Role of individual S4 segments in gating of Cav3.1 T-type calcium channel by voltage.

Contributions of voltage sensing S4 segments in domains I - IV of CaV3.1 channel to channel activation were analyzed. Neutralization of the uppermost charge in individual S4 segments by exchange of arginine for cysteine was employed. Mutant channels with single exchange in domains I - IV, in two adjacent domains, and in all four domains were constructed and expressed in HEK 293 cells. Changes in maximal gating charge Qmax and the relation between Qmax and maximal conductance Gmax were evaluated. Qmax was the most affected by single mutation in domain I and by double mutations in domains I + II and I + IV. The ratio Gmax/Qmax proportional to opening probability of the channel was significantly decreased by the mutation in domain III and increased by mutations in domains I and II. In channels containing double mutations Gmax/Qmax ratio increased significantly when the mutation in domain I was included. Mutations in domains II and III zeroed each other. Mutation in domain IV prevented the decrease caused by the mutation in domain III. Neither ion current nor gating current was observed when channels with quadruple mutations were expressed. Immunocytochemistry analysis did not reveal the presence of channel protein in the cell membrane. Likely, quadruple mutation results in a structural change that affects the channel's trafficking mechanism. Altogether, S4 segments in domains I-IV of the CaV3.1 channel unequally contribute to channel gating by voltage. We suggest the most important role of the voltage sensor in the domain I and lesser roles of voltage sensors in domains II and III.

Modulation of Cav3.2 T-type calcium channel permeability by asparagine-linked glycosylation.

Low-voltage-gated T-type calcium channels are expressed throughout the nervous system where they play an essential role in shaping neuronal excitability. Defects in T-type channel expression have been linked to various neuronal disorders including neuropathic pain and epilepsy. Currently, little is known about the cellular mechanisms controlling the expression and function of T-type channels. Asparagine-linked glycosylation has recently emerged as an essential signaling pathway by which the cellular environment can control expression of T-type channels. However, the role of N-glycans in the conducting function of T-type channels remains elusive. In the present study, we used human Cav3.2 glycosylation-deficient channels to assess the role of N-glycosylation on the gating of the channel. Patch-clamp recordings of gating currents revealed that N-glycans attached to hCav3.2 channels have a minimal effect on the functioning of the channel voltage-sensor. In contrast, N-glycosylation on specific asparagine residues may have an essential role in the conducting function of the channel by enhancing the channel permeability and / or the pore opening of the channel. Our data suggest that modulation of N-linked glycosylation of hCav3.2 channels may play an important physiological role, and could also support the alteration of T-type currents observed in disease states.

Contrasting the roles of the I-II loop gating brake in CaV3.1 and CaV3.3 calcium channels.

Low-voltage-activated CaV3 channels are distinguished among other voltage-activated calcium channels by the most negative voltage activation threshold. The voltage dependence of current activation is virtually identical in all three CaV3 channels while the current kinetics of the CaV3.3 current is one order slower than that of the CaV3.1 and CaV3.2 channels. We have analyzed the voltage dependence and kinetics of charge (Q) movement in human recombinant CaV3.3 and CaV3.1 channels. The voltage dependence of voltage sensor activation (Qon-V) of the CaV3.3 channel was significantly shifted with respect to that of the CaV3.1 channel by +18.6 mV and the kinetic of Qon activation in the CaV3.3 channel was significantly slower than that of the CaV3.1 channel. Removal of the gating brake in the intracellular loop connecting repeats I and II in the CaV3.3 channel in the ID12 mutant channel shifted the Qon-V relation to a value even more negative than that for the CaV3.1 channel. The kinetic of Qon activation was not significantly different between ID12 and CaV3.1 channels. Deletion of the gating brake in the CaV3.1 channel resulted in a GD12 channel with the voltage dependence of the gating current activation significantly shifted toward more negative potentials. The Qon kinetic was not significantly altered. ID12 and GD12 mutants did not differ significantly in voltage dependence nor in the kinetic of voltage sensor activation. In conclusion, the putative gating brake in the intracellular loop connecting repeats I and II controls the gating current of the CaV3 channels. We suggest that activation of the voltage sensor in domain I is limiting both the voltage dependence and the kinetics of CaV3 channel activation.

A Ca(v)3.2/syntaxin-1A signaling complex controls T-type channel activity and low-threshold exocytosis.

T-type calcium channels represent a key pathway for Ca(2+) entry near the resting membrane potential. Increasing evidence supports a unique role of these channels in fast and low-threshold exocytosis in an action potential-independent manner, but the underlying molecular mechanisms have remained unknown. Here, we report the existence of a syntaxin-1A/Ca(v)3.2 T-type calcium channel signaling complex that relies on molecular determinants that are distinct from the synaptic protein interaction site (synprint) found in synaptic high voltage-activated calcium channels. This interaction potently modulated Ca(v)3.2 channel activity, by reducing channel availability. Other members of the T-type calcium channel family were also regulated by syntaxin-1A, but to a smaller extent. Overexpression of Ca(v)3.2 channels in MPC 9/3L-AH chromaffin cells induced low-threshold secretion that could be prevented by uncoupling the channels from syntaxin-1A. Altogether, our findings provide compelling evidence for the existence of a syntaxin-1A/T-type Ca(2+) channel signaling complex and provide new insights into the molecular mechanism by which these channels control low-threshold exocytosis.

The voltage dependence of gating currents of the neuronal CA(v)3.3 channel is determined by the gating brake in the I-II loop.

Low-voltage-activated Ca(v)3 Ca(2+) channels have an activation threshold around -60 mV, which is lower than the activation threshold of other voltage-dependent calcium channels (VDCCs). The kinetics of their activation at membrane voltages just above the activation threshold is much slower than the activation kinetics of other VDCCs. It was demonstrated recently that the intracellular loop connecting repeats I and II of all three Ca(v)3 channels contains a so-called gating brake. Disruption of this brake yields channels that activate at even more hyperpolarized potentials with significantly accelerated kinetics. We have compared gating of a wild-type Ca(v)3.3 channel and a mutated ID12 channel, in which the putative gating brake at the proximal part of the I-II loop was removed. Voltage dependence of the gating current activation was shifted by 34.6 mV towards more hyperpolarized potentials in ID12 channel. ON-charge movement was significantly faster in the ID12 channel, while the kinetics of the off-charge was not altered by the mutation. We conclude that the putative gating brake in I-II loop hinders not only the opening of the conducting pore but also the activating movement of voltage-sensing S4 segments, stabilizing the channel in its closed state.

Removal of the outermost arginine in IVS4 segment of the Ca(V)3.1 channel affects amplitude but not voltage dependence of gating current.

Positively charged amino acids in S4 segments of voltage-dependent Ca(V)3.1 channel form putative voltage sensor. Previously we have shown that exchange of uppermost positively charged arginine in IVS4 segment for cysteine (mutation R1717C) affected deactivation and inactivation, but not activation of macroscopic current. Now we compared gating currents from both channels. Maximal amplitude of charge movement in R1717C channel decreased but voltage-dependent characteristics of charge movement were not significantly altered. We concluded that mutation of R1717C affects the coupling between S4 activation and pore opening, but not the S4 activation itself.

Cysteines in the loop between IS5 and the pore helix of Ca(V)3.1 are essential for channel gating.

The role of six cysteines of Ca(V)3.1 in channel gating was investigated. C241, C271, C282, C298, C313, and C323, located in the extracellular loop between segment IS5 and the pore helix, were each mutated to alanine; the resultant channels were expressed and studied by patch clamping in HEK293 cells. C298A and C313A conducted calcium currents, while the other mutants were not functional. C298A and C313A as well as double mutation C298/313A significantly reduced the amplitude of the calcium currents, shifted the activation curve in the depolarizing direction and slowed down channel inactivation. Redox agents dithiothreitol (DTT) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) shifted the current activation curve of wild-type channels in the hyperpolarizing direction. Activation curve for all mutated channels was shifted in hyperpolarizing direction by DTT while DTNB caused a depolarizing shift. Our study reveals that the cysteines we studied have an essential role in Ca(V)3.1 gating. We hypothesize that cysteines in the large extracellular loop of Ca(V)3.1 form bridges within the loop and/or neighboring channel segments that are essential for channel gating.