Figure 6.
CaV1.1-E29 skipping enhances L-type Ca2+ channel conductance and voltage sensitivity in mouse FDB muscle fibers. (A) Diagram of splice shifting antisense morpholino oligonucleotides used to induce CaV1.1 E29 skipping. The ▵E29 morpholinos target sequences near the 3′ and 5′ splice sites of CaV1.1 E29. (B) RT–PCR assay for CaV1.1-E29 (upper) and Serca1-E22 (lower) splicing 16 and 18 days following a single injection and electroporation of ▵E29 morpholinos into FDB muscle. ‘C’ denotes control FDB muscle that was injected and electroporated with vehicle (saline) alone. RNA for the assay was recovered from FDB fibers remaining after completion of patch-clamp recordings. (C) Representative calcium current (ICa) recordings in control (Ctrl, black) and CaV1.1-E29 skipping (ΔE29, grey) fibers obtained during 200 ms depolarizations to −30, −10, +10 and +30 mV. (D) Average (±SE) ICa–V relationships for control (black circles) and ΔE29-deleted fibers (grey circles) fitted by equation (1) with the following parameters for control: Gmax = 108 nS/nF, V0.5 = 1.1 mV, kg = 6.9 mV, Vrev = 62.2 mV and for ▵E29: Gmax = 135 nS/nF, V0.5 = −21.3 mV, kg = 3.5 mV, Vrev = 59.7 mV. Data were obtained from n = 14 fibers for each group. *P < 0.05, **P < 0.01 and ***P < 0.001. (E) The voltage dependence of fractional inactivation, represented as Iend/Ipeak, plotted against test depolarization voltage. All symbols have the same meaning as in (D). Data are presented as mean ± SEM, 14 fibers were recorded for both control and ▵E29 morpholino-treated fibers.