Two heterozygous Cav3.2 channel mutations in a pediatric chronic pain patient: recording condition-dependent biophysical effects
- 407 Downloads
We report expression system-dependent effects of heterozygous mutations (P769L and A1059S) in the Cav3.2 CACNA1H gene identified in a pediatric patient with chronic pain and absence seizures. The mutations were introduced individually into recombinant channels and then analyzed by means of electrophysiology. When both mutants were co-expressed in tsA-201 cells, we observed a loss of channel function, with significantly smaller current densities across a wide range of voltages (−40 to +20 mV). In addition, when both mutant channels were co-expressed, the channels opened at a more depolarizing potential with a ~5-mV right shift in the half-activation potential, with no changes in half-inactivation potential and the rate of recovery from inactivation. Interestingly, when both mutants were co-expressed in the neuronal-derived CAD cells in a different extracellular milieu, the effect was remarkably different. Although not statistically significant (p < 0.07), current densities appeared augmented compared to wild-type channels and the difference in the half-activation potential was lost. This could be attributed to the replacement of extracellular sodium and potassium with tetraethylammonium chloride. Our results show that experimental conditions can be a confounding factor in the biophysical effects of T-type calcium channel mutations found in certain neurological disorders.
KeywordsCalcium channel T-type Epilepsy Pain Mutation HEK cells
We thank the patient and her family for their cooperation with our research team. We are grateful to Lina Chen for her technical support. We thank Dr. Francois Bernier for the ExAC database search.
Compliance with ethical standards
This work was supported by a grant from the Canadian Institutes of Health Research to GWZ. MAG is supported by a Consejo Nacional de Ciencia y Tecnologia (Mexico) postdoctoral fellowship, and MMW was supported by an Alberta Innovates—Health Solutions summer studentship.
Conflict of interest
The authors declare that they have no conflict of interest.
Informed consent was obtained from the mother for the publication of this article.
- 2.Cain SM, Snutch TP (2013) T-type calcium channels in burst-firing, network synchrony, and epilepsy. Biochimica et Biophysica Acta (BBA) - Biomembranes 1828:1572-1578. doi:http://dx.doi.org/ 10.1016/j.bbamem.2012.07.028
- 11.Bourinet E, Alloui A, Monteil A, Barrere C, Couette B, Poirot O, Pages A, McRory J, Snutch TP, Eschalier A, Nargeot J (2005) Silencing of the Cav3.2 T-type calcium channel gene in sensory neurons demonstrates its major role in nociception. EMBO J 24:315–324. doi: 10.1038/sj.emboj.7600515 CrossRefPubMedPubMedCentralGoogle Scholar
- 12.Francois A, Kerckhove N, Meleine M, Alloui A, Barrere C, Gelot A, Uebele VN, Renger JJ, Eschalier A, Ardid D, Bourinet E (2013) State-dependent properties of a new T-type calcium channel blocker enhance Ca(V)3.2 selectivity and support analgesic effects. Pain 154:283–293. doi: 10.1016/j.pain.2012.10.023 CrossRefPubMedGoogle Scholar
- 13.Garcia-Caballero A, Gadotti VM, Stemkowski P, Weiss N, Souza IA, Hodgkinson V, Bladen C, Chen L, Hamid J, Pizzoccaro A, Deage M, Francois A, Bourinet E, Zamponi GW (2014) The deubiquitinating enzyme USP5 modulates neuropathic and inflammatory pain by enhancing Cav3.2 channel activity. Neuron 83:1144–1158. doi: 10.1016/j.neuron.2014.07.036 CrossRefPubMedGoogle Scholar
- 16.Wen XJ, Li ZJ, Chen ZX, Fang ZY, Yang CX, Li H, Zeng YM (2006) Intrathecal administration of Cav3.2 and Cav3.3 antisense oligonucleotide reverses tactile allodynia and thermal hyperalgesia in rats following chronic compression of dorsal root of ganglion. Acta Pharmacol Sin 27:1547–1552. doi: 10.1111/j.1745-7254.2006.00461.x CrossRefPubMedGoogle Scholar
- 22.Hans M, Luvisetto S, Williams ME, Spagnolo M, Urrutia A, Tottene A, Brust PF, Johnson EC, Harpold MM, Stauderman KA (1999) Functional consequences of mutations in the human α1A calcium channel subunit linked to familial hemiplegic migraine. The Journal of Neuroscience 19:1610–1619PubMedGoogle Scholar
- 23.Tottene A, Fellin T, Pagnutti S, Luvisetto S, Striessnig J, Fletcher C, Pietrobon D (2002) Familial hemiplegic migraine mutations increase Ca(2+) influx through single human CaV2.1 channels and decrease maximal CaV2.1 current density in neurons. Proc Natl Acad Sci U S A 99:13284–13289. doi: 10.1073/pnas.192242399 CrossRefPubMedPubMedCentralGoogle Scholar
- 25.Cummins TR, Aglieco F, Renganathan M, Herzog RI, Dib-Hajj SD, Waxman SG (2001) Nav1. 3 sodium channels: rapid repriming and slow closed-state inactivation display quantitative differences after expression in a mammalian cell line and in spinal sensory neurons. The Journal of Neuroscience 21:5952–5961PubMedGoogle Scholar
- 27.Peloquin JB, Khosravani H, Barr W, Bladen C, Evans R, Mezeyova J, Parker D, Snutch TP, McRory JE, Zamponi GW (2006) Functional analysis of Ca3.2 T-type calcium channel mutations linked to childhood absence epilepsy. Epilepsia 47:655–658. doi: 10.1111/j.1528-1167.2006.00482.x CrossRefPubMedGoogle Scholar
- 30.Powell KL, Cain SM, Ng C, Sirdesai S, David LS, Kyi M, Garcia E, Tyson JR, Reid CA, Bahlo M, Foote SJ, Snutch TP, O'Brien TJ (2009) A Cav3.2 T-type calcium channel point mutation has splice-variant-specific effects on function and segregates with seizure expression in a polygenic rat model of absence epilepsy. J Neurosci 29:371–380. doi: 10.1523/JNEUROSCI.5295-08.2009 CrossRefPubMedGoogle Scholar