Jeffry B. Lansman

Stretch-sensitive channels in developing muscle cells from a mouse cell line.

1. Recordings of single‐channel activity were made from cell‐attached patches on mouse C2 muscle cells at morphologically identifiable stages of myogenesis in vitro. We have identified Ca2(+)‐permeable, cation‐selective channels that are gated by applying suction to the patch electrode and by changes in membrane potential and have analysed single‐channel properties as well as channel expression during myogenesis. 2. Single‐channel activity could be detected when the membrane was held at steady negative potentials. With monovalent cations in the electrode, the single‐channel current‐voltage (i‐V) relations were linear. The channel is permeable to Li+, Na+, K+, Rb+ and Cs+, but is not strongly selective among the monovalent cations as judged by measurements of single‐channel conductance and reversal potential. 3. With 110 mM of either CaCl2 or BaCl2 as the only inward change carrier, slope conductances were approximately 13 and 24 pS and currents reversed at approximately +22 and +17 mV, respectively. The relative permeability of Ca2+ to K+ calculated from the constant‐field equation was PCa/PK = approximately 2. 4. Channel openings occurred as bursts of brief openings and closings separated by much longer closed periods. Closed‐time histograms were best fitted with three exponential components, while histograms of burst duration were best fitted with two exponential components, reflecting the short and long bursts in the single‐channel records. 5. Applying suction to the patch electrode while recording at steady negative membrane potentials produced channel openings to discrete current levels. Mean channel open probability depended linearly on the square of the applied pressure and was greater at positive membrane potentials. The permeability of the channel to monovalent and divalent cations was indistinguishable from the spontaneous activity recorded at steady negative potentials. 6. Channel activity recorded from cell‐attached patches in the absence of applied pressure depended on membrane potential increasing approximately e‐fold per 38 mV with depolarization. Analysis of the kinetics of the response to membrane potential showed that the depolarization reduced the duration of the slowest component of the closed‐time distribution. 7. The lanthanide cation gadolinium (Gd) reduced the amplitude of the unitary currents in a concentration‐dependent manner. The amplitudes of both inward and outward currents were reduced to the same extent suggesting block is voltage‐independent. Gd produced half‐maximal inhibition of the unitary current at approximately 6 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Properties of embryonic and adult muscle acetylcholine receptors transiently expressed in COS cells

We used transient transfection in COS cells to compare the properties of mouse muscle acetylcholine receptors (AChRs) containing alpha, beta, delta, and either gamma or epsilon subunits. gamma- and epsilon-AChRs had identical association rates for binding 125I-alpha-bungarotoxin, and identical curves for inhibition of toxin binding by d-tubocurarine, but epsilon-AChRs had a significantly longer half-time of turnover in the membrane than gamma-AChRs. A myasthenic serum specific for the embryonic form of the AChR reduced toxin binding to gamma-, but not epsilon-AChRs. The gamma-AChRs had channel characteristics of embryonic AChRs, whereas the major class of epsilon-AChR channels had the characteristics of adult AChRs. Two minor channel classes with smaller conductances were also seen with epsilon-AChR. Thus, some, but not all, of the differences between AChRs at adult endplates and those in the extrasynaptic membrane can be explained by the difference in subunit composition of gamma- and epsilon-AChRs.

Changes in mechanosensitive channel gating following mechanical stimulation in skeletal muscle myotubes from the mdx mouse

We studied the effects of membrane stretch and voltage on the gating of single mechanosensitive (MS) channels in myotubes from dystrophin‐deficient mdx mice. In earlier studies of MS channels in mdx myotubes, we found a novel class of stretch‐inactivated channels. In the present experiments, we used a gentle suction protocol to determine whether seal formation damaged the membrane and altered MS channel gating, since dystrophin‐deficiency is known to be associated with an increased susceptibility to mechanically induced damage. In some recordings from mdx myotubes, MS channel open probability gradually increased to levels approaching unity following seal formation. In these recordings, channels remained open for the duration of the recording. In other recordings, MS channel open probability remained low after seal formation and applying weak suction evoked conventional stretch‐activated gating. Applying strong suction or very positive voltages, however, caused some channels to enter a high open probability gating mode. The shift to a high open probability gating mode coincided with the appearance of stretch‐inactivated gating. These findings suggested that mechanical stimulation altered the mechanical properties of the patch causing some MS channels to enter a novel gating mode. In support of this idea, stretch‐activated and stretch‐inactivated channels were not detected in the same membrane patch and channel inactivation occurred at lower pressures than activation (P1/2,=−13 and −26.5 mmHg, respectively). Other experiments showed that stretch‐inactivated gating was not due to a simple loss of MS channel activity from a non‐random process such as vesiculation or bleb formation: channel inactivation by suction was readily reversible, stable over tens of minutes, and followed the predictions of the binomial theorem for independent, randomly gating channels. In addition, the voltage‐dependent gating of stretch‐inactivated channels was similar to that of stretch‐activated channels. The results show that MS channels in dystrophin‐deficient muscle exist in two distinct gating modes and that mechanical stimuli cause an irreversible conversion between modes. We discuss possible mechanisms for the changes in MS channel gating in relation to the known cytoskeletal abnormalities of mdx muscle and its possible implications for the pathogenesis of Duchenne dystrophy.

Voltage-dependent block by zinc of single calcium channels in mouse myotubes.

1. The blocking actions of Zn2+ on currents carried by Ba2+ through single dihydropyridine‐sensitive Ca2+ channels were recorded from cell‐attached patches on myotubes from the mouse C2 cell line. 2. Adding 100 microM‐Zn2+ to the patch electrode containing 110 mM‐BaCl2 produced an increase in the open channel noise, presumably arising from unresolved blocking and unblocking of the open channel by Zn2+. Adding between 200 and 1000 microM‐Zn2+ to the electrode reduced the amplitude of the unitary current in a concentration‐dependent manner. 3. The single‐channel current‐voltage (i‐V) relations showed that Zn2+ reduced the amplitude of the unitary Ba2+ currents at all potentials more negative than 0 mV. A plot of the amplitude of the unitary current in the presence of Zn2+, normalized to the amplitude in its absence, showed that block of the current depended on voltage, decreasing as the patch potential was made more negative. 4. The normalized amplitudes of the unitary currents were plotted as a function of the logarithm of [Zn2+] in the electrode. The relation for currents recorded at different potentials were fitted to an expression for binding to a single site with a KD at 0 mV of approximately 500 microM. The KD changed approximately e‐fold per 83 mV with hyperpolarization. The results suggest Zn2+ binds to a site located at approximately 15% of the potential drop from the surface membrane. 5. Reducing the concentration of Ba2+ in the patch electrode enhanced the steady‐state block of unitary currents by Zn2+. The inverse of the unitary current was plotted as a function of [Ba2+]o in the presence and absence of Zn2+; both were linear and intersected at the ordinate, indicating Ba2+ and Zn2+ compete for a channel site. 6. The kinetics of Zn2+ block of unitary Ba2+ currents were studied by amplitude distribution analysis. As expected for a simple reaction between blocking ion and open channel, the blocking rate depended linearly on the concentration of Zn2+, while the exit rate was independent of concentration. The second‐order rate coefficient for Zn2+ entry in the presence of 110 mM‐BaCl2 at 0 mV was approximately 2.0 X 10(7) M‐1S‐1, while the exit rate was approximately 16000 s‐1. 7. Both entry and exit rates increased as the membrane potential was made more negative. The entry rate increased approximately e‐fold per 66 mV, while the exit rate increased approximately e‐fold per 41 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Open channel block by gadolinium ion of the stretch-inactivated ion channel in mdx myotubes

Currents flowing through single stretch-inactivated ion channels were recorded from cell-attached patches on myotubes from mdx mice. Adding micromolar concentrations of gadolinium to patch electrodes containing normal saline produced rapid transitions in the single-channel current between the fully open and closed states. The kinetics of the current fluctuations followed the predictions of a simple model of open channel block in which the transitions in the current arise from the entry and exit of Gd from the channel pore: histograms of the open and closed times were well fit with single exponentials, the blocking rate depended linearly on the concentration of gadolinium in the patch electrode, and the unblocking rate was independent of the concentration of gadolinium. Hyperpolarizing the patch increased the rate of unblocking (approximately e-fold per 85 mV), suggesting the charged blocking particle can exit the channel into the cell under the influence of the applied membrane field. The rate of blocking was rapid and was independent of the patch potential, consistent with the rate of ion entry into the pore being determined by its rate of diffusion in solution. When channel open probability was reduced by applying suction to the electrode, the blocking kinetics were independent of the extent of inactivation, suggesting that mechanosensitive gating does not modify the structure of the channel pore.

Developmental regulation of mechanosensitive calcium channels in skeletal muscle from normal and mdx mice

Single-channel activity was recorded from cell-attached membrane patches on flexor digitorum brevis fibres acutely isolated from normal and mdx mice at different stages of postnatal development. Recordings from cell-attached patches on both normal and mdx fibres were dominated by the activity of mechanosensitive ion channels with a conductance of approximately 17 pS with 110 mM Ba2+ in the patch electrode. In a small fraction of the patches on mdx fibres from young mice, channels showed very high levels of activity. Channel activity recorded from mdx fibres from older mice was higher than in agematched normal fibres and the level of activity decreased during development. Channel density decreased in normal fibres, whereas it remained relatively constant in mdx fibres, as if channels are down-regulated in normal, but not mdx, fibres during postnatal development. An early step in the dystrophic process may be an alteration of the mechanisms that regulate the expression of functional channels.

Mechanosensitive ion channels in skeletal muscle: a link in the membrane pathology of muscular dystrophy.

1 Mechanosensitive (MS) channels are expressed abundantly in skeletal muscle at all stages of development. In recordings from membrane patches, MS channels are constitutively active at the resting potential. The channels are selective for cations and have a large single‐channel conductance (approximately 25 pS in physiological saline) and a high Ca2+ permeability (relative permeability of Ca2+ to K+ (PCa/PK) = 7). 2 Mechanosensitive channel activity recorded from the surface of myotubes from dystrophic mdx mice was substantially greater than the activity recorded from wild‐type myotubes. Increased channel activity in the mutant results from the induction in a subpopulation of channels of a novel MS gating mode characterized by markedly prolonged channel openings and inactivation in response to membrane stretch. 3 Membrane stretch or a strong depolarization causes an irreversible switch to the stretch‐inactivated gating mode in mdx myotubes. A stretch‐induced shift in MS channel gating mode may contribute to stretch‐induced elevations in [Ca2+]i during the early stages of disease pathogenesis. 4 Abnormalities of MS channel behaviour are also detected in recordings from patches on flexor digitorum brevis fibres acutely isolated from mdx mice. Mechanosensitive channel opening probability is higher in mdx fibres at all developmental stages. In addition, channel numbers are persistently elevated during postnatal development, failing to undergo a normal process of downregulation during the first 3 postnatal weeks. 5 Two distinct mechanisms may contribute to elevations of [Ca2+]i in dystrophin‐deficient skeletal muscle: (i) a membrane stress‐dependent switch of MS channels into to a prolonged opening mode; and (ii) a loss of developmental downregulation leading to persistent MS channel expression during postnatal muscle development.

Reopening of Ca2+ channels in mouse cerebellar neurons at resting membrane potentials during Recovery from Inactivation

Recordings of single-channel activity from cerebellar granule cells show that a component of Ca2+ entry flows through L-type Ca2+ channels that are closed at negative membrane potentials following a strong depolarization, but then open after a delay. The delayed openings can be explained if membrane depolarization drives Ca2+ channels into an inactivated state and some channels return to rest through the open state after repolarization. Whole-cell recordings show that the charge carried by Ca2+ during the tail increases as inactivation progresses, whereas the current during the voltage step decreases. Voltage-dependent inactivation may be a general mechanism in central neurons for enhancing Ca2+ entry by delaying it until after repolarization, when the driving force for ion entry is large. Modifying the rate and extent of inactivation would have large effects on Ca2+ entry through those channels that recover from inactivation by passing through the open state.

Inactivation of calcium currents in granule cells cultured from mouse cerebellum.

1. Cells dissociated from mouse cerebellum were grown in vitro. Ca2+ channel currents were recorded from granule cells with the patch‐clamp technique under conditions which suppressed currents through Na+ and K+ channels and minimized run‐down of current through Ca2+ channels. 2. A strong depolarizing voltage step from a hyperpolarized holding potential produced inward Ca2+ channel current that decayed exponentially to a non‐zero level. Inward current decayed to approximately 40% of its peak amplitude (range 20‐90%). 3. The inward current increased in amplitude when Ca2+ was replaced with Ba2+ or after raising the concentration of extracellular Ba2+, but the rate of decay of current was unaffected. 4. The current‐voltage (I‐V) relation showed that peak or sustained current increased with voltage pulses more positive than approximately ‐30 mV, reached a maximum amplitude near +20 mV and became progressively smaller with larger depolarizations. 5. The tail currents produced after rapidly repolarizing the membrane potential to ‐70 mV from a positive test pulse decayed along a single exponential time course with a time constant of approximately 0.5 ms. The amplitude of tail current measured at a fixed repolarization potential increased as the pre‐pulse was made more positive and reached a maximum with pre‐pulses more positive than +40 mV. A plot of normalized amplitude of the tail current as a function of the pre‐pulse potential was fitted with a Boltzmann relation with V1/2 = approximately + 8 mV and steepness k = 14 mV. 6. Shifting the holding potential to more positive potentials reduced the amplitude of the Ca2+ channel current elicited by the fixed voltage step and abolished the decay of the inward current. The peak current was normalized to the maximum peak current elicited from a very negative holding potential and plotted as a function of holding potential. The points were fitted with a Boltzmann relation for inactivation with V1/2 = approximately ‐57 mV and steepness k = 14 mV. 7. The onset of inactivation was studied in two‐pulse experiments in which the duration of conditioning pre‐pulse was varied. Increasing the duration of a pre‐pulse to a fixed potential reduced the peak inward current evoked by the second test pulse. Plotting normalized current as a function of pre‐pulse duration showed that inactivation developed along a double exponential time course. Both fast and slow time constants decreased as the pre‐pulse potential was made more positive.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence TRPV4 contributes to mechanosensitive ion channels in mouse skeletal muscle fibers.

We recorded the activity of single mechanosensitive (MS) ion channels from membrane patches on single muscle fibers isolated from mice. We investigated the actions of various TRP (transient receptor potential) channel blockers on MS channel activity. 2-aminoethoxydiphenyl borate (2-APB) neither inhibited nor facilitated single channel activity at submillimolar concentrations. The absence of an effect of 2-APB indicates MS channels are not composed purely of TRPC or TRPV1, 2 or 3 proteins. Exposing patches to 1-oleolyl-2-acetyl-sn-glycerol (OAG), a potent activator of TRPC channels, also had no effect on MS channel activity. In addition, flufenamic acid and spermidine had no effect on the activity of single MS channels. By contrast, SKF-96365 and ruthenium red blocked single-channel currents at micromolar concentrations. SKF-96365 produced a rapid block of the open channel current. The blocking rate depended linearly on blocker concentration, while the unblocking rate was independent of concentration, consistent with a simple model of open channel block. A fit to the concentration-dependence of block gave kon = 13 x 106 M−1s−1 and koff = 1609 sec−1 with KD = ~124 µM. Block by ruthenium red was complex, involving both reduction of the amplitude of the single-channel current and increased occupancy of subconductance levels. The reduction in current amplitude with increasing concentration of ruthenium red gave a KD = ~49 µM. The high sensitivity of MS channels to block by ruthenium red suggests MS channels in skeletal muscle contain TRPV subunits. Recordings from skeletal muscle isolated from TRPV4 knockout mice failed to show MS channel activity, consistent with a contribution of TRPV4. In addition, exposure to hypo-osmotic solutions increases opening of MS channels in muscle. Our results provide evidence TRPV4 contributes to MS channels in skeletal muscle.