Abstract
SummaryVoltage-gated calcium channels are exquisitely Ca2+ selective, conferred primarily by four conserved pore-loop glutamate residues contributing to the selectivity filter. There has been little previous work directly measuring whether the trafficking of calcium channels requires their ability to bind Ca2+ in the selectivity filter or to conduct Ca2+. Here, we examine trafficking of neuronal CaV2.1 and 2.2 channels with mutations in their selectivity filter and find reduced trafficking to the cell surface in cell lines. Furthermore, in hippocampal neurons, there is reduced trafficking to the somatic plasma membrane, into neurites, and to presynaptic terminals. However, the CaV2.2 selectivity filter mutants are still influenced by auxiliary α2δ subunits and, albeit to a reduced extent, by β subunits, indicating the channels are not grossly misfolded. Our results indicate that Ca2+ binding in the pore of CaV2 channels may promote their correct trafficking, in combination with auxiliary subunits. Furthermore, physiological studies utilizing selectivity filter mutant CaV channels should be interpreted with caution.
Highlights
Voltage-gated calcium channels (CaV) are exquisitely Ca2+ selective (Hess and Tsien, 1984), and the molecular basis for this key attribute was probed as soon as the first L-type channels were cloned
CaV2.2 Channels with Mutations in Their Selectivity Filters Have Reduced or No Inward Calcium Channel Current Here, we have examined the importance of the CaV2 channel selectivity filter for channel trafficking, taking advantage of our previously described extracellular hemagglutinin (HA)-tagged CaV2.2 construct, in which the tag does not affect function (Cassidy et al, 2014)
We found that the selectivity filter mutant channels, co-expressed with a2d-1 and b1b, were present in the plasma membrane to a much lower extent than WT CaV2.2: a 53.5% reduction for CaV2.2 EIA and a 67.8% reduction for CaV2.2 EI, II, III, IVA (Figures S3A and S3B)
Summary
Voltage-gated calcium channels (CaV) are exquisitely Ca2+ selective (Hess and Tsien, 1984), and the molecular basis for this key attribute was probed as soon as the first L-type channels were cloned. Initial seminal studies with CaV1.2 identified the fundamental role of four conserved selectivity filter glutamates (E) for divalent cation binding in the channel pore and for permeation (Ellinor et al, 1995; Yang et al, 1993). Key evidence for their involvement in the Ca2+ binding sites within the pore came from the finding that they are essential to divalent cation-mediated block of channel permeation by monovalent cations. These studies supported the idea of a single high (mM)-affinity binding site that is occupied by blocking cations such as Cd2+ (Yang et al, 1993)
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