The voltage-dependent structural transitions governing the opening of the human t-type Cav3.1 channel: Insights from voltage-clamp fluorometry.

Abstract

Low-voltage-activated CaV3.1 (T-type) Ca2+ channels contribute to neuronal burst-mode firing and heart pacemaker activity. Similar to other voltage-gated channels, the four Voltage-Sensing Domains (VSDs) are expected to activate before the pore opens. However, previous studies attempting to characterize the CaV3.1 voltage-sensing apparatus in channels blocked by La3+ found that most of the gating charge moves after the channel has opened. To shed light on the molecular mechanisms underlying the unique voltage-dependent gating of CaV3.1, we expressed the human CaV3.1 pore-forming subunit α1G in Xenopus oocytes and used voltage-clamp fluorometry (VCF) to optically track the structural rearrangements of the four VSDs. In conducting channels under physiologically-relevant conditions (2mM [Ca2+]out) we found that the activation curves, F(V), of all VSDs preceded the activation of the ionic conductance, G(V): the half-activation potentials of the four F(V) curves ranged between −61±2 mV and −42±1 mV, while G(V) was V1/2(1) = −36±2 mV; V1/2(2) = 6±3 mV. However, in channels blocked by 200 µM La3+ (as in gating current measurements), the voltage dependence of VSD-I, -III and -IV shifted towards depolarized potentials, to the right of the G(V) curve. Only VSD-II activation was not perturbed by La3+. These results suggest that VSD-I, -III and -IV are energetically coupled with the pore and likely responsible for the voltage-dependent activation. Accordingly, the ataxia-causing mutation R1715H (VSD-IV S4) caused a +20 mV shift in VSD-IV activation and a concomitant +10 mV shift in the G(V). Our findings demonstrate that under conducting conditions all VSDs of Cav3.1 operate in a canonical way: they activate before pore opening. We propose that the impaired activation of CaV3.1 carrying the ataxia-causing mutation is caused by the impaired activation of VSD-IV.