Formation of claudin-10 based tight junctions (TJs) is paramount to paracellular Na⁺ transport in multiple epithelia. Sequence variants in CLDN10 have been linked to HELIX syndrome, a salt-losing tubulopathy with altered handling of divalent cations accompanied by dysfunctional salivary, sweat, and lacrimal glands. Here, we investigate molecular basis and phenotypic consequences of a newly identified homozygous CLDN10 variant that translates into a single amino acid substitution within the fourth transmembrane helix of claudin-10. In addition to hypohidrosis (H), electrolyte (E) imbalance with impaired urine concentrating ability, and hypolacrimia (L), phenotypic findings include altered salivary electrolyte composition and amelogenesis imperfecta but neither ichthyosis (I) nor xerostomia (X). Employing cellular TJ reconstitution assays, we demonstrate perturbation of cis- and trans-interactions between mutant claudin-10 proteins. Ultrastructures of reconstituted TJ strands show disturbed continuity and reduced abundance in the mutant case. Throughout, both major isoforms, claudin-10a and claudin-10b, are differentially affected with claudin-10b showing more severe molecular alterations. However, expression of the mutant in renal epithelial cells with endogenous TJs results in wild-type-like ion selectivity and conductivity, indicating that aberrant claudin-10 is generally capable of forming functional paracellular channels. Thus, mutant proteins prove pathogenic by compromising claudin-10 TJ strand assembly. Additional ex vivo investigations indicate their insertion into TJs to occur in a tissue-specific manner.