Ca ²⁺ currents can be recorded with a voltage clamp

Voltage-gated Ca ²⁺ channels can be studied with the voltage clamp. Whole-cell currents recorded from a neuronal cell body with a voltage clamp are shown in Fig. 8.2 . With K ⁺ channels blocked, a depolarizing voltage clamp step evokes an inward current that has both a fast transient component and a more sustained component. The fast transient component of the current is generated by Na ⁺ channels. When external Na ⁺ is removed, the Na ⁺ channels no longer conduct inward current (because there are no Na ⁺ ions to flow into the cell), and the remaining inward current is carried by Ca ²⁺ ions flowing into the cell through voltage-gated Ca ²⁺ channels ( Fig. 8.2 ). The Ca ²⁺ channels activate more slowly than Na ⁺ channels. Furthermore, the magnitude of the Ca ²⁺ current ( I _Ca ) decreases very slowly over tens of milliseconds. This indicates that Ca ²⁺ channels inactivate slowly (much more slowly than Na ⁺ channels). Inactivation also is incomplete, so that Ca ²⁺ current can continue to flow during maintained depolarization. The maximum amplitude of the Ca ²⁺ current (∼100 μA/cm ² of membrane area) is usually smaller than the Na ⁺ or K ⁺ currents in axons.

Ca ²⁺ and Na ⁺ Currents Recorded from a Snail Neuron During a Voltage Clamp Step ( V _m ) from –50 to –7.5 mV.

With 100 mM [Na ⁺ ] _o there are two current components: (1) a fast, transient inward current that is generated by voltage-gated Na ⁺ channels, and (2) a steady inward current that remains after removal of the external Na ⁺ (0 mM [Na ⁺ ] _o ). Voltage-gated Ca ²⁺ channels generate the second component.

(Data from Kostyuk, P.G., Kristhal, O.A., & Shakhovalov, Y.A. (1977). Separation of sodium and calcium currents in the somatic membrane of mollusc neurones. The Journal of Physiology, 270 (3), 545–568.)

The relationship between Ca ²⁺ channel open probability and V _m is similar to that for voltage-gated Na ⁺ channels, except that the channels activate over a more depolarized voltage range. In some cell types, voltage-gated Ca ²⁺ channels can generate calcium action potentials (Ca ²⁺ APs) in a manner similar to Na ⁺ channels. A depolarizing stimulus opens Ca ²⁺ channels, and Ca ²⁺ ions flow into the cell down their electrochemical gradient. This Ca ²⁺ current further depolarizes the cell and opens more channels. The positive feedback of membrane depolarization on channel opening produces the Ca ²⁺ AP upstroke. Compared with axonal APs generated by Na ⁺ channels, pure Ca ²⁺ APs have slower upstrokes and longer durations because the Ca ²⁺ channels activate more slowly than Na ⁺ channels, and inactivation is slow and incomplete. Ca ²⁺ APs also have lower amplitudes than Na ⁺ APs.

Ca ²⁺ channel blockers are useful therapeutic agents

Various drugs reduce Ca ²⁺ currents by blocking voltage-gated Ca ²⁺ channels. These Ca ²⁺ antagonists include phenylalkylamines (e.g., verapamil and its methoxy derivative known as D600 or gallopamil), benzothiazepines (e.g., diltiazem), and the dihydropyridine derivatives (e.g., nifedipine, nimodipine, and nicardipine). At physiological pH, D600 and diltiazem are protonated and therefore positively charged, whereas nifedipine is uncharged. These drugs selectively block L-type Ca ²⁺ channels ( Box 8.1 ).

Ca ²⁺ channel blockers are widely used as therapeutic agents in the management of coronary artery disease, hypertension, and cardiac arrhythmias ( Box 8.3 ). Some properties of the block produced by Ca ²⁺ antagonists are shown in Fig. 8.3 . The data in Fig. 8.3 A illustrate the effect of repetitive depolarizations on the development of Ca ²⁺ channel block by the positively charged D600. Very little reduction in I _Ca occurs in response to the first stimulus after D600 is added ( Fig. 8.3 A). Subsequent depolarizations, however, produce smaller and smaller Ca ²⁺ currents. This behavior is called use-dependent block of the Ca ²⁺ channels. In contrast, uncharged drugs, such as nifedipine, block Ca ²⁺ channels without requiring depolarization; that is, they produce a significant degree of tonic block and very little use-dependent block. Diltiazem is intermediate between D600 and nifedipine in its use dependence. Use-dependent block by charged drugs is explained by the idea that the activation gate in the channel must open before charged drug molecules can gain access to the blocking site ( Fig. 8.3 B).

Ca ²⁺ Channel Block by D600.

(A) The I _Ca traces were recorded during 120-msec voltage clamp steps to +20 mV. The trace labeled control was recorded before the addition of D600. The remaining traces were obtained in the presence of D600 by use of a series of voltage clamp pulses (to +20 mV) delivered at the rate of three pulses per minute. Little block occurs after the first depolarization, but blockade becomes progressively larger with repeated depolarizations. This type of channel inhibition is called use-dependent block. (B) Use-dependent block by D600 will occur if the drug cannot gain access to its binding site when the channel activation gate is closed. When the gate opens, D600 (D) can enter the inner vestibule of the channel, bind to its receptor, and block the channel.

(A) Data from Lee, K.S., & Tsien, R.W. (1983). Mechanism of calcium channel blockade by verapamil, D600, diltiazem, and nitrendipine in single dialysed heart cells. Nature, 302 (5911), 790–794.)

Ca ²⁺ release-activated Ca ²⁺ (CRAC) channels are opened by depletion of endoplasmic reticulum Ca ²⁺ stores

Store-operated channels (SOCs) are plasma membrane Ca ²⁺ channels that are activated by the depletion of Ca ²⁺ stores in the endoplasmic reticulum (ER). The currents through these channels were initially studied by depleting Ca ²⁺ from the ER, and are referred to as Ca ²⁺ release-activated Ca ²⁺ (CRAC) channel currents. CRAC channels have high Ca ²⁺ selectivity, low single-channel conductance, and are inhibited by lanthanide cations. These properties were ultimately the criteria used to identify the protein ORAI1 as a CRAC channel. ORAI1 is a 33 kDa plasma membrane protein with four transmembrane α helices, and the functional channel may be composed of six ORAI1 subunits. ORAI1 channels are activated by the binding of stromal interaction molecule 1 (STIM1), which is a transmembrane protein located in ER membranes. STIM1 serves two functions in activating ORAI1 channels: it acts as the Ca ²⁺ sensor by detecting depletion of ER Ca ²⁺ stores, and it communicates this information to the surface membrane by binding to, and activating, ORAI1 channels. Homologues, ORAI2, ORAI3, and STIM2, have been identified. CRAC channels serve a variety of physiological functions in different cell types. For example, in lymphocytes the channels play a role in causing the rise in [Ca ²⁺ ] _i that is required for Ca ²⁺ -dependent gene transcription. In skeletal muscle, STIM1 and ORAI1 are located close to one another at triads ( Chapter 15 ), where they allow fast Ca ²⁺ entry during store depletion. Mutant mice lacking STIM1 undergo rapid skeletal muscle fatigue because of the absence of store-operated Ca ²⁺ entry. CRAC channels also play a role in replenishing Ca ²⁺ stores in smooth muscle ( Chapter 15 ).