Saturation effects and rectifier properties of sodium channels in human skeletal muscle

Sodium outward currents were measured in human myoballs with the whole-cell recording method. The electro-chemical gradient of the sodium ions across the cell membrane was modified over a wide range by variations of the clamped membrane potential and of the internal and external soidum concentration. Up to 50 mV positive to the sodium equilibrium potential, E_Na, the current-voltage relation is linear. At a potential 80 mV positive to E_Na the sodium outward current has a maximum and decreases with a further increase in electrochemical gradient. Investigating the instantaneous current change in experiments in which the membrane potential was changed while the channels were already open we could exclude the possibility that the gates of activation or inactivation are responsible for this effect. Therefore we postulate that the sodium channel has a valve-like mechanism producing a negative slope conductance at highly positive membrane potentials, a current saturation with self-inhibition by the intracellular sodium concentration, and a blockade of the channel on reduction of the extracellular sodium concentration.This work was supported by the Deutsche Forschungsgemeinschaft (Ru 138/15-1, 15-2)     

Chimeric study of sodium channels from rat skeletal and cardiac muscle.

Two isoforms of voltage-dependent Na channels, cloned from rat skeletal muscle, were expressed in Xenopus oocytes. The currents of rSkM1 and rSkM2 differ functionally in 4 properties: (i) tetrodotoxin (TTX) sensitivity, (ii) mu-conotoxin (mu-CTX) sensitivity, (iii) amplitude of single channel currents, and (iv) rate of inactivation. rSkM1 is sensitive to both TTX and mu-CTX. rSkM2 is resistant to both toxins. Currents of rSkM1 have a higher single channel conductance and a slower rate of inactivation than those of rSkM2. We constructed (i) chimeras by interchanging domain 1 (D1) between the two isoforms, (ii) block mutations of 22 amino acids in length that interchanged parts of the loop between transmembrane segments S5 and S6 in both D1 and D4, and (iii) point mutations in the SS2 region of this loop in D1. The TTX sensitivity could be switched between the two isoforms by the exchange of a single amino acid, tyrosine-401 in rSkM1 and cysteine-374 in rSkM2 in SS2 of D1. By contrast most chimeras and point mutants had an intermediate sensitivity to mu-CTX when compared with the wild-type channels. The point mutant rSkM1 (Y401C) had an intermediate single-channel conductance between those of the wild-type isoforms, whereas rSkM2 (C374Y) had a slightly lower conductance than rSkM2. The rate of inactivation was found to be determined by multiple regions of the protein, since chimeras in which D1 was swapped had intermediate rates of inactivation compared with the wild-type isoforms.     

Comparison of heterologously expressed human cardiac and skeletal muscle sodium channels.

In this study we have expressed and characterized recombinant cardiac and skeletal muscle sodium channel alpha subunits in tsA-201 cells under identical experimental conditions. Unlike the Xenopus oocyte expression system, in tsA-201 cells (transformed human embryonic kidney) both channels seem to gate rapidly, as in native tissue. In general, hSkM1 gating seemed faster than hH1 both in terms of rate of inactivation and rate of recovery from inactivation as well as time to peak current. The midpoint of the steady-state inactivation curve was approximately 25 mV more negative for hH1 compared with hSkM1. In both isoforms, the steady-state channel availability relationships ("inactivation curves") shifted toward more negative membrane potentials with time. The cardiac isoform showed a minimal shift in the activation curve as a function of time after whole-cell dialysis, whereas hSkM1 showed a continued and marked negative shift in the activation voltage dependence of channel gating. This observation suggests that the mechanism underlying the shift in inactivation voltage dependence may be similar to the one that is causing the shift in the activation voltage dependence in hSkM1 but that this is uncoupled in the cardiac isoform. These results demonstrate the utility and limitations of measuring cardiac and skeletal muscle recombinant Na+ channels in tsA-201 cells. This baseline characterization will be useful for future investigations on channel mutants and pharmacology.     

Opentime heterogeneity during bursting of sodium channels in frog skeletal muscle.

Single voltage-activated Na+ channel currents were obtained from membrane patches on internally dialyzed skeletal muscle segments of adult frogs. The high channel density in these membranes permitted frequent observation of the "bursting mode" of individual Na+ channels during 400-ms records. We examined the opentimes within and between bursts on individual membrane patches. We used a new nonparametric statistical procedure to test for heterogeneity in the opentime distributions. We found that although 80% of all bursts consisted of opentimes drawn from a single distribution, the opentime distribution varied significantly from burst to burst. Significant heterogeneity was also detected within the remaining 20% of individual bursts. Our results indicate that the gating kinetics of individual Na+ channels are heterogeneous, and that they may occasionally change in a single channel.     

Possible roles of exceptionally conserved residues around the selectivity filters of sodium and calcium channels

In the absence of x-ray structures of sodium and calcium channels their homology models are used to rationalize experimental data and design new experiments. A challenge is to model the outer-pore region that folds differently from potassium channels. Here we report a new model of the outer-pore region of the NaV1.4 channel, which suggests roles of highly conserved residues around the selectivity filter. The model takes from our previous study (Tikhonov, D. B., and Zhorov, B. S. (2005) Biophys. J. 88, 184-197) the general disposition of the P-helices, selectivity filter residues, and the outer carboxylates, but proposes new intra- and inter-domain contacts that support structural stability of the outer pore. Glycine residues downstream from the selectivity filter are proposed to participate in knob-into-hole contacts with the P-helices and S6s. These contacts explain the adapted tetrodotoxin resistance of snakes that feed on toxic prey through valine substitution of isoleucine in the P-helix of repeat IV. Polar residues five positions upstream from the selectivity filter residues form H-bonds with the ascending-limb backbones. Exceptionally conserved tryptophans are engaged in inter-repeat H-bonds to form a ring whose π-electrons would facilitate passage of ions from the outer carboxylates to the selectivity filter. The outer-pore model of CaV1.2 derived from the NaV1.4 model is also stabilized by the ring of exceptionally conservative tryptophans and H-bonds between the P-helices and ascending limbs. In this model, the exceptionally conserved aspartate downstream from the selectivity-filter glutamate in repeat II facilitates passage of calcium ions to the selectivity-filter ring through the tryptophan ring. Available experimental data are discussed in view of the models.     

Structural determinants of slow inactivation in human cardiac and skeletal muscle sodium channels.

Skeletal and heart muscle excitability is based upon the pool of available sodium channels as determined by both fast and slow inactivation. Slow inactivation in hH1 sodium channels significantly differs from slow inactivation in hSkM1. The beta(1)-subunit modulates fast inactivation in human skeletal sodium channels (hSkM1) but has little effect on fast inactivation in human cardiac sodium channels (hH1). The role of the beta(1)-subunit in sodium channel slow inactivation is still unknown. We used the macropatch technique on Xenopus oocytes to study hSkM1 and hH1 slow inactivation with and without beta(1)-subunit coexpression. Our results indicate that the beta(1)-subunit is partly responsible for differences in steady-state slow inactivation between hSkM1 and hH1 channels. We also studied a sodium channel chimera, in which P-loops from each domain in hSkM1 sodium channels were replaced with corresponding regions from hH1. Our results show that these chimeras exhibit hH1-like properties of steady-state slow inactivation. These data suggest that P-loops are structural determinants of sodium channel slow inactivation, and that the beta(1)-subunit modulates slow inactivation in hSkM1 but not hH1. Changes in slow inactivation time constants in sodium channels coexpressed with the beta(1)-subunit indicate possible interactions among the beta(1)-subunit, P-loops, and the slow inactivation gate in sodium channels.     

The differing roles of the classical and mannose-binding lectin complement pathways in the events following skeletal muscle ischemia-reperfusion

Complement is an important mediator of the injuries observed after skeletal muscle ischemia and subsequent reperfusion. Although the classical pathway had been assumed to be the major pathway of activation leading to injury, the mannose-binding lectin (MBL) pathway might also play a contributing role. In this study, we found that MBL-deficient mice had significant protection after skeletal muscle reperfusion injury compared with wild-type, classical pathway-specific C1q-deficient mice, or MBL-deficient mice reconstituted with recombinant human MBL. MBL-deficient mice, however, were not protected from permeability edema or secondary lung injury after ischemia-reperfusion. These data indicate that blockade of the classical pathway alone (C1q) is protective against permeability edema and remote pulmonary injury but not protective against histologic muscle injury. In contrast, blocking the MBL pathway alone protects against histological injury but is not protective against permeability edema or lung injury. Thus, the activation of both pathways is likely responsible for the full spectrum of injuries observed after skeletal muscle reperfusion injury.     

Role of charged residues in the S1-S4 voltage sensor of BK channels

The activation of large conductance Ca(2+)-activated (BK) potassium channels is weakly voltage dependent compared to Shaker and other voltage-gated K(+) (K(V)) channels. Yet BK and K(V) channels share many conserved charged residues in transmembrane segments S1-S4. We mutated these residues individually in mSlo1 BK channels to determine their role in voltage gating, and characterized the voltage dependence of steady-state activation (P(o)) and I(K) kinetics (tau(I(K))) over an extended voltage range in 0-50 microM [Ca(2+)](i). mSlo1 contains several positively charged arginines in S4, but only one (R213) together with residues in S2 (D153, R167) and S3 (D186) are potentially voltage sensing based on the ability of charge-altering mutations to reduce the maximal voltage dependence of P(O). The voltage dependence of P(O) and tau(I(K)) at extreme negative potentials was also reduced, implying that the closed-open conformational change and voltage sensor activation share a common source of gating charge. Although the position of charged residues in the BK and K(V) channel sequence appears conserved, the distribution of voltage-sensing residues is not. Thus the weak voltage dependence of BK channel activation does not merely reflect a lack of charge but likely differences with respect to K(V) channels in the position and movement of charged residues within the electric field. Although mutation of most sites in S1-S4 did not reduce gating charge, they often altered the equilibrium constant for voltage sensor activation. In particular, neutralization of R207 or R210 in S4 stabilizes the activated state by 3-7 kcal mol(-1), indicating a strong contribution of non-voltage-sensing residues to channel function, consistent with their participation in state-dependent salt bridge interactions. Mutations in S4 and S3 (R210E, D186A, and E180A) also unexpectedly weakened the allosteric coupling of voltage sensor activation to channel opening. The implications of our findings for BK channel voltage gating and general mechanisms of voltage sensor activation are discussed.     

Modified kinetics and selectivity of sodium channels in frog skeletal muscle fibers treated with aconitine

The effect of the plant alkaloid aconitine on sodium channel kinetics, ionic selectivity, and blockage by protons and tetrodotoxin (TTX) has been studied in frog skeletal muscle. Treatment with 0.25 or 0.3 mM aconitine alters sodium channels so that the threshold of activation is shifted 40-50 mV in the hyperpolarized direction. In contrast to previous results in frog nerve, inactivation is complete for depolarizations beyond about -60 mV. After aconitine treatment, the steady state level of inactivation is shifted approximately 20 mV in the hyperpolarizing direction. Concomitant with changes in channel kinetics, the relative permeability of the sodium channel to NH4,K, and Cs is increased. This altered selectivity is not accompanied by altered block by protons or TTX. The results suggest that sites other than those involved in channel block by protons and TTX are important in determining sodium channel selectivity.     

Blockade by cAMP of native sodium channels of adult rat skeletal muscle fibers

Although theskeletal muscle sodium channel is a good substrate for cAMP-dependentprotein kinase (PKA), no functional consequence was observed for thischannel expressed in heterologous systems. Therefore, we investigatedthe effect of 8-(4-chlorophenylthio)adenosine 3',5'-cyclicmonophosphate (CPT-cAMP), a membrane-permeable cAMP analog, on thenative sodium channels of freshly dissociated rat skeletal musclefibers by means of the cell-attached patch-clamp technique. Externallyapplied CPT-cAMP (0.5 mM) reduced peak ensemble average currents by~75% with no change in kinetics. Single-channel conductance andnormalized activation curves were unchanged by CPT-cAMP. In contrast,steady-state inactivation curves showed a reduction of the maximalavailable current and a negative shift of the half-inactivationpotential. Similar effects were observed with dibutyryl adenosine3',5'-cyclic monophosphate but not with cAMP, which doesnot easily permeate the cell membrane. Incubation of fibers for 1 hwith 10 µM H-89, a PKA inhibitor, did not prevent the effect ofCPT-cAMP. Finally, the -adrenoreceptor agonist isoproterenolmimicked CPT-cAMP when applied at 0.5 mM but had no effect at 0.1 mM.These results indicate that cAMP inhibits native skeletal muscle sodiumchannels by acting within the fiber, independently of PKA activation.

     

Blockade of sodium channels by Bistramide A in voltage-clamped frog skeletal muscle fibres.

The effect of Bistramide A, a toxin isolated from Bistratum lissoclinum Sluiter (Urochordata), on the peak sodium current (INa) of frog skeletal muscle fibres was studied with the double sucrose gap voltage clamp technique. External or internal application of Bistramide A inhibited INa without alteration of the kinetic parameters of the current nor of the apparent reversal potential for Na. The steady-state activation curve of INa was unchanged while the steady-state inactivation curve of INa was shifted towards more negative membrane potentials. Dose-response curves indicated an apparent dissociation constant for Bistramide A of 3.3 microM and a Hill coefficient of 1.2 which suggested a one to one relation between the toxin and Na channel. The inhibition of INa occurred at rest, and was more important at more positive holding potentials. Bistramide A exhibited only a weak frequency-dependent effect. The toxin did not interact with the use-dependent effect of lidocaine. It mainly blocked Na channels at more depolarized holding potentials. The toxin blocked Na channels when it was internally applyed and when the inactivation gating system has been previously destroyed by internal diffusion of iodate. The data suggest that Bistramide A inhibited the Na channel both at rest and in the inactivated state and occupied a site which was not located on the inactivation gate.     

Design of a specific activator for skeletal muscle sodium channels uncovers channel architecture

Gating modifiers of voltage-gated sodium channels (Na(v)s) are important tools in neuroscience research and may have therapeutic potential in medicinal disorders. Analysis of the bioactive surface of the scorpion beta-toxin Css4 (from Centruroides suffusus suffusus) toward rat brain (rNa(v)1.2a) and skeletal muscle (rNa(v)1.4) channels using binding studies revealed commonality but also substantial differences, which were used to design a specific activator, Css4(F14A/E15A/E28R), of rNa(v)1.4 expressed in Xenopus oocytes. The therapeutic potential of Css4(F14A/E15A/E28R) was tested using an rNa(v)1.4 mutant carrying the same mutation present in the genetic disorder hypokalemic periodic paralysis. The activator restored the impaired gating properties of the mutant channel expressed in oocytes, thus offering a tentative new means for treatment of neuromuscular disorders with reduced muscle excitability. Mutant double cycle analysis employing toxin residues involved in the construction of Css4(F14A/E15A/E28R) and residues whose equivalents in the rat brain channel rNa(v)1.2a were shown to affect Css4 binding revealed significant coupling energy (>1.3 kcal/mol) between F14A and E592A at Domain-2/voltage sensor segments 1-2 (D2/S1-S2), R27Q and E1251N at D3/SS2-S6, and E28R with both E650A at D2/S3-S4 and E1251N at D3/SS2-S6. These results show that despite the differences in interactions with the rat brain and skeletal muscle Na(v)s, Css4 recognizes a similar region on both channel subtypes. Moreover, our data indicate that the S3-S4 loop of the voltage sensor module in Domain-2 is in very close proximity to the SS2-S6 segment of the pore module of Domain-3 in rNa(v)1.4. This is the first experimental evidence that the inter-domain spatial organization of mammalian Na(v)s resembles that of voltage-gated potassium channels.     

Probing the role of negatively charged amino acid residues in ion permeation of skeletal muscle ryanodine receptor

Sequence comparison suggests that the ryanodine receptors (RyRs) have pore architecture similar to that of the bacterial K+ channel KcsA. The lumenal loop linking the two most C-terminal transmembrane spanning segments in the RyRs has a predicted pore helix and an amino acid motif (GGGIG) similar to the selectivity filter (TVGYG) of KcsA identified by x-ray analysis. The RyRs have many negatively charged amino acid residues in the two regions linking the GGGIG motif and predicted pore helix with the two most C-terminal transmembrane spanning segments. We tested the role of these residues by generating single-site mutants, focusing on amino acid residues conserved among the mammalian RyRs. Replacement of two acidic residues immediately after the GGGIG motif in skeletal muscle ryanodine receptor (RyR1-D4899 and -E4900) with asparagine and glutamine profoundly affected ion permeation and selectivity. By comparison, mutagenesis of aspartate and glutamate residues in the putative linker regions showed a K+ conductance and selectivity for Ca2+ compared to K+ (P(Ca)/P(K)) close to wild-type. The results show that the negatively charged carboxyl oxygens of D4899 and E4900 side chains are major determinants of RyR ion conductance and selectivity.     

Recombinant human voltage-gated skeletal muscle sodium channels are pharmacologically functional in planar lipid bilayers

Human voltage-gated sodium ion channels are major sites of action for drugs and toxins that modulate cellular excitability, and are therefore key molecular targets for ion channel research, high throughput screening for new drugs, and toxin detection. Protein suitable for these applications must be produced in a functionally active form. We report the successful use of ion metal affinity chromatography (IMAC) to purify C-terminal polyhistidine tagged human skeletal muscle voltage-gated sodium (hSkM1-HT) channels from Sf9 insect cells; hSkM1 channels were pharmacologically functional when reconstituted into liposomes and incorporated into planar bilayer lipid membranes. hSkM1-HT single channel currents activated by veratridine had a conductance of 21 pS and those activated by brevetoxin, 16 pS. Channel activity was inhibited by tetrodotoxin and saxitoxin. This protein is suitable for the development of biosensor and high throughput screening technologies.     

Charge immobilization of skeletal muscle Na+ channels: role of residues in the inactivation linker

We investigated structural determinants of fast inactivation and deactivation in sodium channels by comparing ionic flux and charge movement in skeletal muscle channels, using mutations of DIII-DIV linker charges. Charge altering and substituting mutations at K-1317, K-1318 depolarized the g(V) curve but hyperpolarized the h(infinity) curve. Charge reversal and substitution at this locus reduced the apparent voltage sensitivity of open- and closed-state fast inactivation. These effects were not observed with charge reversal at E-1314, E-1315. Mutations swapping or neutralizing the negative cluster at 1314, 1315 and the positive cluster at 1317, 1318 indicated that local interactions dictate the coupling of activation to fast inactivation. Gating charge was immobilized before channel entry into fast inactivation in hNa(V)1.4 but to a lesser extent in mutations at K-1317, K-1318. These results suggest that charge is preferentially immobilized in channels inactivating from the open state. Recovery of gating charge proceeded with a single, fast phase in the double mutation K-1317R, K-1318R. This mutation also partially uncoupled recovery from deactivation. Our findings indicate that charged residues near the fast inactivation "particle" allosterically interact with voltage sensors to control aspects of gating in sodium channels.     

Orai and TRP channels in skeletal muscle cells

Modern data concerning expression, localization, biophysical properties, involvement in calcium regulation, and physiological functions of TRP and Orai channels in skeletal muscle cells are analyzed. In skeletal muscles TRPC1/2/3/4/5/6/7, TRPV2/4, TRPM2/7 and Orai1/3 channels are expressed. Activities of TRPC1/3 and TRPV4 facilitate maximal muscle contraction during tetanus. Orai1 channels provide recovery of intracellular calcium stores and are obligatory for proliferation of myoblasts and differentiation of skeletal muscles. TRPC1 knockout results in alterations of the development of skeletal muscles. Enhanced calcium influx via the channels is supposed to be a pathogenic factor of myodystrophy.     

Chloride channels in toad skeletal muscle fibers

Chloride currents were measured in short lumbricalis fibers of toads (Bufo arenarum) with voltage and patch clamp techniques. For the availability of chloride currents we applied a double-pulse technique in voltage-clamped fibers. When the test pulse was preceded by a positive prepulse, the initial current was larger than with a negative prepulse and exhibited a different rate of decline to its steady-state value. At the single-channel level we found that in most of the experiments with symmetrical 110 mM NaCl solutions, two levels of conductance, 20 ("small channel") and 360 pS ("maxi channel"), occurred with the highest probabilities. The openings of the maxi channels were more frequent at potentials close to 0 mV, whereas for the small channels the openings were at negative potentials. In contrast with the results with the macroscopic currents, a change of 2 orders of magnitude in the pH, from 7.3 to 5, had only minor effects on the channels' conductance. As with some other anion channels, the selectivity of the channels described here is low, the p(Cl)/p(Na) ratio being 1.9 and 3.7 for the small and maxi Cl(-) channels, respectively. The behavior of these Cl(-) channels with a relative high Na(+) permeability could contribute to the relatively low resting membrane potential of the lumbricalis fibers measured in the standard 110 mM NaCl solution.     

RYR1 and RYR3 have different roles in the assembly of calcium release units of skeletal muscle

Calcium release units (CRUs) are junctions between the sarcoplasmic reticulum (SR) and exterior membranes that mediates excitation contraction (e-c) coupling in muscle cells. In skeletal muscle CRUs contain two isoforms of the sarcoplasmic reticulum Ca(2+)release channel: ryanodine receptors type 1 and type 3 (RyR1 and RyR3). 1B5s are a mouse skeletal muscle cell line that carries a null mutation for RyR1 and does not express either RyR1 or RyR3. These cells develop dyspedic SR/exterior membrane junctions (i.e., dyspedic calcium release units, dCRUs) that contain dihydropyridine receptors (DHPRs) and triadin, two essential components of CRUs, but no RyRs (or feet). Lack of RyRs in turn affects the disposition of DHPRs, which is normally dictated by a linkage to RyR subunits. In the dCRUs of 1B5 cells, DHPRs are neither grouped into tetrads nor aligned in two orthogonal directions. We have explored the structural role of RyR3 in the assembly of CRUs in 1B5 cells independently expressing either RyR1 or RyR3. Either isoform colocalizes with DHPRs and triadin at the cell periphery. Electron microscopy shows that expression of either isoform results in CRUs containing arrays of feet, indicating the ability of both isoforms to be targeted to dCRUs and to assemble in ordered arrays in the absence of the other. However, a significant difference between RyR1- and RyR3-rescued junctions is revealed by freeze fracture. While cells transfected with RyR1 show restoration of DHPR tetrads and DHPR orthogonal alignment indicative of a link to RyRs, those transfected with RyR3 do not. This indicates that RyR3 fails to link to DHPRs in a specific manner. This morphological evidence supports the hypothesis that activation of RyR3 in skeletal muscle cells must be indirect and provides the basis for failure of e-c coupling in muscle cells containing RyR3 but lacking RyR1 (see the accompanying report, ).