Voltage Gated Calcium Channels

In the mammals, three subfamilies of voltage-gated calcium genes (Cav) have been cloned: Cav1, Cav2, and Cav3. Cav1 and Cav2 are also commonly referred to as HVA channels, as they require relatively high-membrane depolarizations (> -30 mV) for activation (Hille, 2001). Cav3 is also called LVA channels because they are activated by lower depolarizations (> -70 mV). The HVA Cav1 subfamily (Cav 1.1-1.4) is also called L-type calcium channels, whereas the HVA Cav2 subfamily consists of P/Q (Cav2.1), N (Cav2.2) and R (Cav2.3). Which type of Cav channels are found in axons? Our focus is on recent discoveries in the mammalian axons, though we are fully cognizant that Cav channels have long been described in the squid giant axons.

Four different approaches have been used to establish the presence of Ca channels on mammalian axons. In the first approach, Ca channel contribution to the waveform of action potentials is unmasked by blocking Na and/or K channels. In the second approach, subtype-specific anti bodies are used in immunohistochemistry to establish the presence of certain Cav channel isoforms. In the third approach, the protective action of calcium channel blockers on ischemic axons is examined, from which the calcium channel subtypes that contribute to axonal damage are inferred (Fern et al., 1995a; Stys et al., 1995). In the fourth and final approach, Ca imaging technique is used to measure directly activity-dependent calcium influx in mammalian axons. In this last approach, which receives more attention here because of the author's current interest, the Ca dyes must be selectively introduced into axons and not the surrounding glial cells. Our laboratory has developed a technique where a tight suction pipette is used to load Ca dyes into thousands of axonal cylinders through the cut end of an optic nerve (Fig. 4A) (Verbny et al., 2002). We use a dextran-conjugated Ca dye that significantly increases its molecular size. The use of this dye has three advantages in analysis of axonal Ca signals. First, the large size of dextran excludes glial cell staining because the dye cannot pass through gap junctions. Second, it allows better retention in axons. Third, a dextran-conjugated Ca dye has low mobility, allowing a more faithful analysis of spatial Ca signals (Gabso et al., 1997) (Sabatini et al., 2001). Fig. 4B shows an image of axons stained with Ca dyes using this technique. Once axons are loaded with Ca indicators, and activity-dependent Ca influx detected, it becomes a straightforward experiment to discern various Cav channel subtypes based on the excellent availability of subtype-specific toxins.

Figure 4 Confocal imaging of calcium fluorescence in axons of mammalian optic nerves. (A) Schematics of the imaging setup. An isolated piece of optic nerve is tightly drawn into a glass pipette on the right filled with calcium indicators. The calcium indicators diffuse into the axonal cylinders. The other nerve end is loosely drawn into a glass stimulation pipette. In this setup, simultaneous recordings of axonal calcium and action potentials can be achieved. (B) Representative images of axons stained with calcium indicators. Bar is 20 |m. (Reproduced from Zhang et al., 2004, with permission.)

Figure 4 Confocal imaging of calcium fluorescence in axons of mammalian optic nerves. (A) Schematics of the imaging setup. An isolated piece of optic nerve is tightly drawn into a glass pipette on the right filled with calcium indicators. The calcium indicators diffuse into the axonal cylinders. The other nerve end is loosely drawn into a glass stimulation pipette. In this setup, simultaneous recordings of axonal calcium and action potentials can be achieved. (B) Representative images of axons stained with calcium indicators. Bar is 20 |m. (Reproduced from Zhang et al., 2004, with permission.)

Results using this approach from our laboratory on the mammalian optic nerves are summarized here.

1. N-type Ca Channels (Cav2.2)

Activity-dependent Ca transients are detected in neonatal optic nerve axons (Fig. 5A, B) (Sun and Chiu, 1999). These Ca transients are reversibly eliminated by removal of external Ca, suggesting that they arise primarily from Ca influx, and are blocked by the wide-spectrum Cav channel blockers Cd and Ni. About 58% of the Ca transients are blocked by raconotoxin-GVIA (Fig. 5C, left), an N-type Cav-specific blocker, demonstrating the presence of axonal N-type Ca channels (Sun and Chiu, 1999). However, the identity of the remaining Ca transients remains unclear, as L-type Ca channel blockers, as well as P/Q specific toxins (Fig. 5C, right), did not affect the Ca transient. Of interest, activation of GABAB receptors by baclofen leads to a rapid downregulation of N-type Ca transients in the neonatal optic nerves (Fig. 5A) (Sun and Chiu, 1999). Neuromodulation of axonal Ca signals might be neuroprotective in pathological axons. Fern et al. (1995b) demonstrated that GABAb receptor activation is linked to protection of white matter during metabolic insults, and it is tempting to speculate that this protection stems from inhibition of N-type Ca channels. However, it is unclear if N-type Ca channels exist in adult myelinated nerves (Brown et al., 2001). As described next, N-type Ca channels are upregulated in nerves of certain demyelinated models.

2. L-Type Calcium Channels

Ischemic studies carried out in adult mammalian white matter show that L-type Ca channel blockers are neuropro-tective, suggesting that L-type Ca channels are present on axons and mediate damaging Ca influx during ischemia (Fern et al., 1995a; Brown et al., 2001; Quardouz et al.,

Effect Baclofen Blood Calcium

Figure 5 N-type calcium channels in neonatal rat optic nerves. (A, B) Simultaneous recordings of axonal calcium transients (A) and action potentials (B) using the setup shown in Fig. 4. Note difference in time scale between the calcium and the action potential recordings. Baclofen rapidly reduces the calcium transient (A) without affecting the action potentials (B). (C) Application of raconotoxin-GVIA, an N-type calcium channel blocker, blocks the calcium transients by ~58% (left). Application of ra-Aga-IVA, a P-type channel blocker, is without effect (right). Note that the baclofen-sensitive component of the calcium transient is identical to the raconotoxin-GVIA-sensitive component, suggesting that N-type calcium channels are coupled to GABAB receptors. (Modified from Sun and Chiu, 1999, with permission.)

Figure 5 N-type calcium channels in neonatal rat optic nerves. (A, B) Simultaneous recordings of axonal calcium transients (A) and action potentials (B) using the setup shown in Fig. 4. Note difference in time scale between the calcium and the action potential recordings. Baclofen rapidly reduces the calcium transient (A) without affecting the action potentials (B). (C) Application of raconotoxin-GVIA, an N-type calcium channel blocker, blocks the calcium transients by ~58% (left). Application of ra-Aga-IVA, a P-type channel blocker, is without effect (right). Note that the baclofen-sensitive component of the calcium transient is identical to the raconotoxin-GVIA-sensitive component, suggesting that N-type calcium channels are coupled to GABAB receptors. (Modified from Sun and Chiu, 1999, with permission.)

2003). Immunohistochemistry suggests that L-type Ca channels, unlike Na channels, have a more diffuse distribution on the axons and are not localized at the nodes of Ranvier (Brown et al., 2001). A more recent immunohisto-chemical study shows that L-type Ca channels in myeli-nated axons form clusters with ryanodine receptors, and that these channel-receptor clusters mediate damaging elevation of intracellular Ca in ischemia (Quardouz et al., 2003). Of interest, L-type Ca channels could not be detected pharmacologically in Ca image analysis of neonatal optic nerves (Sun and Chiu, 1999), suggesting that axonal L-type Ca channels are upregulated during myelinogenesis.

3. Ligand-Gated Ca Channels

Various receptors for neurotransmitters are coupled to an ion channel that exhibits significant permeability to Ca. Ligand-gated Ca channels have been extensively characterized in synapses, notable examples being the NMDA receptor, the AMPA receptor, and the nicotinic acetylcholine receptor (nAChR). Recently, nAChR receptors have been demonstrated in mammalian optic nerve axons in Ca imaging studies (Zhang et al., 2004). In axons loaded with Ca indicators, bath application of nicotine induces a robust Ca elevation in the axons (Fig. 6). The Ca elevation is abolished on removal of bath calcium, indicating that nicotine induces Ca influx into axons. The nicotine response is blocked by various nAChR antagonists including curare, suggesting that the Ca influx is receptor mediated. Further, nicotine abruptly shunts the action potential, consistent with the opening of the cationic nAChR on axons. These observations have led Zhang and co-workers (Zhang et al., 2004) to postulate the presence of functional nAChR on axons of

Figure 6 Calcium-permeable, nicotinic acetylcholine receptors on axons of neonatal mouse optic nerves. Axons were loaded with calcium indicators according to Fig. 4A, and pseudo-color calcium images of axons were monitored before (A) and after (B) 50 |M nicotine was bath applied. (C, D) Computed AF/F from images in A and B, showing the percent calcium change on a pixel-by-pixel basis. Bar is 5 | m. (Reproduced from Zhang et al., 2004, with permission.)

mouse optic nerves. Of interest, the nAChR-mediated calcium response declines as the optic nerve matures, suggesting a downregulation or masking of axonal nAChR as myelin is formed. In the neonatal nerves, repetitive stimulation causes a curare-sensitive shunting of the action potential, suggesting that acetylcholine is released during repetitive activity (Zhang et al., 2004). It is possible that part of the activity-dependent axonal Ca elevation is mediated by endogenous acetylcholine release.

Nicotine Support Superstar

Nicotine Support Superstar

Stop Nicotine Addiction Is Not Easy, But You Can Do It. Discover How To Have The Best Chance Of Quitting Nicotine And Dramatically Improve Your Quality Of Your Life Today. Finally You Can Fully Equip Yourself With These Must know Blue Print To Stop Nicotine Addiction And Live An Exciting Life You Deserve!

Get My Free Ebook


Post a comment