Furthermore, we found that the net sensing charge of the mutant R232H reversibly seemingly decreases over 60% during this process. We concluded that after relaxation, the histidine in position 232 is deprotonated and “trapped” within the VSD without net charge. Similar observations are made using Molecular Dynamics simulation of the Ci-VSP voltage sensor bearing the mutation R232H. We propose electrically driving the VSD back to the resting state is inefficient after neutralizing the histidine R232H. Thus, recovery from relaxation last several seconds, diverging in two orders of magnitude the recovery observed with the native arginine.

Modulation of plant TPC channels by polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) are powerful modulators of several animal ion channels. It is shown here that PUFAs strongly affect the activity of the Slow Vacuolar (SV) channel encoded by the plant TPC1 gene. The patch-clamp technique was applied to isolated vacuoles from carrot taproots and Arabidopsis thaliana mesophyll cells and arachidonic acid (AA) was chosen as a model molecule for PUFAs. Our study was extended to different PUFAs including the endogenous alpha-linolenic acid (ALA). The addition of micromolar concentrations of AA reversibly inhibited the SV channel decreasing the maximum open probability and shifting the half activation voltage to positive values. Comparing the effects of different PUFAs, it was found that the length of the lipophilic acyl chain, the number of double bonds and the polar head were critical for channel modulation.The experimental data can be reproduced by a simple three-state model, in which PUFAs do not interact directly with the voltage sensors but affect the voltage-independent transition that leads the channel from the open state to the closed configuration. The results indicate that lipids play an important role in co-ordinating ion channel activities similar to what is known from animal cells.

The HERG N-Terminal Interacts with Voltage Sensitive Transitions

The channels underlying native human IKr are thought to be formed by homo/heterotetramers of the 2 HERG splice variants HERG1a and HERG1b. HERG1b has a shorter and highly basic 36 amino acid N-terminal, but is otherwise identical to HERG1a. This minor structural difference is sufficient to produce a significant change in gating kinetics: HERG1b has faster activation and deactivation kinetics than HERG1a. HERG activation is a multi-step process involving voltage-dependent and voltage independent transitions. It is unclear whether the difference in activation gating between HERG1a and 1b reflects modification of the voltage sensitive or insensitive steps by the N-terminal domain.We used an envelope of tails protocol to isolate activation from the fast overlapping inactivation for both HERG1a and 1b. Both of these isoforms showed saturation of activation rate at very positive potentials. This suggests that the voltage-independent step is rate limiting at these potentials for both isoforms. At +140 mV the rate of activation of HERG1a was 13.09 ± 0.66 ms (n=3), and HERG1b was 14.25 ± 0.37 ms (n=3). This suggests that the voltage-insensitive step is not affected by the presence or absence of the N-terminal. In contrast, activation at lower potentials was strongly dependent on voltage and was significantly different between isoforms. This indicates that the N-terminal modifies a voltage-sensitive step in the activation pathway. Deactivation is also strongly affected by the presence or absence of the N-terminal. This suggests that the N-terminal affects voltage-dependent transitions near the open state. In conclusion, our data indicate that the HERG N-terminal interacts primarily through modification of voltage-sensitive transitions.

Control of Hv1 Voltage-Gated Proton Channel Opening by Changes in the Transmembrane Voltage and pH Gradients

Hv1 proton channels contain a voltage sensor (VS) domain that is homologous to that of tetrameric voltage-gated cation channels (VGCs) and voltage-sensitive phosphatases (VSPs), but cooperative channel gating by changes in both membrane potential (Vm) and the transmembrane pH gradient (ΔpH = pHO - pHI) is apparently unique to Hv1. The mechanism underlying Vm-ΔpH coupling in Hv1, which results in a linear −40 mV/ΔpH unit shift in the GH+-V relation, is not well understood. In order to isolate ΔpH-dependent steps in the Hv1 activation pathway, we measured the effect of changing ΔpH on expressed H+ currents using patch clamp electrophysiology. We show here that Hv1 opening exhibits both voltage-dependent and voltage-independent kinetic components, but closing is purely voltage-dependent. The forward time constant for the final charge-translocating step in Hv1 activation gating is steeply ΔpH-dependent at acidic pHI but only weakly sensitive to ΔpH at alkaline pHI. In addition, we find that at high pHI, a voltage-independent transition becomes rate-limiting for channel opening. Analysis of the relative contributions of voltage-dependent and -independent Hv1 gating components demonstrates that both are necessary to explain the linear Vm-ΔpH relation between pHI 4.5 and 7.5. The effect of changes in ΔpH on Hv1 gating contrasts with Ci-VSP, indicating that Hv1 is particularly well tuned to respond to small perturbations in ΔpH over the physiological range (pHI ≈ pHO). We propose a new multi-state model to account for complex gating in Hv1 in which VS activation and channel opening occur in distinct transitions and display different sensitivities to changes in ΔpH. Vm-ΔpH coupling in Hv1 thus represents an integrated response to changes in both voltage-dependent and -independent gating steps.

Heteropoda Toxin 2 Interaction with Kv4.3 and Kv4.1 Reveals Differences in Gating Modification

Kv4 (Shal) potassium channels are responsible for the transient outward K(+) currents in mammalian hearts and central nervous systems. Heteropoda toxin 2 (HpTx2) is an inhibitor cysteine knot peptide toxin specific for Kv4 channels that inhibits gating of Kv4.3 in the voltage-dependent manner typical for this type of toxin. HpTx2 interacts with four independent binding sites containing two conserved hydrophobic amino acids in the S3b transmembrane segments of Kv4.3 and the closely related Kv4.1. Despite these similarities, HpTx2 interaction with Kv4.1 is considerably less voltage-dependent, has smaller shifts in the voltage-dependences of conductance and steady-state inactivation, and a 3-fold higher K(d) value. Swapping four nonconserved amino acids in S3b between the two channels exchanges the phenotypic response to HpTx2. To understand these differences in gating modification, we constructed Markov models of Kv4.3 and Kv4.1 activation gating in the presence of HpTx2. Both models feature a series of voltage-dependent steps leading to a final voltage-independent transition to the open state and closely replicate the experimental data. Interaction with HpTx2 increases the energy barrier for channel opening by slowing activation and accelerating deactivation. The greater degree of voltage-dependence in Kv4.3 occurs because it is the voltage-dependent transitions that are most affected by HpTx2; in contrast, it is the voltage-independent step in Kv4.1 that is most affected by the presence of toxin. These data demonstrate the basis for subtype-specificity of HpTx2 and point the way to a general model of gating modifier toxin interaction with voltage-gated ion channels.

Allosteric Features of KCNQ1 Gating Revealed by Alanine Scanning Mutagenesis

Controlled opening and closing of an ion-selective pathway in response to changes of membrane potential is a fundamental feature of voltage-gated ion channels. In recent decades, various details of this process have been revealed with unprecedented precision based on studies of prototypic potassium channels. Though current scientific efforts are focused more on a thorough description of voltage-sensor movement, much less is known about the similarities and differences of the gating mechanisms among potassium channels. Here, we describe the peculiarities of the KCNQ1 gating process in parallel comparison to Shaker. We applied alanine scanning mutagenesis to the S4-S5 linker and pore region and followed the regularities of gating perturbations in KCNQ1. We found a fractional constitutive conductance for wild-type KCNQ1. This component increased significantly in mutants with considerably leftward-shifted steady-state activation curves. In contrast to Shaker, no correlation between V 1/2 and Z parameters was observed for the voltage-dependent fraction of KCNQ1. Our experimental findings are explained by a simple allosteric gating scheme with voltage-driven and voltage-independent transitions. Allosteric features are discussed in the context of extreme gating adaptability of KCNQ1 upon interaction with KCNE β-subunits.

Diversity in Interaction and Gating Modification of Kv4 Channels by Hptx2

HpTx2 is an inhibitor cysteine knot (ICK) toxin that selectively modifies Kv4 gating. Two bulky, hydrophobic amino acids at positions 275 and 276 in the S3b transmembrane segment are required for gating modification by HpTx2; a LV dyad in Kv4.3 and LF in Kv4.1. However, substitutions of other hydrophobic amino acids in Kv4.3 at the same position also allow HpTx2 gating modification, showing that the gross hydrophobic character of the binding site is key for toxin interaction. An S3b mutation, Kv4.3[N280A], reduced the Kd value 10-fold. Surprisingly, Kv4.3[(LV275,N280)A] was as sensitive to HpTx2 as WT Kv4.3. Other hydrophobic amino acid substitutions for N280 showed similar effects. This suggests that these mutations created an additional HpTx2 binding determinant. Gating modification of Kv4.3 is highly voltage-dependent, while that of Kv4.1 is much less so. Swapping four non-conserved S3b amino acids between Kv4.1 and Kv4.3 switched the voltage-dependence of HpTx2 gating modification. To understand this disparity, we modeled the activation gating of Kv4.3 and Kv4.1 and incorporated the effects of HpTx2 into the kinetic parameters of activation. Both models feature four voltage-dependent transitions between closed states followed by a voltage-independent transition to the open state. The disparity in gating modification lies in the relative influence of toxin on the voltage-dependent transitions in the two channels. In Kv4.3, they are more strongly affected by toxin. In contrast the Kv4.1 voltage-independent transition from the closed pre-open to open state is most affected by HpTx2. Therefore, a higher proportion of toxin-bound Kv4.1 channels are in the closed pre-open state; this decreases the voltage dependence of toxin-bound Kv4.1 opening. These data show that amino acids near the HpTx2 binding site play a role in the kinetics of Kv4 channel activation gating.

Open-State Occupancy Prevents Gating Charge Relaxation of N-type (CaV2.2) Calcium Channels

N-type and L-type channels have significant gating differences, and we wondered whether some of these differences are linked to the relationship between charge movement and channel opening. The time constants for N-channel closing ( τ Deact) and Off-gating charge movement ( τQ Off) were compared over a range of voltages. τQ Off was significantly larger than τ Deact at voltages < −10 mV, and the voltage dependence of the τQ Off was less steep than that for τ Deact, which suggests that gating charge relaxation does not limit channel closing. Roscovitine, a drug that slows N-channel closing by holding the channel in a high open-probability state, was found to slow both τQ Off and τ Deact, and thus the time courses of channel closing and gating charge relaxation were similar. Our gating current results were reproduced with the addition of a voltage-independent, closed-closed transition to our previously published two-open-state N-channel model. This work suggests that, like L-type channels, there is a voltage-independent transition along the N-channel activation/deactivation pathway, but this transition occurs between closed states instead of the closed-open states of the L-channel. Also unlike L-type channels, the gating charge appears to be locked into the activated position by the N-channel open state.

HpTx2 Interaction With Kv4.3 and Kv4.1 Reveals Differences in Gating Modification

HpTx2 is an ICK gating modifier toxin specific for Kv4 channels. The Kd of HpTx2 for two similar channels, Kv4.3 and Kv4.1, was 2.4 μM and 7.1 μM, respectively. HpTx2 inhibition of Kv4.3 is highly voltage dependent, shifting the current-voltage relationship to more depolarized potentials; consistent with the classic behavior of gating modifier toxins. However, modification of Kv4.1 gating is much less voltage dependent. Site directed mutagenesis of the S3b interaction site of Kv4.3 and Kv4.1 shows that the same two conserved bulky hydrophobic amino acids are required for HpTx2 interaction with each channel. While the interior of the binding site is conserved between Kv4.3 and Kv4.1, three amino acids adjacent to the binding site are not conserved. Swapping these amino acids between Kv4.3 and Kv4.1 swaps the phenotypic response to toxin, while having minimal effect on gating properties of the channels. We modeled the activation gating of Kv4.3 and Kv4.1 and incorporated the effects of HpTx2 into the kinetic parameters of activation. The model is similar for both channels; it has four voltage-dependent transitions between the closed states followed by voltage-independent transition to an open state. Voltage-dependent transitions in Kv4.3 are more strongly affected by toxin. In Kv4.1, the voltage-independent transition from the closed pre-open to open state is most affected by HpTx2. Therefore, a higher proportion of toxin-bound Kv4.1 channels are in the closed pre-open state, compared to toxin-bound Kv4.3 channels. This decreases the voltage dependence of toxin-bound Kv4.1 opening. The model closely recapitulates our experimental data. These data show that amino acids near the HpTx2 binding site play a role in the kinetics of Kv4 channel activation gating.

Activation and inactivation kinetics of a Ca2+-activated Cl- current: photolytic Ca2+ concentration and voltage jump experiments

The activation kinetics of the endogenous Ca(2+)-activated Cl(-) current (I (Cl,Ca)) from Xenopus oocytes was investigated in excised "giant" membrane patches with voltage and Ca(2+) concentration jumps performed by the photolytic cleavage of the chelator DM-nitrophen. Currents generated by photolytic Ca(2+) concentration jumps begin with a lag phase followed by an exponential rising phase. Both phases show little voltage dependence but are Ca(2+)-dependent. The lag phase decreases from about 10 ms after a small Ca(2+) concentration jump (0.1 microM) to less than 1 ms after a saturating concentration jump (55 microM). The rate constant of the rising phase is half-maximal at about 5 microM. At saturating Ca(2+) concentrations, the rate constant is 400 to 500 s(-1). The Ca(2+) dependence of the stationary current can be described by the Hill equation with n=2.3 and K (0.5)=0.5 microM. The amplitude of the stationary current decreases after the excision of the membrane patch with t (1/2) approximately 5 min (run-down). The activation kinetics of the current elicited by a Ca(2+) concentration jump is not affected by the run-down phenomenon. At low Ca(2+) concentration (0.3 microM), voltage jumps induce a slowly activating current with voltage-independent time-course. Activation is preceded by an initial transient of about 1-ms duration. At saturating Ca(2+) levels (1 mM), the initial transient decays to a stationary current. The transient can be explained by a voltage-dependent inactivation process. The experimental data reported here can be described by a linear five-state reaction model with two sequential voltage-dependent Ca(2+)-binding steps, followed by a voltage-independent rate-limiting transition to the open and a voltage-dependent transition to a closed, inactivated state.

Distinct kinetics of cloned T‐type Ca2 + channels lead to differential Ca2 + entry and frequency‐dependence during mock action potentials

Voltage-dependent activity around the resting potential is determinant in neuronal physiology and participates in the definition of the firing pattern. Low-voltage-activated T-type Ca2 + channels directly affect the membrane potential and control a number of secondary Ca2 + -dependent permeabilities. We have studied the ability of the cloned T-type channels (alpha1G,H,I) to carry Ca2 + currents in response to mock action potentials. The relationship between the spike duration and the current amplitude is specific for each of the T-type channels, reflecting their individual kinetic properties. Typically the charge transfer increases with spike broadening, but the total Ca2 + entry saturates at different spike durations according to the channel type: 4 ms for alpha1G; 7 ms for alpha1H; and > 10 ms for alpha1I channels. During bursts, currents are inhibited and/or transiently potentiated according to the alpha1 channel type, with larger effects at higher frequency. The inhibition may be induced by voltage-independent transitions toward inactivated states and/or channel inactivation through intermediate closed states. The potentiation is explained by an acceleration in the channel activation kinetics. Relatively fast inactivation and slow recovery limit the ability of alpha1G and alpha1H channels to respond to high frequency stimulation ( > 20 Hz). In contrast, the slow inactivation of alpha1I subunits allows these channels to continue participating in high frequency bursts (100 Hz). The biophysical properties of alpha1G, H and I channels will therefore dramatically modulate the effect of neuronal activities on Ca2 + signalling.

Nifedipine inhibits movement of cardiac calcium channels through late, but not early, gating transitions.

L-type Ca2+ channels were studied in guinea pig ventricular myocytes by examining how photoinactivation of nifedipine affected the Ca2+ current (ICa) and the Ca2+ channel gating current (Ig). ICa, blocked by nifedipine, reappeared in qualitatively different phases (immediate and delayed) following photoinactivation of nifedipine. Immediate recovery was attributed to unblock of closed Ca2+ channels, while delayed recovery was attributed to unblock of inactivated channels. In contrast to the ICa results, photoinactivation of nifedipine produced only delayed recovery of Ig. Analysis of these results suggests the following conclusions. First, the actions of inhibitory dihydropyridines can be attributed to binding to either the inactivated or the closed conformation, but only binding to the inactivated state is associated with reduction of Ig. Second, the action of inhibitory dihydropyridines on closed channels is to retard their movement through a final, voltage-independent transition to the open state. This effect seems to be the converse of a major action of stimulatory dihydropyridines and thus is the principal mechanistic difference between stimulatory and inhibitory dihydropyridines.

Two types of A-channels in Lymnaea neurons.

The gating mechanism of A-channels of Lymnaea neurons and the effect of tetraethylammonium (TEA) on these channels were studied using macroscopic recording techniques. Along with the fast-inactivating A-current (Iaf) described earlier we found a slow-inactivating A-current (Ias) in some neurons of the visceral ganglion. Both currents have revealed similar activation kinetics, but differ in the inactivation kinetics and mechanisms. The inactivation kinetics of Ias were satisfactorily described by a sum of two exponentials with rate constants (tau -1) of 28 s-1 and 4.5 s-1 at V = -20 mV. Intracellular TEA reduced the peak amplitudes of Iaf and Ias and slowed the rate of the fast phase of inactivation of Iaf. This resulted in a crossover of the current traces in the presence and absence of TEA, as though it competes with the binding of the inactivating particle. The mechanism of the fast phase of inactivation of Iaf is similar to that of fast inactivation of the Shaker K+ channels which appears to be due to a ball-and-chain mechanism. The slow phases of inactivation of Iaf and Ias reveal properties characteristic of C-type inactivation shown in Shaker K+ channels. A partially coupled model including three pathways for transition of a channel from the closed to open states accurately reproduces all of the experimental data. It has voltage-independent transitions to the inactivation states indicating that inactivation of A-current is not associated with charge movement through the membrane. The results suggest that Lymnaea A-channels seem to be heteromultimeric.

Modeling state-dependent inactivation of membrane currents

Inactivation of many ion channels occurs through largely voltage-independent transitions to an inactivated state from the open state or from other states in the pathway leading to opening of the channel. Because this form of inactivation is state-dependent rather than voltage-dependent, it cannot be described by the standard Hodgkin-Huxley formalism used in virtually all modeling studies of neuronal behavior. Using two examples, cumulative inactivation of the Kv3 potassium channel and inactivation of the fast sodium channel, we extend the standard formalism for modeling macroscopic membrane currents to account for state-dependent inactivation. Our results provide an accurate description of cumulative inactivation of the Kv3 channel, new insight into inactivation of the sodium channel, and a general framework for modeling macroscopic currents when state-dependent processes are involved. In a model neuron, the macroscopic Kv3 current produces a novel short-term memory effect and firing delays similar to those seen in hippocampal neurons.

Voltage-gated transient currents in bovine adrenal fasciculata cells. I. T-type Ca2+ current.

The whole cell version of the patch clamp technique was used to identify and characterize voltage-gated Ca2+ channels in enzymatically dissociated bovine adrenal zona fasciculata (AZF) cells. The great majority of cells (84 of 86) expressed only low voltage-activated, rapidly inactivating Ca2+ current with properties of T-type Ca2+ current described in other cells. Voltage-dependent activation of this current was fit by a Boltzmann function raised to an integer power of 4 with a midpoint at -17 mV. Independent estimates of the single channel gating charge obtained from the activation curve and using the "limiting logarithmic potential sensitivity" were 8.1 and 6.8 elementary charges, respectively. Inactivation was a steep function of voltage with a v1/2 of -49.9 mV and a slope factor K of 3.73 mV. The expression of a single Ca2+ channel subtype by AZF cells allowed the voltage-dependent gating and kinetic properties of T current to be studied over a wide range of potentials. Analysis of the gating kinetics of this Ca2+ current indicate that T channel activation, inactivation, deactivation (closing), and reactivation (recovery from inactivation) each include voltage-independent transitions that become rate limiting at extreme voltages. Ca2+ current activated with voltage-dependent sigmoidal kinetics that were described by an m4 model. The activation time constant varied exponentially at test potentials between -30 and +10 mV, approaching a voltage-independent minimum of 1.6 ms. The inactivation time constant (tau i) also decreased exponentially to a minimum of 18.3 ms at potentials positive to 0 mV. T channel closing (deactivation) was faster at more negative voltages; the deactivation time constant (tau d) decreased from 8.14 +/- 0.7 to 0.48 +/- 0.1 ms at potentials between -40 and -150 mV. T channels inactivated by depolarization returned to the closed state along pathways that included two voltage-dependent time constants. tau rec-s ranged from 8.11 to 4.80 s when the recovery potential was varied from -50 to -90 mV, while tau rec-f decreased from 1.01 to 0.372 s. At potentials negative to -70 mV, both time constants approached minimum values. The low voltage-activated Ca2+ current in AZF cells was blocked by the T channel selective antagonist Ni2+ with an IC50 of 20 microM. At similar concentrations, Ni2+ also blocked cortisol secretion stimulated by adrenocorticotropic hormone. Our results indicate that bovine AZF cells are distinctive among secretory cells in expressing primarily or exclusively T-type Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)

On the activation—inactivation coupling in Shaker potassium channels

The ‘ball-and-chain’ model suggests the existence of a negative site which may attract the positively charged inactivation ball to occlude the pore when the channel is in the open state. For Shaker K + channels, we propose that the state-dependent negative site be tryptophan-435, which becomes negatively charged after receiving an electron from tyrosine-445. The kinetic scheme for the channel's activation-inactivation coupling as derived from the YW-gated model resembles a successful ‘scheme 8’ proposed by Zagotta and Aldrich. Our model suggests that the final rapid voltage-independent transition to the open state is due to the deprotonation of tyrosine-445.

Inactivation of the sodium channel. I. Sodium current experiments.

Inactivation of sodium conductance has been studied in squid axons with voltage clamp techniques and with the enzyme pronase which selectively destroys inactivation. Comparison of the sodium current before and after pronase treatment shows a lag of several hundred microseconds in the onset of inactivation after depolarization. This lag can of several hundred microseconds in the onset of inactivation after polarization. This lag can also be demonstrated with double-pulse experiments. When the membrane potential is hyperpolarized to -140 mV before depolarization, both activation and inactivation are delayed. These findings suggest that inactivation occurs only after activation are delayed. These findings suggest that inactivation occurs only after activation; i.e. that the channels must open before they can inactivate. The time constant of inactivation measured with two pulses (tau(c)) is the same as the one measured from the decay of the sodium current during a single pulse (tau(h)). For large depolarizations, steady-state inactivation becomes more incomplete as voltage increases; but it is relatively complete and appears independent of voltage when determined with a two- pulse method. This result confirms the existence of a second open state for Na channels, as proposed by Chandler and Meves (1970. J. Physiol. [Lond.]. 211:653-678). The time constant of recovery from inactivation is voltage dependent and decreases as the membrane potential is made more negative. A model for Na channels is presented which has voltage-dependent transitions between the closed and open states, and a voltage-independent transition between the open and the inactivated state. In this model the voltage dependence of inactivation is a consequence of coupling to the activation process.