Spinocerebellar ataxia type 29 (SCA29) is autosomal dominant congenital ataxia characterized by early-onset motor delay, hypotonia, and gait ataxia. Recently, heterozygous missense mutations in an intracellular Ca²⁺ channel, inositol 1,4,5-trisphosphate (IP₃) receptor type 1 (IP₃R1), were identified as a cause of SCA29. However, the functional impacts of these mutations remain largely unknown. Here, we determined the molecular mechanisms by which pathological mutations affect IP₃R1 activity and Ca²⁺ dynamics. Ca²⁺ imaging using IP₃R-null HeLa cells generated by genome editing revealed that all SCA29 mutations identified within or near the IP₃-binding domain of IP₃R1 completely abolished channel activity. Among these mutations, R241K, T267M, T267R, R269G, R269W, S277I, K279E, A280D, and E497K impaired IP₃ binding to IP₃R1, whereas the T579I and N587D mutations disrupted channel activity without affecting IP₃ binding, suggesting that T579I and N587D compromise channel gating mechanisms. Carbonic anhydrase-related protein VIII (CA8) is an IP₃R1-regulating protein abundantly expressed in cerebellar Purkinje cells and is a causative gene of congenital ataxia. The SCA29 mutation V1538M within the CA8-binding site of IP₃R1 completely eliminated its interaction with CA8 and CA8-mediated IP₃R1 inhibition. Furthermore, pathological mutations in CA8 decreased CA8-mediated suppression of IP₃R1 by reducing protein stability and the interaction with IP₃R1. These results demonstrated the mechanisms by which pathological mutations cause IP₃R1 dysfunction, i.e., the disruption of IP₃ binding, IP₃-mediated gating, and regulation via the IP₃R-modulatory protein. The resulting aberrant Ca²⁺ homeostasis may contribute to the pathogenesis of cerebellar ataxia.

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