Skeletal muscle has a high resting Cl ⁻ permeability

In skeletal muscle, the resting V _m (∼–90 mV) is much more negative than in neurons (∼–70 mV) owing to the relatively high permeability of the sarcolemma to both K ⁺ and Cl ⁻ . Because of the high resting Cl ⁻ permeability, a relatively large stimulus is normally necessary to bring the V _m to mechanical threshold. In some skeletal muscle diseases, loss-of-function mutations in skeletal muscle Cl ⁻ channels drastically reduce the Cl ⁻ permeability of the sarcolemma. As a result, the skeletal muscle AP threshold can be reached more easily and the muscle becomes hyperexcitable ( Box 15.1 ).

A single action potential causes a brief contraction called a twitch

The temporal relationship between the skeletal muscle AP and the force generated by the muscle is illustrated in Fig. 15.2 . The skeletal muscle AP is similar to the nerve axon AP ( Chapter 7 ) in that it is generated by the activity of voltage-gated Na ⁺ and K ⁺ channels. The development of force by the myocyte begins several milliseconds after the AP. The transient contraction produced by a myocyte in response to a single AP is called a twitch . During the twitch, the contractile force rises to a peak in 30 to 50 milliseconds and then declines over the next 50 to 100 milliseconds. The duration of the twitch is approximately 100-fold longer than the duration of the AP.

The Temporal Relationship Between the AP and a Twitch Contraction in a Frog Skeletal Muscle Fiber.

The trace labeled V _m shows the time course of the skeletal muscle AP. The twitch contraction (trace labeled Force) begins a few milliseconds after the AP. Peak twitch force is reached in approximately 30 milliseconds, and the muscle then relaxes over the next 50 milliseconds. After the peak of the AP, the sarcolemma initially repolarizes rapidly. This is followed by a prolonged period of much slower repolarization called an afterdepolarization, which is caused by accumulation of K ⁺ in the lumen of the T-tubule during fast repolarization, which transiently increases E _K .

(From Lüttgau, H.C, & Stephenson, G.D. (1986). Ion movements in skeletal muscle in relation to the activation of contraction. In Andreoli, T.E., Hoffman, J.F., Fanestil, D.D. & Schultz, S.G. (eds.) Physiology of membrane disorders. New York, Plenum, 449–468.)

How does depolarization increase intracellular [Ca ²⁺ ] in skeletal muscle?

In Chapter 14 we learned that an increase in [Ca ²⁺ ] _i activates contraction in all types of muscle cells. Therefore the central question in E-C coupling is, “How does depolarization of the sarcolemma bring about an increase in [Ca ²⁺ ] _i ?” A related, but more subtle, question is, “How does the [Ca ²⁺ ] _i increase fast enough throughout the muscle fiber so that myofibrils deep inside the fiber can contract synchronously with those close to the surface?”

In skeletal muscle, depolarization of the T-tubule membrane is required for excitation-contraction coupling

Is diffusion of a soluble factor from the surface membrane (e.g., Ca ²⁺ ions entering the cell through voltage-gated Ca ²⁺ channels) to the interior of the cell sufficiently fast to explain the rapid activation of skeletal muscle? A molecule would take about a second to diffuse to the center of a 100-μm diameter skeletal muscle cell ( Chapter 2 ). This is much too slow to account for the development of force during a twitch contraction, which begins just a few milliseconds after the AP ( Fig. 15.2 ).

How, then, does the AP in the sarcolemma rapidly activate the myofibrils in the center of the cell? The critical clue was the discovery that “hot spots” distributed over the sarcolemma (later shown to be T-tubule openings) provide a pathway for depolarization to spread from the surface into the interior of the fiber. T-tubules form an intricate network that extends throughout the skeletal muscle cell ( Fig. 15.3 ). Thus the T-tubule lumen is a narrow extension of the extracellular space into the interior of the muscle cell. The T-tubule membrane contains voltage-gated Na ⁺ and K ⁺ channels, and APs from the surface are propagated along the T-tubule membrane into the interior of the cell.

The Extensive Network of T-tubules in Skeletal Muscle.

A Golgi stain was used to infiltrate the T-tubule system from the extracellular space. The resulting black areas in this electron micrograph clearly delineate the extensive network of tubules. Longitudinal elements of the T-tubules interconnect transverse elements. The enlarged areas of the tubules are regions of functional coupling between the T-tubule and sarcoplasmic reticulum membranes.

(Courtesy D. Appelt and C. Franzini-Armstrong.)

In skeletal muscle, extracellular Ca ²⁺ is not required for contraction

The T-tubule membrane of skeletal muscle contains receptors for the dihydropyridine derivatives that block L-type voltage-gated Ca ²⁺ channels ( Chapter 8 ). Voltage clamp studies in skeletal muscle demonstrate that inward Ca ²⁺ currents are generated by depolarization ( Fig. 15.4 ) and that these currents are blocked by dihydropyridines. Thus the dihydropyridine receptors (DHPRs) in the T-tubule membrane are L-type Ca ²⁺ channels. Therefore we might reasonably expect that the Ca ²⁺ entry through these channels raises [Ca ²⁺ ] _i and activates contraction, but this is not the case. Skeletal muscle continues to contract normally when bathed in a solution containing no Ca ²⁺ ions. This finding indicates that Ca ²⁺ entry is not essential for skeletal muscle contraction and that all the Ca ²⁺ required for activating the contractile machinery is derived from intracellular sources. This is in marked contrast to the mechanism of E-C coupling in cardiac muscle, where Ca ²⁺ entry through voltage-gated Ca ²⁺ channels is required (see later). Even though Ca ²⁺ entry through DHPRs is not required for E-C coupling in skeletal muscle, the DHPRs do play a crucial role in this mechanism.

Depolarization Activates a Slow Inward Ca ²⁺ Current ( I _Ca ) in Skeletal Myocytes.

A voltage clamp was used to record I _Ca after current flow through other channels was eliminated. In response to a voltage clamp step from −90 to −24 mV, an inward I _Ca developed slowly, peaking in approximately 200 milliseconds. Surprisingly, although L-type Ca ²⁺ channels generate I _Ca in skeletal muscle, their activation is nearly two orders of magnitude slower than in other cell types ( Fig. 8.2 ).

(Data from Sánchez, J.A., & Stefani, E. (1983). Kinetic properties of calcium channels of twitch muscle fibres of the frog. The Journal of Physiology, 337 , 1–17.)

In skeletal muscle, the sarcoplasmic reticulum stores all the Ca ²⁺ needed for contraction

The SR is an extensive intracellular membrane-enclosed system of sacs and tubules that envelopes the myofibrils in skeletal muscle cells ( Fig. 14.1 ). That the SR surrounds all myofibrils has long suggested that it plays an important role in skeletal muscle contraction. The SR is a specialized ER with copious expression of three proteins: SERCA ( Chapter 11 ); the ryanodine receptor (RyR) , which is the Ca ²⁺ release channel in the SR membrane; and calsequestrin . Virtually all the Ca ²⁺ released during a contraction is rapidly and efficiently transported back into the SR by the extremely abundant SERCA in the SR membrane. The rapid removal of Ca ²⁺ from the cytoplasm causes relaxation. Because essentially all the Ca ²⁺ is recycled in this way, extracellular Ca ²⁺ is not required in the short term, and skeletal muscle contraction can proceed even in the absence of extracellular Ca ²⁺ . The capacity of the SR to store Ca ²⁺ is greatly enhanced by calsequestrin, the Ca ²⁺ -binding protein that is highly expressed in the SR lumen and acts as a Ca ²⁺ buffer.

The SR Ca ²⁺ release channels are called RyRs because they avidly bind ryanodine, a paralyzing plant alkaloid that blocks the channels. There are three RyR isoforms: RyR1 in adult skeletal muscle; RyR2 in cardiac muscle; and RyR3 in embryonic skeletal muscle, brain (in ER), and other tissues. Each Ca ²⁺ release channel is a tetramer of four identical RyR protein subunits. Malignant hyperthermia is a genetic disease of skeletal muscle caused by mutations in RyR1 ( Box 15.2 ).

The triad is the structure that mediates excitation-contraction coupling in skeletal muscle

The SR and T-tubule membranes in skeletal muscle are closely apposed at specialized junctions called triads ( Fig. 15.5 ). The triad consists of a T-tubule that is flanked on either side by enlarged terminal sacs of the SR, called terminal cisterns . The RyRs are located in the membrane of the terminal cisterns. A large cytoplasmic domain of the RyR spans the narrow gap between the T-tubule and SR membranes. These domains are visible by electron microscopy and have been termed junctional feet ( Fig. 15.5 ). At the triads, the T-tubule membrane contains clusters of DHPRs that are located in precise register with the RyR subunits in the underlying SR membrane ( Fig. 15.6 ). The DHPRs are grouped into tetrads, and each of the four DHPR molecules in the T-tubule membrane is aligned with, and apposed to, one subunit of the RyR tetramer in the SR membrane. Alternate RyR tetramers are, however, unapposed by DHPRs ( Fig. 15.6 ). This precise spatial organization of RyRs and DHPRs, which does not occur in cardiac muscle, is the structural basis of the E-C coupling mechanism in skeletal muscle.

A Triad in Skeletal Muscle Consists of a T-tubule Sandwiched Between Two Terminal Cisterns of the SR.

This longitudinal electron microscopic section shows the three elements of the triad. The flattened, oval elements are cross-sectional views of T-tubules. On either side of each T-tubule is a terminal cistern of the SR. Junctional “feet” ( arrows ) span the space between the T-tubule and SR membranes.

(Courtesy C. Franzini-Armstrong.)

The Three-Dimensional Structure of a Triad.

Four RyR subunits in the SR membrane form the Ca ²⁺ release channel. These subunits are the “foot” proteins Fig. 15.5 ) that connect the SR terminal cistern membrane to the cytoplasmic face of the T-tubule. In the T-tubule membrane, DHPRs are grouped into tetrads, and each member of the tetrad is apposed to an RyR subunit. Every second RyR tetramer, however, lacks the opposing tetrad of DHPRs. Calsequestrin and SERCA are also shown.

(Modified from Block, B.A., Imagawa, T., Campbell, K.P., & Franzini-Armstrong, C. (1988). Structural evidence for direct interaction between the molecular components of the transverse tubule/sarcoplasmic reticulum junction in skeletal muscle. The Journal of Cell Biology, 107 (6 Pt 2), 2587–2600.)

In skeletal muscle, excitation-contraction coupling is mechanical

In response to depolarization of the T-tubule membrane, the RyRs open to allow Ca ²⁺ to flow out of the SR, down its concentration gradient, into the cytoplasm, where it binds to TnC and activates contraction. The central question in E-C coupling is this: How does depolarization of the T-tubule membrane open the SR Ca ²⁺ release channel? The finding that the SR membrane is electrically isolated from the T-tubule membrane rules out the possibility that depolarization spreads directly from the T-tubule to the SR. Two classes of indirect coupling models have been proposed. First, T-tubule membrane depolarization could generate a chemical messenger that diffuses to and activates the RyRs. Alternatively, the coupling mechanism could be a mechanical link between a voltage sensor in the T-tubule membrane and the gating mechanism of the RyR in the SR membrane. The available evidence supports the mechanical coupling model in skeletal muscle.

The DHPR is homologous to the voltage-gated Na ⁺ channel. Like the Na ⁺ channel ( Chapter 5 ), the DHPR has a positively charged, membrane-spanning segment (S4 region) that acts as the voltage sensor. In the T-tubules of skeletal muscle the DHPR also acts as the voltage sensor for E-C coupling. Importantly, its function as a voltage sensor is independent of its ability to conduct Ca ²⁺ ions. The movement of the positive charges in the S4 segment of the DHPR in response to depolarization generates a transient capacitive current that can be measured with a voltage clamp ( Box 15.3 ). This charge movement is an essential first step in E-C coupling in skeletal muscle. The relationships among voltage-dependent activation of DHPRs, charge movement, and E-C coupling have been demonstrated in animals expressing mutated DHPRs ( Box 15.4 ). These studies suggest that the movement of the voltage sensor in the DHPR is mechanically linked to the opening of the RyR Ca ²⁺ release channel ( Fig. 15.7 ). The main elements of E-C coupling in skeletal muscle are illustrated in Fig. 15.8 .

BOX 15.3

Nonlinear Charge Movement Reflects Movement of the Voltage Sensor in Skeletal Muscle

In skeletal muscle, the voltage clamp can be used to measure a nonlinear component of the capacitive current that is qualitatively similar to the Na ⁺ channel gating current ( Box 7.3 ). A depolarizing voltage step generates an outward current. This current reflects the movement of charged amino acid residues in the S4 segment of the dihydropyridine receptors (DHPRs) that act as the voltage sensors for excitation-contraction coupling (E-C coupling) in skeletal muscle. Several lines of evidence indicate that this charge movement is related to E-C coupling. For example, the magnitude of the charge movement increases with the size of the depolarization and this occurs over the same range of potentials where muscle force increases ( Fig. 15.1 ). Furthermore, Ca ²⁺ release from the sarcoplasmic reticulum (SR) is tightly correlated with charge movement. Finally, no experimental or physiological conditions have been found that eliminate charge movement while maintaining depolarization-induced SR Ca ²⁺ release in skeletal muscle.

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