A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function.
Bottom Line: Neutralization of four arginine residues eliminated plasma membrane binding.Patch clamp recordings revealed facilitated opening of Cav1.2 channels containing these mutations, weaker inhibition by phospholipase C activation, and reduced expression of channels (as quantified by ON-gating charge) at the plasma membrane.Our data provide new evidence for a membrane binding motif within the I-II linker of LTCC α1-subunits essential for stabilizing normal Ca(2+) channel function.
Affiliation: From the Institute of Pharmacy, Department of Pharmacology and Toxicology, and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria.Show MeSH
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Mentions: All four mutant constructs conducted inward Ca2+ currents. Current-voltage relationships revealed a significant 6–10 mV shift of V0.5 in the hyperpolarizing direction for both mutants (Table 1 and Fig. 6A). This was due to a significant increase in the slope without changes in activation threshold (Table 1), suggesting more efficient coupling of pore opening to membrane depolarization. The mutations had little effect on the kinetics of ICa inactivation during 300-ms depolarizing pulses to the voltage corresponding to the peak of the I-V relationship (Table 2). The apparent reversal potential was also unchanged (Table 1). Differences between the mutants and wild-type channels were observed when we studied the relationship between the size of ON-gating charges (as a measure of active channels on the cell surface; enlarged ON-gating currents are shown in the insets of Fig. 6B) and the size of ionic tail currents (Fig. 6B). As shown by us and others previously, this ratio provides an estimate for the channel's open probability (61–63). The two wild-type constructs served as an internal control because a higher open probability was reported earlier for CaV1.2S (59, 61). This is evident as a statistically significant difference of the ITail/QON ratio in our experiments (Fig. 6B; see Table 1 for statistics). This is also evident from the steeper slopes of the regression lines of plots of ITailversus QON (Fig. 6C). In CaV1.2L, the 4R/4A mutation caused a 64% increase in the ITail/QON ratio. A similar increase was also observed in the CaV1.2S mutants despite higher basal open probability. An even larger effect was seen for the CaV1.2L4E and Cav1.2S4E mutants, which more than doubled the open probability. We also found that all four arginine mutant constructs significantly reduced QON in both CaV1.2L and CaV1.2S, with the reduction again more pronounced for the two 4R/4E mutations. Our data demonstrate that positive charges located in proximity to the transmembrane segment IIS1 within the I-II linker are important determinants of normal CaV1.2 Ca2+ channel function independent of the length of their C-terminal tails and basal open probabilities. The negative shift in V0.5 and higher open probability both indicate a tighter coupling between the voltage sensor and the pore. Based on our membrane targeting analysis, we propose that we have identified a site that is involved in the membrane lipid-dependent stabilization of channel function as well as the regulation of the expression of functional channels at the plasma membrane.
Affiliation: From the Institute of Pharmacy, Department of Pharmacology and Toxicology, and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria.