Terrance Preston Snutch
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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2019)
T-type voltage-gated calcium channels are expressed throughout the central and peripheral nervous systems as well as in several non-neuronal tissues and contribute to variety of functions such as neuronal excitability, intracellular calcium influx, shaping action potentials, pace-making activity, hormone secretion, and neurotransmitter release. Of the three T-type channel isoforms, Cav3.2 is uniquely sensitive to redox modulation with oxidizing reagents inhibiting and reducing compounds enhancing channel activity. This modulation has been shown to alter firing patterns of reticular thalamic neurons and to affect the nociceptive threshold in vivo suggesting that redox modulation of Cav3.2 may play an important role in regulating neuronal activity. A potential source of oxidizing molecules in vivo is neuronal nitric oxide synthase (nNOS), a calcium dependent enzyme which synthesizes nitric oxide (NO) from arginine. Interestingly, the carboxyl terminus of Cav3.2 possesses a putative PDZ-3 binding ligand which is compatible with the PDZ-3 domain of nNOS. I hypothesize that Cav3.2 and nNOS physically interact via the PDZ-3 binding ligand of Cav3.2 and that this physical interaction mediates a functional interaction whereby Cav3.2 activity stimulates nNOS to produce NO which, in turn, inhibits Cav3.2 activity. Cav3.2 and nNOS were expressed in a heterologous system which allowed us to examine the putative PDZ-3 mediated interactions between the two proteins. Immunoprecipitation experiments using Cav3.2 specific antibodies demonstrate that Cav3.2 and nNOS can interact via the carboxyl PDZ-3 ligand of Cav3.2 and that this interaction is disrupted when the PDZ-3 ligand is mutated. Utilizing a NO sensitive fluorometric assay we show that Cav3.2 activity can stimulate nNOS to produce NO and that disruption the PDZ-3 interaction precludes nNOS activation. We also demonstrate that the PDZ-3 mediated physical interaction facilitates the inhibition of Cav3.2 by nNOS derived NO.Disruption of the Cav3.2/nNOS interaction in vivo using intraperitoneal injection of membrane permeable peptides designed to competitively disrupt the PDZ-3 interaction produces an exaggerated respiratory response to changes in available oxygen and a blunted response in the hyperoxic response test. These results indicate that Cav3.2 and nNOS physically and functionally interact to contribute to normal physiological processes.
T-type calcium (Ca²⁺) channels contribute to the normal development of the heart and are also implicated in pathophysiological states such as cardiac hypertrophy. Functionally distinct Cav3 T-type Ca²⁺ channel isoforms can be generated by alternative splicing from each of three different Cav3 genes (Cav3.1, Cav3.2 and Cav3.3), although it remains to be described whether specific splice variants are associated with developmental stages and pathological conditions. Using full length cDNA generated from rat cardiac tissues, this study identified ten major regions of alternative splicing and systematically identified alternative splice variants of cardiac Cav3.2 channels. Quantitative real-time PCR analysis on the mRNA expression of the most common variants revealed preferential expression of Cav3.2(-25) splice variant channels in the newborn rat heart, whereas in the adult heart approximately equal levels of expression of both (+25) and (-25) exon variants was observed. In the adult stage of hypertensive rats, an increase in overall Cav3.2 mRNA expression and a shift towards the expression of Cav3.2(+25) containing channels as the predominant form was observed. This is the first evidence to show that cardiac Cav3.2 is subject to considerable splicing. Moreover, this thesis is also the first study to show developmental and pathological changes in expression of specific splice variants of the Cav3.2 channels. The biophysical characteristics of cloned Cav3.2 splice variants and T-type Ca²⁺ currents from dissociated cultured newborn ventricular myocytes were investigated using whole cell patch clamp analysis. This study showed variant-specific voltage-dependent facilitation (VDF) of Cav3.2 channels attributed to the exclusion of exon 25 in Cav3.2 transcripts. Lastly, this thesis is the first to provide evidence on VDF of T-type currents in rat ventricular myocytes.
Cav2.1 calcium (Ca²⁺) channels are expressed throughout the mammalian central nervous system where they mediate P/Q-type Ca²⁺ currents essential for neurotransmitter release at most fast synapses. In humans, naturally occurring mutations in the CACNA1A gene encoding Cav2.1 are associated with several severe congenital disorders including familial hemiplegic migraine type 1 (FHM-1).Alternative splicing of the Cav2.1 transcript generates multiple functionally distinct channel variants with unique spatial and temporal expression patterns. Yet, whether different Cav2.1 splice variants have distinct responses to FHM-1 missense mutations that relate to the localized, episodic nature of the FHM-1 phenotype has not been explored. Using recombinant Cav2.1 channels, we systematically compared the biophysical effects of three FHM-1 mutations in two prevalent Cav2.1 splice variants. All three FHM-1 mutations caused differential effects on voltage-dependent and kinetic properties when expressed in the short carboxyl terminus variant (Cav2.1 Δ47) compared to the long variant (Cav2.1 +47). Our findings provide important insight concerning the role of Cav2.1 alternative splicing and the pathophysiology of FHM-1.Ca²⁺-dependent facilitation (CDF) of Cav2.1 channels is a powerful means of channel control proposed to play a role in short-term facilitation of synaptic release during repetitive action potentials (APs). However, empirical evidence to support CDF of Cav2.1 as a relevant mechanism of synaptic facilitation in the CNS is limited. As such, short-term facilitation of synaptic release is generally attributed to enhanced vesicle release due to residual Ca²⁺ binding to sensor proteins that directly mediate vesicle fusion and transmitter release. However, we found that two FHM-1 mutations occluded CDF of Cav2.1 in both recombinant and native systems and cause a corresponding attenuation in short-term synaptic facilitation at the cerebellar parallel fibre to Purkinje synapse. This is the first evidence that presynaptic Ca²⁺ at this fast central synapse also enhances Ca²⁺ influx through Cav2.1 by means of CDF and acts as an additional required mechanism for short-term plasticity. Thus, the data supports the notion that CDF of Cav2.1 underlies key aspects of short-term plasticity in the CNS and provides the first evidence that FHM-1 mutations directly affect Cav2.1 CDF.
T-type voltage-gated calcium (Ca2+) channels play critical roles in controlling neuronal excitability, firing patterns, and synaptic plasticity, although the mechanisms and extent to which T-type Ca2+ channels are modulated by G-protein coupled receptors (GPCRs) remains largely unexplored. Investigations into T-type modulation within native neuronal systems have been complicated by the presence of multiple GPCR subtypes and a lack of pharmacological tools to separate currents generated by the three T-type isoforms; Cav3.1, Cav3.2, and Cav3.3. We hypothesize that specific Cav3 subtypes play unique roles in neuronal physiology due to their differential functional coupling to specific GPCRs. Co-expression of T-type channel subtypes and GPCRs in a heterologous system allowed us to identify the specific interactions between muscarinic acetylcholine (mAChR) or metabotropic glutamate (mGluR) GPCRs and individual Cav3 isoforms. Perforated patch recordings demonstrated that activation of Galpha
-coupled GPCRs had a strong inhibitory effect on Cav3.3 T-type Ca2+ currents but either no effect or a stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. Further study of the inhibition of Cav3.3 channels by a specific Galpha
-coupled mAChR (M1) revealed that this reversible inhibition was associated with a concomitant increase in inactivation kinetics. Pharmacological and genetic experiments indicated that the M1 receptor-mediated inhibition of Cav3.3 occurs specifically through a Galpha
signaling pathway that interacts with two distinct regions of the Cav3.3 channel. As hypothesized, the potentiation of Cav3.1 channels by a Galpha
-coupled mGluR (mGluR1) initially characterized in the heterologous system was also observed in a native neuronal system: the cerebellar Purkinje cell (PC). In recordings on PCs within acute cerebellar slices, we demonstrated that the potentiation of Cav3.1 currents by mGluR1 activation is strongest near the threshold of T-type currents, enhancing the excitability of PCs. Ultrafast two-photon Ca2+ imaging demonstrated that the functional coupling between mGluR1 and T-type transients occurs within dendritic spines, where synaptic integration and plasticity occurs. A subset of these experiments utilized physiological synaptic activation and specific mGluR1 antagonists in wild-type and Cav3.1 knock-out mice to show that the mGluR1-mediated potentiation of Cav3.1 T-type currents may promote synapse-specific Ca2+ signaling in response to bursts of excitatory inputs.
Master's Student Supervision (2010 - 2018)
P/Q-type voltage-gated calcium channels are essential for Ca2+ influx and neurotransmitter release in hippocampal synaptic transmission. Through alternative splicing, the exclusion or inclusion of the NP splice variant determines the classification of the P-type or Q-type channels, respectively, which differ in their sensitivity to the peptide toxin ω-Agatoxin IVA (Aga-IVA). Familial hemiplegic migraine type-1 (FHM-1) is an autosomal dominant subtype of migraine caused by gain-of-function missense mutations in the CaV2.1 subunit of P/Q-type channels. The S218L FHM-1 mutation is associated with a particularly severe clinical syndrome which includes ataxia, generalized seizures and fatal cerebral edema, and causes a hyperpolarizing shift in channel activation resulting in an increased proportion of P/Q-type channels being open at the resting membrane potential and is predicted to increase glutamate release. Increased sensitivity of Aga-IVA has been observed in synaptic signaling in CA1 hippocampal neurons of a S218L FHM-1 mouse model although the underlying mechanism is not known. Here, performing subunit and splice-variant specific quantitative real-time PCR on mouse hippocampal regions, I demonstrate that CaV channel subunits and P/Q-type splice variants are not differentially expressed between WT and S218L mice. Using whole-cell patch-clamp electrophysiology on CA1 neurons in mouse brain slices, I further show that the contribution of P/Q-, N- and R-type channels to excitatory miniature release in WT and S218L mice is highly variable. Examining the contribution of P/Q-type channels to evoked release, I show that P/Q-type channels are an important contributor towards the rate of excitatory spontaneous action potential evoked release in WT neurons. Further, that WT CA1 neurons exhibit a large unitary EPSC response evoked by paired-pulse stimulation and was reduced when P/Q-type channels were blocked. In contrast, EPSC amplitude in S218L neurons tended to be smaller compared to EPSC amplitudes from WT although this effect was not consistent. Together, these data suggest that in CA1 neurons P/Q-type channels are predominant in evoked synaptic transmission in WT neurons and that the S218L mutation appears to cause decreased action potential evoked Ca2+ influx. Further investigation is required to determine whether other VGCCs act to compensate evoked release in S218L neurons.
Cav2.1 P/Q-type voltage-gated calcium channels are essential for neurotransmission in many regions of the mammalian central nervous system (CNS). Alternative splicing generates functional diversity between Cav2.1 splice isoforms and is thought to be a mechanism by which fine-tuning and complexity of Cav2.1-mediated activities occur. The Cav2.1 +SSTR splice variant, located in the S3-S4 linker of domain III, has been identified in rodent brain although its effects on the biophysical and pharmacological properties of Cav2.1 have not been previously studied. Here, by performing splice variant-specific quantitative real-time PCR on selected regions of the rat CNS I demonstrate that +SSTR variant channels are differentially expressed spatially with predominant expression in the brainstem, reticular thalamus and spinal cord. Using whole-cell patch-clamp electrophysiology performed on transfected HEK 293 cells I have shown that compared to ΔSSTR channels, +SSTR variants exhibit faster activation kinetics and a hyperpolarizing shift in the voltage-dependence of activation and inactivation. Additionally, the +SSTR and ΔSSTR variants respond differently to increasing durations of action potential waveforms (APWs) with the charge transference through +SSTR channels being significantly less sensitive to APW broadening than ΔSSTR channels. Together, these data suggest that the unique biophysical properties of the Cav2.1 splice variants contribute to distinct roles in CNS synaptic physiology by relaying different types of action potential-encoding synaptic information. Lastly, I examined whether the +SSTR variant affected the sensitivity of Cav2.1 to the gating modifier peptide toxin ω-Agatoxin-IVA. Using whole-cell patch-clamp electrophysiology I found that the effects of ω-Agatoxin-IVA on current block did not significantly differ between the +SSTR and ΔSSTR splice variants suggesting that SSTR insertion does not affect the binding of ω-Agatoxin-IVA to Cav2.1 channels. The differential expression of Cav2.1 splice variants and their unique channel properties provides insight into the mechanisms by which complexity of P/Q-type calcium channel-mediated signaling contributes to CNS physiology.