Voltage Sensor Operation in the Embryonic Splice Variant of Skeletal CaV1.1 Channels

Abstract

Two splice variants of the voltage-gated skeletal CaV1.1 channel (embryonic and adult forms) have been recently reported by Tuluc and colleagues (2009 Biophys J). The embryonic CaV1.1 channel lacks exon 29, which encodes for 19 amino acids within the S3-S4 loop of the fourth Voltage-Sensing Domain (VSD-IV), and exhibits a left-shifted voltage dependence of activation (G(V)) compared to adult channels, in the rabbit isoform. To elucidate the molecular mechanisms underlying the enhanced voltage-sensitivity of the embryonic variant, we expressed human CaV1.1 channels in Xenopus oocytes and used the voltage-clamp fluorometry technique to optically track the operation of the individual, homologous but not identical CaV1.1 VSDs. We found that the voltage-dependent activation of human embryonic channels was facilitated, compared to the adult form (adult: Vhalf= 30±1mV, z= 2.1±0.04e0, N=10; embryonic: Vhalf= 18±2mV, z= 1.5±0.02e0, N=5). Fluorometry data revealed that VSD-IV operation was drastically modified: in embryonic channels, VSD-IV activated at more depolarized potentials, approaching the G(V) curve, and with steeper voltage dependence (adult: Vhalf= −38±1mV, z= 0.68±0.01e0, N=6; embryonic: Vhalf= 6.2±1.0mV, z= 1.3±0.02e0, N=3); kinetically, VSD-IV activation was ∼1 order of magnitude slower than in adult channels. Furthermore, VSD-I exhibited a decreased voltage-sensitivity by ∼1e0 (adult: Vhalf= 4.2±0.8mV, z= 3.1±0.05e0, N=6; embryonic: Vhalf= −2.1±1.9mV, z= 1.9±0.07e0, N=3). VSDs II and III are currently under investigation. In conclusion, in human embryonic CaV1.1 channels, a shorter S3-S4 loop (lack of exon 29) profoundly remodeled VSD-IV, which acquires kinetics and voltage-dependence compatible with those of pore opening. Surprisingly, this structural change in VSD-IV also altered VSD-I operation, possibly through the pore interaction. We are analyzing the data with a structurally-relevant allosteric model to quantify the coupling energies between each VSD and the pore.