P/Q-type (Cav2.1) calcium channels mediate Ca2+ influx essential for neuronal excitability and synaptic transmission. The CACNA1A gene, encoding the Cav2.1 pore forming subunit, is highly expressed throughout the mammalian central nervous system. Alternative splicing of Cav2.1 pre-mRNA generates diverse channel isoforms with distinct biophysical properties and drug affinities, which are differentially expressed in nerve tissues. Splicing variants can also affect channel function under pathological conditions although their phenotypic implication concerning inherited neurological disorders linked to CACNA1A mutations remains unknown. Here, we quantified the expression of Cav2.1 exon 24 (e24) spliced transcripts in human nervous system samples, finding different levels of expression within discrete regions. The corresponding Cav2.1 variants, differing by the presence (+) or absence (Δ) of Ser-Ser-Thr-Arg residues (SSTR) in the domain III S3-S4 linker, were functionally characterized using patch clamp recordings. Further, the + /ΔSSTR isoforms were used to demonstrate the differential impact of the Familial Hemiplegic Migraine Type 1 (FHM-1) S218L mutation, located in the domain I S4-S5 linker, on the molecular structure and electrophysiological properties of Cav2.1 isoforms. S218L has a prominent effect on the voltage-dependence of activation of +SSTR channels when compared to ΔSSTR, indicating a differential effect of the mutation depending on splice-variant context. Structural modeling based upon Cav2.1 cryo-EM data provided further insight reflecting independent contributions of amino acids in distant regions of the channel on gating properties. Our modelling indicates that by increasing hydrophobicity the Leu218 mutation contributes to stabilizing a structural conformation in which the domain I S4-S5 linker is oriented alongside the inner plasma membrane, similar to that occurring when S4 is translocated upon activation.The SSTR insertion appears to exert an influence in the local electric field of domain III due to an change in the distribution of positively charged regions surrounding the voltage sensing domain, which we hypothesize impacts its movement during the transition to the open state. In summary, we reveal molecular changes correlated with distinct functional effects provoked by S218L FHM-1 mutation in hCav2.1 splice isoforms whose differential expression could impact the manifestation of the neurological disorder.