We used a human embryonic kidney cell line, tsA201, to express cloned neuronal CaV
2.2 N-type calcium ion channel chosen for its established role in regulating neurite outgrowth, transmitter release, and neuronal signaling.[17
] This technique avoids current from other types of channels and thus isolates the behavior of the CaV
2.2 N-type calcium ion channel. Calcium currents originating from synchronous activation of CaV
2.2 channels were recorded using the whole-cell patch clamp recording method (, and Supplemental
). We exposed cells to physiological solutions containing different concentrations of water soluble (aryl-sulfonate functionalized[19
]) arc synthesized single-walled carbon nanotubes (SWNTs) and monitored the magnitude and voltage-dependence of calcium currents.
Figure 1 SWNT sample A inhibits calcium ion channels, while SWNT sample B does not. a: Schematic of whole cell patch clamp experiment. b: Time course of calcium current under constant voltage step exposed to 100 μg/ml SWNT A. Horizontal bar indicates the (more ...)
Calcium currents were inhibited rapidly when cells were exposed to SWNT-containing solutions (; sample “A”). Inhibition was dose-dependent () and apparent at all voltages without altering the voltage-dependence of channel activation (). The magnitude and speed of inhibition was unexpected and indicative of direct inhibitory effects on the calcium ion channel (). We removed the SWNTs from solution through centrifugal ultrafiltration and, surprisingly, observed an almost equivalent level of inhibitory activity (), which was similar in time course to the action of the original SWNT-containing solution (). This inhibition occurs in the absence of the original nanotubes.
The inhibitory action of the supernatant indicates a mechanism involving nanotube-induced alteration of the fluid medium, rather than direct interaction between tubular graphene and the ion channel or other cellular targets. High-surface-area SWNTs have been shown to significantly alter cell culture medium through adsorption of folic acid[20
] and molecular probes[21
] and can release soluble metal forms[16
] through oxidative attack on metal catalyst residues that are not fully encapsulated by graphenic carbon shells[16
]. To assess the role of metals, we therefore tested the effects of SWNT B, a sample determined to be unusually well purified with respect to free metal (). Physiological solutions containing SWNT B had no detectable inhibitory activity on voltage-gated calcium channels up to 100 μg/ml (). Since the supernatant of SWNT A had similar inhibitory capability to SWNT A containing solutions, and the unusually well purified sample SWNT B did not have any inhibitory action, this strongly suggests a metals effect rather than an alteration of the buffer composition through adsorption.
Figure 2 Bioavailable nickel and yttrium in SWNT samples. a, b: Metal catalyst nanoparticles visible by TEM. Scale bar: 100 nm. Arrows point to catalytic particles. The total metal mass percentages by digestion and ICP-ES are 23.3% nickel and 5.77% yttrium for (more ...)
The nanotubes in our studies were fabricated using a nickel-yttrium catalyst, and abundant metal nanoparticles are visible in both SWNT samples by TEM (). Previous studies have reported that a small portion of CNT-imbedded metal is typically fluid accessible through defective carbon shells, and can become solubilized in physiological buffers by slow oxidation[16
]. This “bioavailable” metal fraction is not always eliminated by current purification protocols, and does not correlate well with total metal content[16
]. As both SWNT samples contain visible metal nanoparticles, we hypothesized that nickel and yttrium are released and solubilized into the recording solution in sufficient quantities to inhibit the channels from SWNT A but not B. We used inductively coupled plasma- atomic emission spectrometry (ICP-AES) to measure levels of bioavailable nickel and yttrium () in the recording solution (See Supplemental
). shows that both SWNT samples contain bioavailable nickel, but only SWNT A contains detectable quantities of bioavailable yttrium.
We next tested the sensitivity of N-type calcium channels to nickel and yttrium cations in control salt solutions. Both metals inhibited calcium current rapidly, but yttrium was 300-fold more potent than nickel (Nickel IC50 = 219 ± 40 μM; Yttrium IC50 = 0.76 ± 0.15 μM; n = 3; ). Similar inhibitory effects of yttrium have been documented on native calcium channels and calcium-dependent processes[25
] consistent with inhibitory effects of other ions of the lanthanide series at protein calcium binding sites[25
]. Of the trivalent ions in the lanthanide series, yttrium is the most potent inhibitor of high voltage-activated calcium channels[27
]. Yttrium like cadmium, another potent inhibitor of high voltage-activated calcium channels, is thought to inhibit calcium flow by competing for calcium binding sites of the ion selectively filter of the pore[26
] (see ). To characterize the factors governing yttrium release from nanotubes, we included two additional SWNT samples (C and D) and studied the location, form, and behavior of CNT-associated yttrium. SWNT C has been commercially functionalized with carboxylate groups. SWNT D was included to show that significant concentrations of bioavailable metal may remain in vendor purified samples. A majority of CNT-associated metal in these samples is in the form of metal-rich nanoparticles encapsulated by thin carbon shells of variable thickness (). Partial oxidation of SWNT D was carried out to simulate oxidative purification processes for removal of amorphous carbon by heating. Air oxidation attacks carbon shell structures (See Supplemental
), greatly increasing the mobilization of Y and Ni into media (). The dissolved Y:Ni ratio is much higher than the initial condensed-phase Y:Ni ratio of 1:7. This indicates preferential oxidation and solution release of Y over Ni, consistent with the higher oxidation potential of Y (2.37 V[29
]) relative to Ni (0.25 V). While SWNT D has been “purified” by the supplier, it nevertheless contains sufficient free Y to inhibit channel function, even before air oxidation is employed to remove carbon shells and increase metal bioavailability (~ 10 μM at zero carbon loss in , which by , is 13 times the IC50 of Y).
Figure 3 Control experiments with soluble salts isolate the effects of Ni and Y on neuronal voltage-gated calcium ion channels. a-c: Nickel effects. a: Time course showing decreasing calcium current with increasing nickel dose. Solid bars represent time and dose (more ...)
Figure 5 Summary of proposed mechanism through which carbon nanotube suspensions inhibit neuronal calcium ion channels. Nickel-yttrium catalyst nanoparticles (Purple) are oxidatively corroded by fluid-phase attack through defects or cracks in the surrounding carbon (more ...)
Figure 4 Yttrium phases in SWNTs and mechanisms of yttrium ion release and recapture by SWNT surface functional groups. a: Typical metal catalyst morphology in arc-synthesized SWNT (sample D): Ni/Y nanoparticles are encapsulated in thin (2 – 10 nm) carbon (more ...)
In samples subjected to acid purification, yttrium salt re-deposition on surface functional groups is also possible and may be an unappreciated source of bioavailable metal in nanotubes. shows that sulfonate and carboxylate functional groups introduced on CNTs can bind soluble yttrium from solution. The adsorption isotherms in panel 4e were used to derive fundamental equilibrium constants for yttrium binding to CNT-carboxylate (See Supplemental
) and the competitive effects of Na+
binding on yttrium adsorption from saline solutions. We report dissociation constants: Kd
= 1.2 μM (for Y3+
); and Kd
= 9060 μM (for Na+
). The low Kd
for soluble Y is consistent with the expected strong binding of the hard Lewis acid Y3+
to the hard carboxylate anion. This strong binding allows significant Y3+
adsorption to occur even in the presence of Na+
, which is the major ion in physiological saline (~1500 factor higher concentration than yttrium). The Kd
/SWNT-COO-) is 26.5 μM[24
], which is also much higher than Kd
(1.2 μM), implying that Y3+
has the potential to replace Ca2+
on carboxylic binding sites.