Voltage Dependence Of Charge Movement Research Articles

Biophysical reviews top five: voltage-dependent charge movement in nerve and muscle.

The discovery of gating currents and asymmetric charge movement in the early 1970s represented a remarkable leap forward in our understanding of the biophysical basis of voltage-dependent events that underlie electrical signalling that is vital for nerve and muscle function. Gating currents and charge movement reflect a fundamental process in which charged amino acid residues in an ion channel protein move in response to a change in the membrane electrical field and therefore activate the specific voltage-dependent response of that protein. The detection of gating currents and asymmetric charge movement over the past 50 years has been pivotal in unraveling the multiple molecular and intra-molecular processes which lead to action potentials in excitable tissues and excitation-contraction (EC) coupling in skeletal muscle. The recording of gating currents and asymmetric charge movement remains an essential component of investigations into the basic molecular mechanisms of neuronal conduction and muscle contraction.

On the frequency response of prestin charge movement in membrane patches

Outer hair cell (OHC) nonlinear membrane capacitance derives from voltage-dependent sensor charge movements within the membrane protein prestin (SLC26a5) that drive OHC electromotility. The ability of the protein to influence hearing depends on its reaction to membrane receptor potentials across auditory frequency. Estimates of prestin’s frequency response have been evaluated by several groups out to tens of kHz in voltage-clamped macro-patches of OHC membrane. The response is a power function of frequency that is down 40 dB at 77 kHz. Despite these observations, concerns remain that the macro-patch approach is flawed due to mechanical constraints of pipette solution column load or patch size itself. In the absence of these influences, prestin’s frequency response is posited by some to be ultrasonic in nature. Here we evaluate the influence of these putative confounding factors on prestin’s frequency response. We show that neither pipette column height nor negative or positive pipette pressure substantially influence total sensor charge frequency response. Additionally, patch surface area has negligible influence. We conclude that the speed of voltage-driven conformational changes in prestin within the plasma membrane is accurately assessed with the macro-patch technique, permitting investigations of membrane characteristics that can substantially alter prestin’s performance bandwidth. We illustrate significant alterations in bandwidth by perturbation of membrane fluidity and chloride anion concentration. Finally, we speculate that OHC membrane characteristics may differ along the tonotopic axis of the cochlea to tune nonlinear membrane capacitance frequency cutoffs.

Two-Dimensional Brain Microtubule Structures Behave as Memristive Devices

Microtubules (MTs) are cytoskeletal structures that play a central role in a variety of cell functions including cell division and cargo transfer. MTs are also nonlinear electrical transmission lines that produce and conduct electrical oscillations elicited by changes in either electric field and/or ionic gradients. The oscillatory behavior of MTs requires a voltage-sensitive gating mechanism to enable the electrodiffusional ionic movement through the MT wall. Here we explored the electrical response of non-oscillating rat brain MT sheets to square voltage steps. To ascertain the nature of the possible gating mechanism, the electrical response of non-oscillating rat brain MT sheets (2D arrays of MTs) to square pulses was analyzed under voltage-clamping conditions. A complex voltage-dependent nonlinear charge movement was observed, which represented the summation of two events. The first contribution was a small, saturating, voltage-dependent capacitance with a maximum charge displacement in the range of 4 fC/μm2. A second, major contribution was a non-saturating voltage-dependent charge transfer, consistent with the properties of a multistep memristive device. The memristive capabilities of MTs could drive oscillatory behavior, and enable voltage-driven neuromorphic circuits and architectures within neurons.

Chloride Anions Regulate Kinetics but Not Voltage-Sensor Qmax of the Solute Carrier SLC26a5

In general, SLC26 solute carriers serve to transport a variety of anions across biological membranes. However, prestin (SLC26a5) has evolved, now serving as a motor protein in outer hair cells (OHCs) of the mammalian inner ear and is required for cochlear amplification, a mechanical feedback mechanism to boost auditory performance. The mechanical activity of the OHC imparted by prestin is driven by voltage and controlled by anions, chiefly intracellular chloride. Current opinion is that chloride anions control the Boltzmann characteristics of the voltage sensor responsible for prestin activity, including Qmax, the total sensor charge moved within the membrane, and Vh, a measure of prestin’s operating voltage range. Here, we show that standard narrow-band, high-frequency admittance measures of nonlinear capacitance (NLC), an alternate representation of the sensor’s charge-voltage (Q-V) relationship, is inadequate for assessment of Qmax, an estimate of the sum of unitary charges contributed by all voltage sensors within the membrane. Prestin’s slow transition rates and chloride-binding kinetics adversely influence these estimates, contributing to the prevalent concept that intracellular chloride level controls the quantity of sensor charge moved. By monitoring charge movement across frequency, using measures of multifrequency admittance, expanded displacement current integration, and OHC electromotility, we find that chloride influences prestin kinetics, thereby controlling charge magnitude at any particular frequency of interrogation. Importantly, however, this chloride dependence vanishes as frequency decreases, with Qmax asymptoting at a level irrespective of the chloride level. These data indicate that prestin activity is significantly low-pass in the frequency domain, with important implications for cochlear amplification. We also note that the occurrence of voltage-dependent charge movements in other SLC26 family members may be hidden by inadequate interrogation timescales, and that revelation of such activity could highlight an evolutionary means for kinetic modifications within the family to address hearing requirements in mammals.

Functional Analysis of CaV1.1 L-Type Calcium Channels Expressed in tsA201 Cells

Expression of modified CaV1.1 constructs in dysgenic (CaV1.1 null) myotubes has been useful in the determination of regions of the CaV1.1 that are important for skeletal muscle excitation-contraction (EC) coupling, however the use of this system to probe CaV1.1 channel gating has been problematic because dysgenic myotubes express a variety of other ion channels which conduct Ca2+ and contribute to intramembrane gating charge movement. This obstacle can be overcome by recording from tsA201 cells coexpressing CaV1.1 α1S, β1a, α2δ1 and Stac3 subunits. Our group has previously characterized, in dysgenic myotubes, a CaV1.1 mutation (R174W) which affects the innermost basic residue of the voltage-sensing S4 α-helix of Repeat I; R174W abolished activation of the L-type Ca2+ current in response to depolarization without affecting the magnitude or voltage-dependence of charge movement. Using tsA201 cells, we show that this dysfunction arises from an impaired gating transition of the RI voltage-sensor that is not detectable with short (20 ms) depolarizations. Specifically, we were unable to recruit the documented slow-moving gating charge component attributable to RI with longer (200 ms) test potentials. We next assessed the effects of corresponding mutations in Repeats II (K537W), III (R906W) and IV (K1245W) in order to determine the way in which translocation of these voltage-sensors affects channel opening. Currents and intramembrane charge movements produced by R906W were both smaller and shifted (∼10 mV) to more depolarizing test potentials. In contrast to the severe loss of function observed with R174W and the somewhat impaired gating of R906W, the K537W and K1245W mutants produced L-type currents with enormous peak amplitudes almost three times greater than wild-type CaV1.1 and with greatly impaired deactivation. Supported by Conacyt 169006 (UM), the Boettcher Foundation (RAB), and NIH AR055104, NIH AR052354 and MDA 277475 (KGB).

Neuronal ClC-3 Splice Variants Differ in Subcellular Localizations, but Mediate Identical Transport Functions

ClC-3 is a member of the CLC family of anion channels and transporters, for which multiple functional properties and subcellular localizations have been reported. Since alternative splicing often results in proteins with diverse properties, we investigated to what extent alternative splicing might influence subcellular targeting and function of ClC-3. We identified three alternatively spliced ClC-3 isoforms, ClC-3a, ClC-3b, and ClC-3c, in mouse brain, with ClC-3c being the predominant splice variant. Whereas ClC-3a and ClC-3b are present in late endosomes/lysosomes, ClC-3c is targeted to recycling endosomes via a novel N-terminal isoleucine-proline (IP) motif. Surface membrane insertion of a fraction of ClC-3c transporters permitted electrophysiological characterization of this splice variant through whole-cell patch clamping on transfected mammalian cells. In contrast, neutralization of the N-terminal dileucine-like motifs was required for functional analysis of ClC-3a and ClC-3b. Heterologous expression of ClC-3a or ClC-3b carrying mutations in N-terminal dileucine motifs as well as WTClC-3c in HEK293T cells resulted in outwardly rectifying Cl(-) currents with significant capacitive current components. We conclude that alternative splicing of Clcn3 results in proteins with different subcellular localizations, but leaves the transport function of the proteins unaffected.

The Amino Terminus Controls Sorting and Voltage Dependence of CLC 3

ClC-3 is a member of the CLC family of anion channels and anion-proton exchangers. So far, no agreement has been reached its biophysical properties and its subcellular localization. Some groups reported ClC-3 being an intracellular transporter, whereas other experimental evidence supports anion channel function and plasmalemmal localization. Since alternative splicing might result in distinct ClC-3 proteins with diverse functions we decided to compare biophysical properties and subcellular localization of known ClC-3 splice variants, ClC-3A and ClC-3B, and to compare them with the well characterized short isoform ClC-3short (ACS Chem Neurosci 4, 994-1003). Both isoforms target exclusively to intracellular compartments, ClC-3A to the endosomal/lysosomal system and ClC-3B to Golgi apparatus. ClC-3short contains a dileucine-like motif in its amino terminus, and we here identified two additional dileucine-like motifs in this region of ClC-3A/B. These motifs interact with clathrin, and their disruption permit surface membrane insertion and electrophysiological analysis of ClC-3A/B. Heterologous expression of ClC-3A/B26/41/71 in HEK293T cells results in outwardly rectifying Cl- currents with amplitudes up to 2 nA. Similar to the short isoform, ClC-3A26/41/71 and ClC-3B26/41/71 exhibit significant capacitive current components indicating that there is a high probability of non-complete transport cycles under our conditions. For ClC-3A/B26/41/71 the ratio of transport currents and gating charge is significantly lower than for the short one. Moreover, the voltage dependence of charge movement was shifted to more positive potentials compared to the short isoform. The kinetic of the gating charge movements was faster for ClC-3A26/41/71 than the ClC-3B26/41/71. Our results show that all known ClC-3 splice variants are transporters, with significant capacitive currents and pronounced outward rectification. N-terminal alternative splicing does not only affect trafficking, but also influences the voltage dependence of ClC-3, permitting adjustment of transport functions to distinct physiological roles.

Effects of Substituting Tryptophan for Basic Residues in the S4 Voltage-Sensing Helices of CaV1.1

In skeletal muscle, CaV1.1 serves dual functions as the voltage sensor for excitation-contraction (EC) coupling and as an L-type Ca2+ channel. It has been long established that L-type Ca2+ current activates at potentials ∼20 mV more depolarized than intramembrane charge movement or EC coupling. We recently characterized a CaV1.1 mutation (R174W) which affects the innermost basic residue of the voltage-sensing S4 helix of Repeat I and results in malignant hyperthermia susceptibility in humans. Interestingly, R174W abolishes activation of the L-type current in response to 200 ms depolarizations without affecting the voltage dependence of intramembrane charge movement or of SR Ca2+ release. In this study, we have made corresponding mutations in Repeats II (K537W), III (R906W) and IV (K1245W) and expressed these mutants in dysgenic (CaV1.1 null) myotubes. Confocal imaging of affixed YFP tags indicated that all the mutants were correctly targeted to membrane-SR junctions. In striking contrast to the virtual loss of channel function observed with R174W, the K537W and K1245W mutants produced enormous L-type currents with peak amplitudes nearly 8-fold greater than wild-type CaV1.1 and greatly impaired deactivation. Even though these two gain of channel function mutants activated at substantially more hyperpolarized potentials (∼20 mV shift), the voltage-dependence of charge movement and SR Ca2+ release were largely unaffected. On the other hand, R906W produced currents of similar amplitude to wild-type CaV1.1 but with a consistent ∼15 mV depolarizing shift in activation. We are currently investigating the effects of the R906W mutation on the voltage dependence of charge movement and SR Ca2+ release in order to determine whether RIII plays a more critical role in engaging EC coupling than the other conserved Repeats of CaV1.1. Supported by NIH AR055104 (KGB) and AG038778 (RAB), and MDA176448 (KGB).

The V499G/Y501H Mutation Impairs Fast Motor Kinetics of Prestin and Has Significance for Defining Functional Independence of Individual Prestin Subunits

Outer hair cells (OHCs) are a mammalian innovation for mechanically amplifying sound energy to overcome the viscous damping of the cochlear partition. Although the voltage-dependent OHC membrane motor, prestin, has been demonstrated to be essential for mammalian cochlear amplification, the molecular mechanism by which prestin converts electrical energy into mechanical displacement/force remains elusive. Identifying mutations that alter the motor function of prestin provides vital information for unraveling the energy transduction mechanism of prestin. We show that the V499G/Y501H mutation does not deprive prestin of its voltage-induced motor activity, but it does significantly impair the fast motor kinetics and voltage operating range. Furthermore, mutagenesis studies suggest that Val-499 is the primary site responsible for these changes. We also show that V499G/Y501H prestin forms heteromers with wild-type prestin and that the fast motor kinetics of wild-type prestin is not affected by heteromer formation with V499G/Y501H prestin. These results suggest that prestin subunits are individually functional within a given multimer.

Dissection of Proton Inhibition Mechanism in the H+/Cl− Exchanger, CLC-7

ClC-7 is a voltage-activated 1H+:2Cl− exchanger that serves a critical yet somewhat uncertain role in lysosomal membranes of diverse mammalian cell types. Despite trafficking to acidic vesicles in the cell, proton inhibition is a functional characteristic of mammalian ClC H+/Cl− exchangers in general and of ClC-7 in particular. We sought to dissect the mechanism of proton inhibition in ClC-7 function. Leisle L, Ludwig CF, et. al recently utilized a method to robustly redirect ClC-7 to the plasma membrane by mutating lysosomal sorting motifs at the amino terminus of ClC-7 and by co-expressing an essential auxiliary subunit called Ostm-1 (Leisle, Ludwig, et al, 2011 EMBO J, 30,2140). using this strategy, we describe extracellular [H+]-dependent inhibition of ClC-7 mediated ionic current in comparison with published data for ClC-5 from experiments by Picollo, Malvezzi, and Accardi (2010, JGP, 135, 653). In the tested conditions, ClC-7 is more sensitive than ClC-5 to extracellular H+ (pK(0 mV) = 7.9 estimated for ClC-7/Ostm-1 and pK(0 mV) = 6.6 reported for ClC-5 expressed in oocytes). Further, we evaluate effects of increasing extracellular [H+] and varying intracellular [Cl−] on voltage-dependent transient current intrinsic to a transport deficient mutant of ClC-7 (E314A). Increasing [H+]ext shifts the half-activating voltage of voltage-dependent charge movement in ClC-7 (mean V1/2 ≥ +20 mV shift per pH unit increase), but does not drastically change the apparent charge valence (mean z = 0.85 to 0.95 qe at all [H+]ext tested). This result suggests that the voltage-dependent transient current does not arise from protonation of ClC-7, but that extracellular pH changes the work required for the electric field to elicit charge movement.

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Tonotopic Relationships Reveal the Charge Density Varies along the Lateral Wall of Outer Hair Cells

Outer hair cells amplify and improve the frequency selectivity of sound within the mammalian cochlea through a sound-evoked receptor potential that induces an electromechanical response in their lateral wall membrane. We experimentally show that the membrane area and linear membrane capacitance of outer hair cells increases exponentially with the electrically evoked voltage-dependent charge movement (QT) and peak membrane capacitance (Cpeak). We determine the size of the different functional regions (e.g., lateral wall, synaptic basal pole) of the polarized cells from the tonotopic relationships. We then establish that QT and Cpeak increase with the logarithm of the lateral wall area (ALW) and determine from the functions that the charge (σLW, pC/μm2) and peak (ρLW, pF/μm2) densities vary inversely with ALW (σLW = 1.3/ALW and ρLW = 9/ALW). This shows contrary to conventional wisdom that σLW and ρLW are not constant along the length of an individual outer hair cell.

Depolarization induces a conformational change in the binding site region of the M2 muscarinic receptor.

G protein-coupled receptors play a central role in signal transduction and were only known to be activated by agonists. Recently it has been shown that membrane potential also affects the activity of G protein-coupled receptors. For the M(2) muscarinic receptor, it was further shown that depolarization induces charge movement. A tight correlation was found between the voltage-dependence of the charge movement and the voltage-dependence of the agonist binding. Here we examine whether depolarization-induced charge movement causes a conformational change in the M(2) receptor that may be responsible for the voltage-dependence of agonist binding. Using site-directed fluorescence labeling we show a voltage-dependent fluorescence signal, reflecting a conformational change, which correlates with the voltage-dependent charge movement. We further show that selected mutations in the orthosteric site abolish the fluorescence signal and concomitantly, the voltage-dependence of the agonist binding. Surprisingly, mutations in the allosteric site also abolished the voltage-dependence of agonist binding but did not reduce the fluorescence signal. Finally, we show that treatments, which reduced the charge movement or hindered the coupling between the charge movement and the voltage-dependent binding, also reduced the fluorescence signal. Our results demonstrate that depolarization-induced conformational changes in the orthosteric binding site underlie the voltage-dependence of agonist binding. Our results are also unique in suggesting that the allosteric site is also involved in controlling the voltage-dependent agonist binding.

Dissecting the electromechanical coupling mechanism of the motor-protein prestin.

Prestin, which is a member of the solute carrier 26 anion transporter family (SLC26A5), is a voltage-dependent membrane-based motor protein that confers electromotility on mammalian cochlear outer hair cells (OHCs).1 OHCs are a mammalian innovation, their presence2 and their endowment with functional prestin is essential for normal hearing of mammals.3 In order to clarify the molecular mechanism underlying the voltage-dependent motility of prestin, precise description of the relation between voltage-induced prestin-associated charge movement and the resulting cell displacement is essential. By simultaneously measuring voltage-dependent charge movement, which is manifested in the nonlinear capacitance (NLC) of the cell membrane, and voltage-induced OHC displacement, we provided compelling experimental evidence that prestin-associated charge movement and the resulting electromotility are fully coupled, and that prestin has at least two voltage-dependent conformational transition steps. These findings provide a basis for understanding the molecular mechanism of prestin. Here we discuss the relevance of our finding in the elucidation of the voltage-dependent motor mechanism of prestin, and speculate about possible voltage sensing mechanisms of the molecule.

Dissecting the electromechanical coupling mechanism of the motorprotein prestin

Prestin, which is a member of the solute carrier 26 anion transporter family (SLC26A5), is a voltagedependent membrane-based motor protein that confers electromotility on mammalian cochlear outer hair cells (OHCs).1 OHCs are a mammalian innovation, their presence2 and their endowment with functional prestin is essential for normal hearing of mammals.3 In order to clarify the molecular mechanism underlying the voltage-dependent motility of prestin, precise description of the relation between voltage-induced prestin-associated charge movement and the resulting cell displacement is essential. By simultaneously measuring voltage-dependent charge movement, which is manifested in the nonlinear capacitance (NLC) of the cell membrane, and voltage-induced OHC displacement, we provided compelling experimental evidence that prestin-associated charge movement and the resulting electromotility are fully coupled, and that prestin has at least two voltage-dependent conformational transition steps. These findings provide a basis for understanding the molecular mechanism of prestin. Here we discuss the relevance of our finding in the elucidation of the voltage-dependent motor mechanism of prestin, and speculate about possible voltage sensing mechanisms of the molecule.

Evidence That Prestin Has at Least Two Voltage-dependent Steps

Prestin is a voltage-dependent membrane-spanning motor protein that confers electromotility on mammalian cochlear outer hair cells, which is essential for normal hearing of mammals. Voltage-induced charge movement in the prestin molecule is converted into mechanical work; however, little is known about the molecular mechanism of this process. For understanding the electromechanical coupling mechanism of prestin, we simultaneously measured voltage-dependent charge movement and electromotility under conditions in which the magnitudes of both charge movement and electromotility are gradually manipulated by the prestin inhibitor, salicylate. We show that the observed relationships of the charge movement and the physical displacement (q-d relations) are well represented by a three-state Boltzmann model but not by a two-state model or its previously proposed variant. Here, we suggest a molecular mechanism of prestin with at least two voltage-dependent conformational transition steps having distinct electromechanical coupling efficiencies.

A highly expressing Tet-inducible cell line recapitulates in situ developmental changes in prestin's Boltzmann characteristics and reveals early maturational events

Prestin is the motor protein within the lateral membrane of outer hair cells (OHCs), and it is required for mammalian cochlear amplification. Expression of prestin precedes the onset of hearing in mice, and it has been suggested that prestin undergoes a functional maturation within the membrane coincident with the onset of hearing. We have developed a tetracycline-inducible prestin-expressing cell line that we have used to model prestin's functional maturation. We used prestin's voltage-dependent nonlinear charge movement (or nonlinear capacitance) as a test of function and correlated it to biochemical measures of prestin expressed on the cell surface. An initial stage of slow growth in charge density is accompanied by a rapid increase in our estimate of charge carried by an individual motor. A rapid growth in charge density follows and strongly correlates with an increasing ratio between an apparently larger and smaller monomer, suggesting that the latter exerts a dominant-negative effect on function. Finally, there is a gradual depolarizing shift in the voltage of peak capacitance, similar to that observed in developing OHCs. This inducible system offers many opportunities for detailed studies of prestin.

Combinatorial Cysteine Mutagenesis Reveals a Critical Intramonomer Role for Cysteines in Prestin Voltage Sensing

Prestin is a member of the SLC26 family of anion transporters and is responsible for electromotility in outer hair cells, the basis of cochlear amplification in mammals. It is an anion transporting transmembrane protein, possessing nine cysteine residues, which generates voltage-dependent charge movement. We determine the role these cysteine residues play in the voltage sensing capabilities of prestin. Mutations of any single cysteine residue had little or no effect on charge movement. However, using combinatorial substitution mutants, we identified a cysteine residue pair (C415 and either C192 or C196) whose mutation reduced or eliminated charge movement. Furthermore, we show biochemically that surface expression of mutants with markedly reduced functionality can be near normal; however, we identify two monomers of the protein on the surface of the cell, the larger of which correlates with surface charge movement. Because we showed previously by Förster resonance energy transfer that monomer interactions are required for charge movement, we tested whether disulfide interactions were required for dimerization. Using Western blots to detect oligomerization of the protein in which variable numbers of cysteines up to and including all nine cysteine residues were mutated, we show that disulfide bond formation is not essential for dimer formation. Taken together, we believe these data indicate that intramembranous cysteines are constrained, possibly via disulfide bond formation, to ensure structural features of prestin required for normal voltage sensing and mechanical activity.

Voltage Clamping a Supported Bilayer

Supported bilayer has many advantages over conventional black lipid bilayer. A supported bilayer is highly stable and can be made with a large surface area. Membrane proteins can be incorporated in very large numbers without disruption of the bilayer, thereby allowing robust macroscopic recordings. A very important advantage is that incorporated proteins are immobile. This lack of mobility is essential for the study of conformational changes with single molecule fluorescence. We are interested in studying gating charge movement and conformational changes in voltage dependent membrane proteins such as a voltage dependent K+ channel. For this purpose, we have developed an essentially electrostatic voltage clamp system for a supported bilayer that allows measurement of intramembrane transient currents but not DC currents. The supported bilayer is formed by liposome fusion on top of a semiconductor substrate that serves as the reference electrode. Electrolyte solution is present above the bilayer where an Ag/AgCl electrode serves as the active electrode. The electrode pair is connected to conventional voltage clamp electronics that imposes the voltage and measures the current. We verified supported bilayer formation by the decrease of the total capacitance. Furthermore, we have verified that a voltage is imposed across the bilayer by using voltage dependent fluorescent membrane probes. The electrolyte/supported bilayer/electrode system is essentially linear across a voltage range of −300 to +300 mV. We have seen that direct incorporation of the voltage dependent protein KvAP into the supported bilayer modifies the kinetics of the transient currents as well as the voltage dependence of charge movement. This method opens the possibility for studies of simultaneous gating charge movement electrically and voltage dependent conformational change spectroscopically in purified membrane proteins. Support: NIH GM030376.

The α1S III-IV Loop Influences 1,4-Dihydropyridine Receptor Gating but Is Not Directly Involved in Excitation-Contraction Coupling Interactions with the Type 1 Ryanodine Receptor

In skeletal muscle, coupling between the 1,4-dihydropyridine receptor (DHPR) and the type 1 ryanodine receptor (RyR1) underlies excitation-contraction (EC) coupling. The III-IV loop of the DHPR alpha(1S) subunit binds to a segment of RyR1 in vitro, and mutations in the III-IV loop alter the voltage dependence of EC coupling, raising the possibility that this loop is directly involved in signal transmission from the DHPR to RyR1. To clarify the role of the alpha(1S) III-IV loop in EC coupling, we examined the functional properties of a chimera (GFP-alpha(1S)[III-IVa]) in which the III-IV loop of the divergent alpha(1A) isoform replaced that of alpha(1S). Dysgenic myotubes expressing GFP-alpha(1S)[III-IVa] yielded myoplasmic Ca(2+) transients that activated at approximately 10 mV more hyperpolarized potentials and that were approximately 65% smaller than those of GFP-alpha(1S). A similar reduction was observed in voltage-dependent charge movements for GFP-alpha(1S)[III-IVa], indicating that the chimeric channels trafficked less well to the membrane but that those that were in the membrane functioned as efficiently in EC coupling as GFP-alpha(1S). Relative to GFP-alpha(1S), L-type currents mediated by GFP-alpha(1S)[III-IVa] were approximately 40% smaller and activated at approximately 5 mV more hyperpolarized potentials. The altered gating of GFP-alpha(1S)[III-IVa] was accentuated by exposure to +/-Bay K 8644, which caused a much larger hyperpolarizing shift in activation compared with its effect on GFP-alpha(1S). Taken together, our observations indicate that the alpha(1S) III-IV loop is not directly involved in EC coupling but does influence DHPR gating transitions important both for EC coupling and activation of L-type conductance.