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Perspectives

Inactivation of N-type Ca²⁺ channels: Ca²⁺ vs. voltage

Stephen W. Jones

The question of Ca²⁺-dependent inactivation of Ca²⁺ channels just won't go away. Two new papers, one in this issue (Shirokov, 1999) and one in the Biophysical Journal (Jones et al. 1999), present divergent views of the mechanism of inactivation of N-type Ca²⁺ channels. These channels are important for neurotransmitter release, so their inactivation is of considerable physiological interest.

It is generally accepted that L-type Ca²⁺ channels of cardiac and smooth muscle inactivate primarily by a rapid Ca²⁺-dependent process, based on several criteria (Eckert & Chad, 1984). (1) Inactivation is faster when the charge carrier is Ca²⁺ (vs. Ba²⁺ or monovalent cations). (2) High extracellular [Ca²⁺] speeds inactivation. (3) The voltage dependence of inactivation is U-shaped, decreasing at strongly depolarized voltages, thus paralleling Ca²⁺ entry. (4) Buffering of intracellular Ca²⁺ reduces inactivation. This last criterion requires special comment. The slow Ca²⁺ buffer EGTA prevented Ca²⁺-dependent inactivation, in neurons of invertebrates (Eckert & Chad, 1984). However, EGTA was ineffective, and the fast Ca²⁺ buffer BAPTA only partially reduced Ca²⁺-dependent inactivation, in cardiac and smooth muscle. The explanation was that Ca²⁺ acts near the inner mouth of an open Ca²⁺ channel, where local [Ca²⁺] is high (>1 µM) and only partially reduced by BAPTA. Ca²⁺ appears to act via calmodulin, bound to a cytoplasmic domain of the Ca²⁺ channel (discussed by Ehlers & Augustine, 1999).

Ca²⁺-independent inactivation is also well established. It is often called 'voltage-dependent inactivation', but (as for Na⁺ and K⁺ channels) such inactivation depends primarily on the state of the channel, rather than on voltage directly. For T-type Ca²⁺ channels, or for L-channels with Ba²⁺ as the charge carrier, inactivation usually increases monotonically with depolarization, saturating at voltages where channels are fully activated.

The primary controversy regards other Ca²⁺ channels, notably N-channels of neurons. A decade ago, the voltage dependence of inactivation was found to be U-shaped, but other tests for Ca²⁺ dependence failed (Jones & Marks, 1989). EGTA or BAPTA did reduce inactivation, but the BAPTA-resistant component was still U-shaped. Most tellingly, BAPTA-resistant inactivation was similar with Ca²⁺ and Ba²⁺, and was unaffected when the current was changed 10-fold by varying [Ba²⁺]. Other studies emphasized Ca²⁺-dependent inactivation of N-channels in the absence of BAPTA (Cox & Dunlap, 1994), although that inactivation was slower than in muscle. Also, inactivation was reduced with monovalent ions as charge carrier (vs. Ca²⁺ or Ba²⁺).

Recently, this controversy seemed to be resolved in favour of state-dependent inactivation. First, U-shaped inactivation was associated with rapid cumulative inactivation, in response to repetitive depolarizations (Patil et al. 1998). Physiologically, that suggested that N-channels might inactivate significantly during a train of action potentials. Mechanistically, it suggested preferential inactivation from 'partially activated' closed states (Patil et al. 1998). Most voltage-dependent channels activate with a sigmoidal delay, implying that multiple closed states are traversed before the channel actually opens. If those intermediate closed states are especially subject to inactivation, repeated depolarizations will produce cumulative inactivation, and weak depolarizations will also populate those states, giving a U-shaped voltage dependence. One precedent for this inactivation mechanism is the Kv2.1 delayed rectifier K⁺ channel (Klemic et al. 1998).

Closed-state inactivation of N-channels seemed to be confirmed by a study of gating currents (Jones et al. 1999). Conformational changes in channels produce tiny 'gating currents' as the voltage-sensing structures move through the membrane's electrical field. Gating currents are detectable only in the absence of the large ionic currents that flow through open channels. Jones et al. (1999) reported that the U-shaped voltage dependence of inactivation was reflected in 'immobilization' of gating current: if inactivation prevents or greatly slows return of the voltage sensors to their resting position, they appear to be immobilized. The U-shape was observed at the reversal potential for the ionic current, or when ionic currents were blocked by replacement of Ca²⁺ with La³⁺ + Mg²⁺.

However, a paper in this issue of The The Journal of Physiology vigorously reopens the debate. First, Shirokov (1999) found that gating charge was not immobilized, but could move rapidly at strongly negative voltages. That is well known for inactivation of Na⁺, K⁺ and certain Ca²⁺ channels, but was not observed by Jones et al. (1999) for N-channels. Second, blockade of ionic current (with Co²⁺ + Gd³⁺) decreased inactivation, and eliminated the U-shaped voltage dependence, in contrast to Jones et al. (1999). Finally, in an especially brave experiment, cells were dialysed with 10-15 µM [Ca²⁺]. That had surprisingly little effect on the N-current amplitude, but inactivation was 2-fold faster. This suggested that 10-15 µM Ca²⁺ does not bind appreciably to the resting closed state, but is comparable to the [Ca²⁺] sensed by the open channel during Ca²⁺-dependent inactivation. Such high concentrations occur only in the immediate vicinity of the pore. Shirokov (1999) suggests that Ca²⁺ acts within the permeation pathway.

We now have two very attractive, but very different, pictures of N-channel inactivation. In one (Shirokov, 1999), Ca²⁺-dependent inactivation occurs in a BAPTA-inaccessible space in the pore. In the other (Patil et al. 1998; Jones et al. 1999), inactivation occurs from partially activated closed states. The main experimental discrepancy is whether the voltage dependence of inactivation is U-shaped in the absence of Ca²⁺ influx. Future studies will be required to resolve this issue.

Cox, D. H. & Dunlap, K. (1994). Journal of General Physiology 104, 311-336	[Abstract]
Eckert, R. & Chad, J. E. (1984). Progress in Biophysics and Molecular Biology 44, 215-267	[Medline]
Ehlers, M. D. & Augustine, G. J. (1999). Nature 399, 105-108	[Medline]
Jones, L. P., DeMaria, C. D. & Yue, D. T. (1999). Biophysical Journal 76, 2530-2552	[Abstract/Full Text]
Jones, S. W. & Marks, T. N. (1989). Journal of General Physiology 94, 169-182	[Abstract]
Klemic, K. G., Shieh, C. C., Kirsch, G. E. & Jones, S. W. (1998). Biophysical Journal 74, 1779-1789	[Abstract/Full Text]
Patil, P. G., Brody, D. L. & Yue, D. T. (1998). Neuron 20, 1027-1038	[Medline]
Shirokov, R. (1999). The Journal of Physiology 518, 697-703.	[Abstract/Full Text]

Email: swj{at}po.cwru.edu

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This Article Abstract Full Text (PDF) Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jones, S. W. Search for Related Content PubMed PubMed Citation Articles by Jones, S. W. Related Collections Perspectives