Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC116
Species variations in TRPC4 properties
M. Kustov1, K. Otsuguro2, A. Bavencoffe3, M. X. Zhu3, A. V. Zholos1
1. Centre for Vision & Vascular Science, Queen's University Belfast, Belfast, United Kingdom. 2. Laboratory of Pharmacology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan. 3. Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, Texas, United States.
Transient receptor potential (TRP) channels are a large superfamily of nonselective cation channels, most of which are permeable to calcium. They form ubiquitous receptor- and store-operated channels, as well as more specialised receptors in some cell types (e.g. neuronal thermosensors) (Venkatachalam & Montell, 2007). TRPC4 and related TRPC5 are receptor-operated channels mediating both membrane depolarisation and intracellular Ca2+ increases involved in the regulation of vascular tone and endothelial permeability, neurotransmitter release, and gastrointestinal smooth muscle cholinergic excitation. We have previously investigated mouse TRPC4 isoforms, the full-length mTRPC4α and shorter mTRPC4β, and noted differences in their regulation by PIP2 (Otsuguro et al., 2008). We have now cloned three TRPC4 isoforms from the guinea-pig designated cpTRPC4α, β and γ. Mouse and guinea-pig TRPC4 channels are 95% identical, while most differences are localised to their C-termini. Investigation of these natural differences may provide new insights into TRPC4 structure-function relations and their species variations. The above described TRPC4 isoforms were stably expressed in HEK293 cells grown under culture conditions and recorded using symmetrical Cs+ solutions (125 mM) with intracellular Ca2+ “clamped” at 100 nM with 10 mM BAPTA (Otsuguro et al., 2008). Channels were activated by infusion of GTPγS (0.2 mM) via patch pipette. All TRPC4 isoforms formed functional channels, although current densities at maximal activation (HP=-40 mV) varied from about 30 pA/pF (mTRPC4β) to 80 pA/pF (mTRPC4α and cpTRPC4γ). Voltage-dependent properties were investigated in detail by measuring steady-state I-V relationships (6 s ramps from +80 to -120 mV), converting them into the conductance curves which can be approximated by the Boltzmann relation. Although the voltage dependence was identical in all isoforms (slope of 15-19 mV), the potential of half-maximal activation (V1/2) varied between mouse and guinea pig isoforms. Most interestingly, activation of mTRPC4 did not involve a change in V1/2, while cpTRPC4γ showed a negative shift of ~25 mV of its V1/2 during increased G protein activity. At the same time, the initial V1/2 value (i.e. at low G protein activation) was more negative in mTRPC4α (-65.3±2.1 mV, n=10) compared to cpTRPC4γ ( 47.9±6.8 mV, n=8) and mTRPC4β (-47.3±5.3 mV, n=6) (P<0.02). These differences prompted us to create and investigate two chimeric channels in which the C-termini in mTRPC4β and cpTRPC4β were swapped. The cpTRPC4 with the mouse C-terminus chimera mimicked most closely the V1/2 behaviour seen in wild-type mTRPC4, while the mTRPC4 with the guinea pig C-terminus chimera mimicked that of cpTRPC4γ. The results suggest that the C-terminus of TRPC4 mainly determines the position of the activation curve on the voltage axis and, likely, its regulation via G protein activation.
Where applicable, experiments conform with Society ethical requirements